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Nitrogen fertilization as a way to sequester carbon in forests of British Columbia Yolova, Veneta Dimitrova 2007

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N I T R O G E N F E R T I L I Z A T I O N A S A W A Y T O S E Q U E S T E R C A R B O N I N F O R E S T S OF B R I T I S H C O L U M B I A by V E N E T A D I M I T R O V A Y O L O V A B.Sc. University of Forestry (Bulgaria), 2003 A THESIS S U B M I T T E D FN P A R T I A L F U L F I L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R OF S C I E N C E in T H E F A C U L T Y O F G R A D U A T E S T U D I E S (Forestry) T H E U N I V E R S I T Y OF B R I T I S H C O L U M B I A M a y 2007 © Veneta Dimitrova Yolova , 2007 ABSTRACT Greenhouse gases (GHG) , especially carbon dioxide (CO2), are believed to be the major cause of global warming today. The increased concentration of atmospheric CO2 after the Industrial Revolution (late 18 t h and early 19 t h century) is attributed mostly to fossil-fuel combustion and deforestation. Consumption of fossil fuels and unsustainable use of forests are not likely to cease soon, hence alternative strategies for removal of atmospheric CO2 are sought. This study provides information on the potential of forest fertilization with nitrogen (N) to sequester additional carbon (C) in tree biomass and soil. Two tree species (lodgepole pine and western hemlock), which are economically important in the province of British Columbia (BC) , were examined. The amounts of C sequestered in tree biomass, understory vegetation, and soil after fertilization with N -alone (or N+B), and complete ( N + other nutrients) were measured and compared with the amounts of C sequestered in non-fertilized plots. The effect of N on cellulolytic and ligninolytic enzyme activity was also examined. Fertilization increased the individual and stand-level tree biomass; although the stand-level tree biomass did not significantly differ between treatments. Understory vegetation biomass was not significantly affected by N fertilization; although there were some differences in species composition between treatments. Fertilization increased the soil C concentration (%) in the lodgepole pine stand and the soil C content (t/ha) in the western hemlock stand. Although there was greater average total ecosystem C stored in biomass and soil (to 40 cm depth) in fertilized plots, the differences between treatments were not statistically significant. Enzyme activity was not significantly affected by fertilization, except for one of the ligninlytic enzymes (peroxidase) for which activity increased after the N+B treatment. The results i i suggest that forest fertilization with N w i l l accelerate C storage in these forests. However the decision as to whether this practice is the most beneficial way to sequester C should be made only after a detailed cost-benefit analysis and consideration of multiple desired outcomes for the forest in question. 111 T A B L E O F C O N T E N T S Page A B S T R A C T i i T A B L E O F C O N T E N T S iv LIST O F T A B L E S v i LIST O F F I G U R E S v i i A C K N O W L E D G E M E N T S v i i i 1 I N T R O D U C T I O N 1 Rationale 1 Background 2 Greenhouse gases 2 Accumulation of carbon 3 Forest fertilization 6 Effect of N on microbial communities 8 Objectives 13 Hypotheses 13 2 M A T E R I A L S A N D M E T H O D S 14 Study sites 14 Kenneth Creek 14 SCff lRP 16 Sampling description 19 Biomass estimation 21 Enzyme assays 22 iv Statistical analyses 23 3 R E S U L T S 24 Biomass gains 24 Changes in soil C and N content 29 Ecosystem response to N additions 33 Changes in enzyme activity 35 4 D I S C U S S I O N 41 5 C O N C L U S I O N S 51 R E F E R E N C E S 53 v LIST O F T A B L E S Page Table 2.1 Soil profile description at the Kenneth Creek site 16 Table 2.2 Typical soil profile description of the C H (cedar-hemlock) ecosystem at S C H I R P 18 Table 3.1. Stand-level aboveground biomass + C content of lodgepole pine and western hemlock to fertilization treatments: Control; N B = nitrogen and boron; N = nitrogen, and C O M = complete 26 Table 3.2 Stand-level aboveground biomass + C content of western redcedar, total understory vegetation and salal at the S C H I R P site to fertilization treatments: Control; N = nitrogen, and C O M = complete 27 Table 3.3 Estimated belowground-biomass and C content (t/ha) o f lodgepole pine and western hemlock at the Kenneth Creek and S C H I R P sites in the fertilization treatments: Control; N B = nitrogen and boron; N = nitrogen, and C O M = complete 28 Table 3.4 Carbon concentration (%), bulk density (t/m 3), dry mass (t/ha), and C content (t/ha) of soils at the Kenneth Creek site 30 Table 3.5 Carbon concentration (%), bulk density (t/m 3), dry mass (t/ha), and C content (t/ha) of soils at the S C H I R P site 31 Table 3.6 Nitrogen concentration (%) in different soil layers at the Kenneth Creek and S C H I R P sites 32 v i LIST O F F I G U R E S Page Figure 3.1 Aboveground-biomass response of lodgepole pine at the Kenneth Creek site to fertilization treatments: Control; N B = nitrogen and boron; C O M = complete 25 Figure 3.2 Aboveground-biomass response of western hemlock at the S C H I R P site to fertilization treatments: Control; N = nitrogen; C O M = complete 26 Figure 3.3 Total C content (t/ha) at the Kenneth Creek site in response to fertilization treatments: Control; N B = nitrogen and boron, and C O M = complete 33 Figure 3.4 Total C content (t/ha) at the S C H I R P site in response to fertilization treatments: Control; N = nitrogen, and C O M = complete 34 Figure 3.5 Phenol oxidase activity in different soil layers at the Kenneth Creek site 36 Figure 3.6 Peroxidase activity in different soil layers at the Kenneth Creek site 36 Figure 3.7 Phenol oxidase activity in different soil layers at the S C H I R P site 37 Figure 3.8 Peroxidase activity in different soil layers at the S C H I R P site 37 Figure 3.9 P-glucosidase activity in different soil layers at the Kenneth Creek site 38 Figure 3.10 Cellobiohydrolase activity in different soil layers at the Kenneth Creek site. 38 Figure 3.11 p-glucosidase activity in different soil layers at the S C H I R P site 39 Figure 3.12 Cellobiohydrolase activity in different soil layers at the S C H I R P site 39 Figure 3.13 Significant positive correlation between peroxidase activity and N concentration (%) in the forest floor at Kenneth Creek 40 Figure 3.14 Significant negative correlation and regression modal between glucosidase activity and N concentration in H I layer at S C H I R P 40 v i i ACKNOWLEDGEMENTS First and foremost I would like to thank my supervisor, Dr. Cindy Prescott, for giving me the opportunity to work on this project and for guiding me patiently and encouraging me through my whole educational process as a M S c . student at U B C . I would also like to thank my committee members Dr. Sue Grayston and Dr. Maja Krz i c , as well as my non-departmental examiner Dr. Andrew Black for reviewing my thesis and giving very useful advice which greatly improved my work. I would like to acknowledge Rob Brockley and Dr. Paul Sanborn who kindly provided data and wil l ingly shared their broad professional experience and knowledge in forest fertilization and soil science. I am very grateful to many people at U B C , especially in the Faculty of Forestry, for their friendship and support. Special thanks to Sara, Denise, Nate, Jason and Pedro who always found time to discuss and answer my plentiful "quick questions" :). I am happy to have worked and shared so much fiin with my office mates Sara, Shannon, Rachelle, Lucie, Jocelyn, Amer, Jason and Pedro. Last but not least, many thanks to my special people - my lovely husband, brother, parents and Bulgarian friends for their endless encouragement, love and support. v in 1 INTRODUCTION Rationale Atmospheric concentrations of greenhouse gases (GHG), including CO2, are a major environmental issue today. Scientists believe that fossil-fuel combustion and cement production along with land-use change are responsible for the increase in concentrations of CO2 in recent decades (Melillo, 1996). Society's consumption of fossil-fuel energy and deforestation are not likely to cease soon, so alternative strategies for reducing G H G concentrations are needed. Sequestration of atmospheric CO2 into vegetation and soil is another possible means of reducing atmospheric CO2 concentration, and means of sequestering additional CO2 are sought. In this study I examine the potential of forest fertilization with nitrogen (N) to sequester atmospheric CO2 into living vegetation and soil. I examine two economically important tree species which are often operationally fertilized (lodgepole pine and western hemlock). I compare the amounts of C sequestered in tree biomass, understory vegetation and soil in plots fertilized with N and other nutrients and non-fertilized plots during the 12-18 years after fertilization. The study provides information on the potential C sink-strength of these forests and offers some suggestions for further research. 1 Background Greenhouse gases Greenhouse gases are those which trap longwave (infrared) radiation emitted by the Earth's surface and hence contribute to the increase of temperatures on the planet, i.e. "global warming" (Baede et al., 2001). Greenhouse gases include: water vapour, carbon dioxide (CO2), methane (CH4), nitrous oxide (N2O), ozone (O3), sulfur hexafluoride (SF6). About 60% of the observed global warming is thought to be due to the increased CO2 concentration in the atmosphere (Grace, 2004). Carbon dioxide is common in the atmosphere, related to natural processes of respiration, decomposition of organic matter, and volcanic activity, but the rapid increase in CO2 concentration in the air after the Industrial Revolution (late 18 t h and early 19 t h century) has been attributed to human activities. The amount of CO2 has increased by more than 30% since pre-industrial times and is still increasing at an unprecedented rate of 0.4% per year (Baede et al, 2001). The main sources of CO2 emissions are combustion of fossil fuels, cement production, and tropical land use changes (Meli l lo et al, 1996). A n obvious solution to the problem would be to reduce emissions by reducing and/or replacing fossil fuels use with bioenergy (Pussinen et al, 1997; Marland et al, 1997) or other renewable sources (e.g., sun, wind, water) and managing land in a more sustainable manner. Although the introduction of renewable energy has supporters, use of fossil fuel is not likely to disappear soon - either in developing countries or in industrialized ones. For example in E U countries in 2000, energy use was 41% from oi l , 23% from gas, 15% from coal, 15% from nuclear, and 6% from renewable - of which 3.7% was from biomass. Unsustainable land management, particularly deforestation, is 2 also unlikely to cease soon, as land is needed for cropland, fuel-wood, grazing areas, migrating and landless people (Cannell, 2003). Another means of reducing CO2 concentrations in the air is to use methods which remove CO2 from the atmosphere and sequester it in other pools. Carbon (C) sequestration is a process of removal and long-term storage of CO2 from the atmosphere through the use of natural sinks. Natural sinks could be living vegetation (especially forests), soils, geological formations, oceans, and wood products. Accumulation of carbon Carbon is a basic element for all l iving organisms. It represents between 45% and 52% of tree dry biomass (45 or 50% are usually used) (Nabuurs et al, 1997). In order to be sequestered in tree biomass, C must be added in their structure. This happens through the process of photosynthesis during which plants capture CO2 from the atmosphere and convert it into organic compounds which are used in building tissues. The accumulation rate of organic matter depends on the tree's growth rate. When plants take up more CO2 through photosynthesis, they increase their size and hence store more C. Tree growth requires adequate amounts of nutrients and water, and a favorable environment. Growth characteristics also vary between species, varieties and even between parts of the same tree (Kozlowski , 1979, 1971). Tree age also affects growth - aging is associated with reduced metabolism, reduced tissue growth, increased number o f dead branches, and lower resistance towards pathogens (Kozlowski, 1971). Tree growth is the result of complex interactions between physiological processes controlled by both plant heredity and surrounding conditions (Kozlowski and Pallardy, 1997). Therefore, i f we wish to manipulate the rate of tree growth by altering the natural conditions, we should be aware 3 of consequent changes in the ecosystem (Kozlowski , 1979); by changing C sequestration capacity we may alter ecosystem structure and functioning (Pussinen et al, 1997). The storage period of the accumulated C is also important, which brings up the question of harvest rotation. Not all tree species can be logged at the same age - some fast-growing trees may reach a particular size in 20-40 years, while others could take more than 100 years to reach the same size. Harvesting methods and forest management procedures can affect the C storage period. Silviculture practices like thinning, pruning, or selective logging may increase growth rate, but may also cause damage and consequent mortality of some of the remaining trees and hence reduce stand biomass production (Masera et al, 2003). Mortality of tropical forests due to selective logging can reach up to 20% of the remaining basal area (Alder and Silva, 2000). Mortality is related to the intensity of logging, density of the stand, years after the harvesting operation, methods used and technology (Masera et al, 2003; Pinard and Putz, 1996). On the other hand, selective logging can supply harvestable biomass without losing the entire C sequestered (Marland et al, 1997). The fate of the trees after harvesting also affects carbon sequestration. They may be used for fuel, or converted into paper products, or become furniture. The life-span of the forest products depends on the end use, on the longevity of wood itself, and the social status and behavior of the consumers (Masera et al, 2003). Fortunately, tree biomass is not the only sink of C. After the deep oceans, soil is the second largest C reservoir (Raich and Potter, 1995; Schimel, 1995). Soil is an important reservoir because in some ecosystems humus accumulation can take place over millennia (Paul, 1984; Wardle et al, 1997). If there are no major disturbances, the 4 amount of forest floor can reach 109 kg/m2 in temperate ecosystems (Berg et al, 1993) or 49 kg/m2 in boreal forest after 3000 years (Wardle et al, 1997). Schimel (1995) states that the amount of C in boreal forest soils could be three times more than that accumulated in plants. Currently, the total Canadian C stocks are estimated to be 88 Gt (12 Gt of standing biomass and 76 Gt in soil and peat) (Fluxnet Canada website). However, the rate and amount of C storage vary considerably among ecosystems. Build-up of a stable humus, and hence C sequestration, depends on microbial decomposition and mineralization (Berg and McClaugherty, 2003). Decomposition is a process of physical breakdown and mass loss of organic matter by soil microbial communities, while mineralization refers to the conversion of organic compounds into inorganic forms, which could be taken up by plants (Prescott, 2005). Decomposition rate is controlled by a diversity of factors such as temperature, moisture, chemical and physical characteristics of the litter and humus, nutrient availability, soil texture, site-specific factors, microbial communities, and human activity (Prescott, 2005; Berg and McClaugherty, 2003). Heterogeneity of litter composition and its interaction with the factors mentioned above makes organic-matter decay an extremely complex process, which can follow variable scenarios (Berg and McClaugherty, 2003). However, decomposition can be generalized into a process with two (Melillo et al, 1989) or three phases (Berg and McClaugherty, 2003). Berg and McClaugherty (2003) distinguish an early stage, late stage, and humus-near stage. However, a clear border between the late stage and humus-near stage is hard to define and "near-humus" and true humus have some common properties. Nevertheless, the model appears to be appropriate for different types of foliar and root litter and also for needle litter of many pine species. 5 The decay of newly-shed litter starts with decomposition of solubles (e.g., sugars, phenolics, hydrocarbons and glycerides [note: phenolics are highly variable regarding their solubility]), cellulose and hemicelluloses. The late stage can be recognized with litter containing mostly lignin and some remains of cellulose and hemicellulose enclosed and protected by lignin. In the humus-near stage, decay almost stops or goes so slowly that it may be described by an asymptote or as a limit value for the decomposition process. The limit value for foliar litter is somewhere between 50 and 100% mass loss. The initial N concentration of litter is believed to be negatively correlated with the limit value, or in other words, more N-r ich litter w i l l reach its limit value sooner and w i l l remain less decomposed (Berg and McClaugherty, 2003). The productivity of forests can be limited by insufficient supply of one or more nutrients (Cannel et al, 1976; Binkley, 2001). A l l terrestrial plants need at least 16 chemical elements which they can obtain either from the air and water (e.g., C , H and O), or from the soil solution (e.g., inorganic ions such as Ca or NO3 ). Each of these nutrients takes part in specific biophysical and/or biochemical processes; therefore the lack of even one can disturb the normal development of plants (Glass, 1989). Forest fertilization Forest fertilization is one of the most common and successful ways to correct mineral deficiency and improve plant growth (Kozlowski and Pallardy, 1997). Nutritional management has had different purposes over time. In the beginning, the goal was to increase the survival and growth of seedlings and newly established stands (Ingerslev et al, 2001). Since the 1950s, fertilizers have been applied to middle-aged and mature stands in order to enhance the production of high-value sawlogs (Kukkola and Saramaki, 6 1983; Ingerslev et al, 2001). During the 1970s and 1980s, air pollution and acid rain became a "hot topic" and scientists started to think of fertilization as a way to counteract soil acidification and nutrient depletion (Ingerslev et al, 2001). Currently, fertilization has multiple functions - increasing wood productivity, and improving forest vitality and ecosystem stability (Saarsalmi and Malkdnen, 2001). Fertilization may also provide an alternative strategy for reducing air pollution by sequestering more CO2 in tree biomass and soil (Makipaa, 1995; Olsson et al, 2005). Nitrogen (N) is an essential nutrient for living organisms. It is a constituent of all plant proteins (Glass, 1989), and is required by plants in larger amounts than other essential elements (Lousier et al, 1991). Nitrogen, together with low temperatures, are the most important growth-limiting factors in boreal and temperate forests (Makipaa, 1995; Lousier et al, 1991; Carreiro et al, 2000). The low N mineralization rates and hence N deficiency in boreal forests are attributed to the low soil p H , low temperatures and poor litter quality (Paavolainen, 1999). Common ways to add N to an ecosystem are the use o f N-f ixing plants, municipal or industrial wastes, or most often an application of synthetic fertilizers (Chappell et al, 1992). Currently only four elements are operationally applied as fertilizers to forests in British Columbia (BC), namely: N , S (sulphur), P (phosphorus), and B (boron). The last three nutrients are usually added to the fertilizer recipe in order to prevent nutrient deficiencies and misbalances of other elements due to the larger amounts of N taken up by trees (Anonymous, 1995). The main N sources used for forest fertilization are urea [(TSfFL^CO] and ammonium nitrate (NH4NO3) which differ in physical and chemical properties (e.g. melting point, % N , p H of 1 M solution) (Nason and Myrold , 1992). Another major difference can be seen in 7 their breakdown products - ammonium nitrate hydrolyzes to N H 4 and NO3 ions, both of which can be absorbed by plants, while urea hydrolyzes only to N r L ; + which may be oxidized to NO2 and NO3 and can cause different environmental effects (Lousier et al, 1991). The choice of fertilizer seems to be a compromise from the known pros and cons of the N source for a specific situation (Hauck, 1967). Currently urea is the main fertilizer broadly used for operational applications in B C due to its good response history, simple requirements for storage, availability, higher N content (46% N in urea vs. 34% N in ammonium nitrate) and lower cost (Anonymous, 1995). The efficiency of forest fertilization depends on the rate of plant uptake, which can be less than 10% (Preston et al, 1990) or up to 30% (Nason, 1989; Bauer et al, 2004) depending on the weather, site and stand conditions, and fertilizer properties. What happens to the rest of N ? Generally N does not remain in soil in inorganic forms for a long time. It is either assimilated by plants or heterotrophs; or involved in reactions with soil particles, or nitrifiers and denitrifiers (Johnson, 1995). Effect of N on microbial communities Changes in microbial community structure and function can be expected after human-driven N deposition in terrestrial ecosystems (Frey et al, 2004). Soderstrom et al. (1983) found decreased microbial biomass and activity, in both field study and laboratory experiment, after application of N fertilizers (NH4NO3 and urea). Nohrstedt (1988) also detected decreased microbial activity and biomass per gram of C in fertilized plots. Fog (1988) reported that N additions had no effect or sometimes a markedly negative effect on microbial activity. Sjoberg (2003) confirmed that long-term N addition reduced the decomposition rate of soil organic matter. Bowden et al. (2004) found suppressed soil 8 respiration in both coniferous and mixed deciduous stands after 13 years of continuous N fertilization. Similar results were obtained by Olsson et al. (2005) - decreased heterotrophic and autotrophic respiration in fertilized compared to non-fertilized plots despite the much higher aboveground production in the fertilized plots. Soderstrom et al. (1983) and Agren et al. (2001) hypothesized that inhibition of CO2 production in fertilized plots is due to formation of recalcitrant compounds between the organic matter and the added N (ammonia and amino compounds). Sjdberg (2003) could not detect any heterocyclic N compounds either in N-fertilized or in non-fertilized forest floors. He concluded that condensation processes of this kind could happen, but not to an extent which would be significant for the humification process. We should also keep in mind that these reactions between ammonia and phenolic compounds occur at high p H (Nommik, 1970), whereas forest floors of boreal forests usually have low p H values. Another explanation for the slower decomposition rates in N-fertilized plots could be a reduced enzyme activity of white-rot fungi responsible for degradation of lignin (Keyser et al, 1978; Carreiro et al, 2000; Frey et al, 2004). Enzymes are believed to control major soil biological processes, such as organic matter degradation, mineralization, and nutrient cycling (Marx et al, 2001). They are proteins which participate in specific types of chemical reactions by causing them to proceed at faster rates. Enzyme sources could be microbial organisms, or plant and animal residues. Regarding the form under which they exist in the substrate, we can distinguish three types of enzymes: free enzymes (e.g., exoenzymes released from living cells), endoenzymes (released from disintegrating cells), and enzymes stuck to cell constituents 9 (e.g., disintegrating cells, cell fragments, and alive but non-proliferating cells) (Tabatabai, 1994). A s an integral part of soil biochemical processes, enzymes could be used as functional indicators (Marx et al., 2001). Some of the factors affecting rates of enzyme activity are temperature, moisture, p H , and availability of inhibitors. Enzyme inhibition could be provoked by a variety of organic and inorganic chemicals; hence, compounds such as fertilizers, pesticides, municipal and industrial wastes can influence enzyme activity (Tabatabai, 1994). Degradation of the main biopolymers in litter, namely cellulose and lignin, is an important step in the C cycle and C sequestration. For long-term C storage in soil, lignin has greater potential than cellulose because lignin is usually degraded more slowly than many other litter components and is a large contributor to the residues of the terrestrial biomass (Kogel-Knabner, 2002; Berg and McClaugherty, 2003). Lignin is highly resistant to microbial decomposition, and is insoluble in water, and many organic solvents and strong acids ( M c C o l l and Gressel, 1995). Understanding the mechanisms controlling lignin degradation may help us predict the rate and amount of organic matter accumulation in soil. Although lignin could be degraded by other groups of decomposers (e.g., soft-rot, brown-rot fungi, bacteria), white-rots are the most studied because they have the potential to mineralize lignin structures to CO2 and H 2 0 by producing phenol oxidase and other enzymes important for complete lignin degradation (Carreiro et al., 2000; Berg and McClaugherty, 2003). Ligninolytic activity could be suppressed by several factors. Carreiro et al. (2000) found reduced phenol-oxidase activity in N-enriched oak litter, although it was not clear i f these results were due to suppressed enzyme production or to decreased abundance of 10 white-rot species relative to other fungal communities in the N-enriched substrate. Based on an experiment with Phanerochaete chrisosporium (wood-rotting hymenomycete), Keyser et al. (1978) concluded that efficient degradation of lignin in nature by this species, and maybe by white-rot fungi in general, could occur only in N-limiting environments. Contrarily, other studies have shown that some fungal species isolated from N-rich environments are not as sensitive to N enrichment (Freer and Detroy, 1982; Leatham and Kirk, 1983). These results suggest that suppression of lignin mineralization does not always occur in N-enriched substrate - some fungi are actually tolerant to N addition and by applying more N we could even increase their activity (Fog, 1988; Collins and Dobson, 1997; Berg and McClaugherty, 2003). The third hypothesis for the decreased C mineralization is explained by a shift of microbial communities in favor of microorganisms with higher N assimilation efficiency (Agren et al, 2001). Frey et al. (2004) found changes in microbial community composition, based on significantly lower fungal: bacterial biomass ratio. There was also reduced phenol-oxidase activity, but increased P-glucosidase (cellulose-degrading enzyme) activity in fertilized plots compared to non-fertilized plots. This shift of microbial communities could be explained by the fact that bacteria have a lower C/N ratio (Griffin, 1985). The average C-to-N ratio of bacteria is ~6 and that of fungi is -15. This means that bacteria have a higher N demand than fungi and N-rich environments may be more favorable for bacterial communities (Wallenstein et al, 2006). However, Compton et al. (2004) counted fungi and bacteria by direct microscopy and culture techniques and could not find any clear response to N additions. DeForest et al. (2004) also found no changes in the PLFAs proportion of the bacteria, actinomycetes, fungi and 11 protozoa in soil due to chronic NO3 deposition, but significantly lower microbial biomass. Frey et al. (2004) detected differences in fungal diversity between fertilized and non-fertilized plots showing that the most abundant species in control plots decreased in fertilized ones. Reduced fungal richness with increasing N inputs was also observed by Lilleskov et al. (2002). However, there is an important contrast between the Frey et al. (2004) and Lilleskov et al. (2002) data. Whereas Frey et al. (2004) showed a decline of one E M F (ectomycorrhizal fungi) species due to the applied N in Harvard forest, Lil leskov et al. (2002) found the opposite response for the same fungus - Lactarius theiogalus, in fertilized plots in Alaska. Moreover, Wright (2006, pers. comm.) found no long-term negative effect of fertilization (N-alone, and N+P) on overall E M F biodiversity. There was high species richness in all treatments, and the N+P treatment had significantly higher abundance. These contradictory findings support the necessity of additional studies which examine the whole ecological mechanism (biotic and abiotic factors) responsible for potential community shifts after N enrichment (Frey et al, 2004). The question of whether the decreased rate of organic matter decomposition during the late stage of decay is due to lignin, N , or both together is still unanswered (Berg and McClaugherty, 2003). But the potential for N deposition to influence the mineralization rate is worth investigating as a potential means of manipulating soil C retention and accurately predicting ecosystem C sequestration (Carreiro et al, 2000; Waldrop et al, 2004). B y altering the dynamics of humus formation through N fertilization, we may be able to increase the C sink strength o f northern forests (Magi l l and Aber, 1998). 12 Objectives The objective of this research was to investigate the potential of N fertilization to increase C sequestration in B C forests. I examined two economically important species, which are often operationally fertilized in B C - lodgepole pine (Pinus contorta Dough var. latifolia Engelm.) and western hemlock (Tsuga heterophylla (Rafh.) Sarg.). M y specific objectives were: 1. To quantify the amount of additional C in tree biomass and soil sequestered after forest fertilization with N ; 2. To compare enzyme activity of lignin- and cellulose-degrading microorganisms in fertilized and non-fertilized plots. Hypotheses 1. N fertilization w i l l increase stem biomass, stand-level biomass, and belowground biomass; 2. N fertilization w i l l be associated with less complete litter decay and larger accumulation of humus and soil organic matter. The reasons for this could be: • suppressed enzyme activity among white-rot fungi responsible for degradation of lignin; • altered microbial composition and assimilation efficiency; • formation of recalcitrant phenolic compounds. 13 2 MATERIALS AND METHODS Study sites Kenneth Creek The Kenneth Creek installation is located approximately 75 km east of Prince George, B C within the Wi l low variant of the wet cool subzone of the Sub-Boreal Spruce Biogeoclimatic Zone ( S B S w k l ) (Pojar et al, 1986). The climate of the SBS zone is continental, and is characterized by seasonal extremes of temperature; severe, snowy winters; relatively warm, moist, and short summers. The mean annual temperature for this zone is between 1.7 and 5°C and the mean annual precipitation is between 440-900 mm of which 25-50% falls as snow (Meidinger et al, 1991). Soi l and vegetation description indicates the site belongs to the submesic Sxw-Huckleberry-Highbush cranberry (05) site series (DeLong, 1993). Derived from thick, well-sorted glaciofluvial outwash parent material, the soil is well-drained and stone-free, with a fine-to-medium loamy sand texture. The soil is classified as an Eluviated Dystric Brunisol (Soil Classification Working Group, 1998). A detailed description of the soil profile is given in Table 2.1. The site was logged in 1980, broadcast burned in 1982, and planted in the spring of 1983 with lodgepole pine 1+0 container stock. The plantation was chemically brushed in 1986. A t the time of trial establishment in 1993, the stand was 12 years old and had an average density of approximately 1360 stems per hectare. A l l treatment plots were thinned to a uniform density of 1100 stems per hectare during plot establishment (Brockley and Simpson, 2004; Sanborn et al, 2005). 14 The site is a part of Experimental Project (EP) 886.13 titled "Maximizing the Productivity of Lodgepole Pine and Spruce in the Interior of British Columbia" which has been designed and implemented by the B C Ministry of Forests and Range: http://vvww.for.gov.bc.ca/hfd/pubs/Docs/Tr/Tr018.htm We looked at the effect of three treatments: • Control - non-fertilized • N B - fertilized every 6 years with 200 kg N/ha, and 1.5 kg B/ha. • Complete - fertilized every 6 years with 200 N , 100 P, 100 K , 50 S, 25 M g , 1.5 B (kg/ha). Each treatment was replicated in three 0.164 ha plots. Nitrogen was added as urea (46% N) , and B as granular borate (15% B) . The treatments were assigned under a completely randomized experimental design. Each treatment plot was divided into 16 equal-sized sectors prior to fertilizer application and pre-measured amounts of the specified fertilizer blend were uniformly broadcast-applied by hand to each of the 16 sectors (Brockley and Simpson, 2004). 15 Table 2.1 Soi l profile description at the Kenneth Creek site. Horizon Depth (cm) Description L - H 2-0 Litter and semi-decomposed organic material; abrupt, wavy boundary; extremely acid: pH (CaCI2) = 3.58, and pH (H 20) = 4.23. Ae 0-5 Light brownish grey (10YR 6/2 m); sandy loam; 0 % coarse fragments; weak fine platy; very friable; plentiful fine and medium horizontal roots; abrupt wavy boundary; 3-8 cm thick; extremely acid pH (CaCI2) = 3.44, and pH (H 20) = 4.20. Bf 5-12 Strong brown (7.5YR 4/6 m); loamy sand; 0 % coarse fragments; massive; friable; plentiful fine and medium, few coarse, horizontal roots; gradual wavy boundary; 4-13 cm thick; extremely acid: pH (CaCI2) = 4.35, and pH (H 20) = 4.84. Bfj 12- 27 Brown (10YR 4/3 m); sand; 0% coarse fragments; massive; friable; plentiful fine and medium, few coarse, horizontal roots; gradual wavy boundary; 9-15 cm thick; very strongly acid: pH (CaCI2) = 5.01, and pH (H 20) = 5.49. Bm 27 - 60 Olive brown (2.5Y 4/4 m); sand; 0 % coarse fragments; single grain; very friable; few fine and medium oblique roots; diffuse wavy boundary; 30-40 cm thick; very strongly acid: pH (CaCI2) = 4.95, and pH (H 20) = 5.67. B C 60- 100 Greyish-brown (2.5Y 5/2 m); sand; 0 % coarse fragments; single grain; very friable; no roots; diffuse wavy boundary; 40-50 cm thick; strongly acid: pH (CaCI2) = 5.11, and pH (H 20) = 5.98. C 100 -125+ Dark greyish brown (2.5Y 4/2 m); sand; 0% coarse fragments; single grain; very friable; no roots; very strongly acid: pH (CaCI2) = 4.99, and pH (H 20) = 5.82. Source: Adopted from Sanborn et al., 2005: Table 7. Morphological description of representative Eluviated Dystric Brunisol pedon, Kenneth Creek site; and Table 8. Physical and chemical characteristics of Eluviated Dystric Brunisol, Kenneth Creek site. S C H I R P The second site is a part of S C H I R P (Salal Cedar Hemlock Integrated Research Program) - a research collaboration between Western Forest Products Ltd. , B C Ministry of Forests and Range, Canadian Forest Service, and the University of British Columbia (UBC) . The study area is on northern Vancouver Island, between the towns of Port Hardy and Port 16 M c N e i l l , B C . The area is within the very wet maritime subzone of the Coastal Western Hemlock ( C W H ) biogeoclimatic zone (Pojar et al, 1986), and has a maritime climate with mild winters and cool moist summers. Mean annual precipitation is about 1700 mm, 65% of which occurs between October and February, and almost all is rain. Mean annual temperature is 7.9°C and daily averages range from 2.4°C in January to 13.8°C in August. The surface geological material is deep unconsolidated morainal and fluvial outwash material overlying three types of bedrock: gently dipping sedimentary rocks of the Cretaceous Nanaimo formation, relatively soft volcanics of the Bonanza group and a small area of harder Karmutsen formation basalt which protrudes through the moranial cover (Prescott and Weetman, 1994). The soils are imperfectly to moderately-well drained Duric Humo-Ferric Podzols with coarse-to-medium textured materials. The B f horizon is of firm consistency and only moderately deep due to a highly cemented t i l l layer which occurs at a depth of 40 to 50 cm (Germain, 1985). They have mor humus, 10-25 cm deep (Prescott, 1996). A detailed description of the soil profile is given in Table 2.2. The former stand was a normal density stand of hemlock and cedar over 141 years of age and 30 to 40 m in height. The stand was logged in 1979 and burned in the fall of 1980. The cutover was then planted in 1981 with 2-0 western hemlock plugs at 3 x 3 m spacing or 1030 stems per ha (Barker et al, 1987). Fertilizer was applied by hand in 1987 and again in 1997.1 looked at the effects of three treatments, replicated three times: • Control - non-fertilized; • N200 - 200 kg N/ha; 17 • Complete - N 200, P 99, S 50, C u 1.5, K 102, Fe 9.0, B 1.5, Ca 129, Z n 3.5, M o 1.0, M g 51, and M n 3.75 (kg/ha). The plot size was 0.025 ha. Nitrogen was added as urea, and P as triple super phosphate. The experimental design was randomized complete block design (Barker et al, 1987). Table 2.2 Typical soil profile description of the C H (cedar-hemlock) ecosystem at SCHIRP . Horizon Depth (cm) Description LF 27 - 26 Mixture of coniferous and salal litter and mosses; loose consistency; no roots; abrupt wavy boundary to, H 26-0 Reddish black, dark reddish brown; massive; abundant roots all sizes; abrupt wavy boundary to, Ae 0-4 Grey to brown; medium subangular blocky; friable; few fine and medium roots; clear, broken to, Bhf 4-19 Red, dark brown to brown; sandy loam; weak medium subangular blocky; firm when moist; non-sticky and slightly plastic, wet; plentiful fine roots, few medium and coarse roots; abrupt wavy boundary to, Bf 19-34 Yellowish red, yellowish brown; sandy loam; medium subangular blocky; firm when moist; non-sticky and non-plastic, wet; few fine roots; abrupt wavy boundary to, Bfgj 34 - 48 Yellowish brown, brownish yellow; gravelly sandy loam; weak, medium and coarse subangular blocky, extremely firm when moist, non-sticky and non-plastic; no roots; common faint mottles, seepage water present; abrupt wavy boundary to, BCc 48+ Strongly cemented to indurated gravelly sandy loam. Source: Adapted from: Germain, 1985: Table 3. Soi l profile description of the CH-phase. 18 Sampling description To check my first hypothesis, forest floor and mineral soil samples were collected from fertilized and non-fertilized plots at each installation. Stratified-random sampling was used. The number o f samples was 16 at Kenneth Creek (within an area of 0.164 ha) and nine at S C H I R P (within an area of 0.025 ha). These numbers were decided arbitrarily which is a usual practice when the variability is not known or cannot be estimated and the required number of samples cannot be determined according to a specified degree of accuracy by means of equations (Cline, 1944). Considering our area as homogeneous with respect to soil type, plant growth and treatment and using recommendations of other authors (Anonymous, 1978), the number of samples should be adequate given the limits of time, costs and objectives. The forest floor was sampled with a wooden frame (20 x 20 cm inside opening) at both Kenneth Creek and S C H I R P sites. With the help of a knife, the forest floor (L and F horizons together) was cut around the inside of the frame and collected into a pre-labeled plastic bag. Humus (H layer) was put into separate bags. Fertilized plots at S C H I R P had humus formed from litter of the present stand (HI) , as well as residual humus from the previous stand (H2), so the two H layers were collected separately. From the samples were excluded: • A l l green stems and leaves of l iving herbaceous plants • Lichens and green upper portions of mosses • Branches, twigs, slash fragments, woody debris, charcoals, cones • Roots > 1 mm. 19 I sampled mineral soil only at S C H I R P ; data for Kenneth Creek was available from our collaborators Paul Sanborn ( U N B C ) and Rob Brockley ( B C Ministry of Forests and Range, Vernon). Two samples from 0 - 2 0 and 20 - 40 cm depths were taken for estimation of total C and N contents. Two separate samples per plot in 20-cm increments were taken to estimate bulk density using the soil core method (Blake and Hartge, 1986). Samples were stored in a refrigerator at 4°C until further processed. The samples were oven-dried at 50°C to a constant weight. Oven-drying may cause some C to be lost due to oxidation of organic matter, but drying at elevated temperatures may also cause destruction of a number of microorganisms (Tan, 1996). Moreover, for routine soil-testing, all Canadian provinces recommend drying of samples before analysis, and for B C this drying temperature is 55°C (Carter, 1993). Dry forest-floor samples were reduced in size by quartering in order to save time and labor grinding the whole sample. The sample was spread uniformly over a flat clean surface and divided into four equal parts (special attention was taken when the sample was heterogeneous in particle size in order to be split properly). Two of the portions were collected and the remainder was discarded. The collected material was ground in a Wiley mi l l to pass through a 1-mm sieve for C and N analysis. Five to 10 g of each soil sample and 2 - 3 g of each forest floor sample was analyzed for total C and N concentrations by the dry combustion method using a L E C O CN-2000 analyzer at the U B C Soil Science Laboratory (Matejovic, 1993; Jimenez and Ladha, 1993). Diameter at breast height (DBH) and height of all trees within each of the nine plots were measured at S C H I R P in July 2005. Data for Kenneth Creek from 2005 measurements were provided by Rob Brockley. A t S C H I R P three samples of understory 20 vegetation per plot were taken at representative locations in each plot. The percentage of total understory vegetation cover per plot was determined visually and was used to adjust the estimations of total understory vegetation on area basis (t/ha). A l l vegetation within the sampled area of 0.5 m 2 was clipped at the base. Samples were stored at 4°C and transported to U B C , where they were oven-dried and measured. Kenneth Creek's understory vegetation was not sampled because there were no obvious differences between the fertilized and non-fertilized plots neither in species variety nor in their growth rate (Brockley, 2005, pers. comm.). Two composite (n = 3) samples per plot from the forest floor and mineral soil layers, at both Kenneth Creek and S C H I R P , were taken for enzyme assays. Samples were kept frozen until further processed. Biomass estimation The aboveground tree biomass was estimated by equations established for British Columbia tree species (Standish et al, 1985): y = a + b D D H , where y = total aboveground biomass (dry mass); a, b = coefficients (lodgepole pine: a = 34.7, b = 160.8; western hemlock: a = 29.8, b = 155.8); D = diameter at breast height in meters; H = height in meters. The belowground tree biomass was estimated using equations developed for the Carbon Budget Model of the Canadian Forest Sector (L i et al, 2003). : R B = 0.222AB, 21 P f = 0.072 + 0.354 e"UU0UKB, where, RB and AB = root and aboveground biomass, respectively; Pf- fine-root proportion of the total root biomass (RB). Enzyme assays Enzyme assays were performed to test the second hypothesis and to check for differences in the microbial activity between fertilized and non-fertilized plots. The activity of ligninolytic enzymes (phenol-oxidase and peroxidase) was tested colourimetrically, while the activity of cellulolytic enzymes (P-glucosidase and cellobiohydrolase) was tested fluorimetrically. L-3,4-dihydroxyphenylalanine (DOPA) was the substrate used for the measurement of phenol-oxidase and peroxidase activities, while 4-methylumbelliferone(MUB)-P-D-glucoside and 4-MUB-P-D-cellobioside were the substrates used for the measurement of P-glucosidase and cellobiohydrolase activities, respectively. Sample sizes were 0.02 g of forest floor and mineral soil for cellulolytic and 0.25 g for ligninolytic enzyme assays. After incubation in the dark (P-glucosidase - 3 hours, cellobiohydrolase - 7 hours, phenol-oxidase - 18 hours, and peroxidase - 5 hours) the activities of phenol-oxidase and peroxidase were quantified by microplate spectrometer which measured absorbance at 450 nm; and the activities of P-glucosidase and cellobiohydrolase were measured using a microplate fluorometer with 360 nm excitation and 450 nm emission. All enzyme activities were expressed in units of nmol/h/g. The procedure was adopted in protocols for the use of B E G (Belowground Ecosystem Group, UBC) and is fully described in Sinsabaugh et al. (2003). 22 Sta t i s t i ca l analyses Analysis of variance in a completely randomized design at Kenneth Creek and in a randomized complete block design at S C H I R P was used to test for differences between fertilized and non-fertilized plots in terms of individual and stand-level tree biomass (above- and belowground), understory vegetation, soil C and N concentration (%) and content (t/ha), and enzyme activity. Treatments (at Kenneth Creek and SCHIRP) and blocks (at SCHIRP) were considered fixed factors. Further analyses were conducted by Scheffe's multiple comparisons to test for differences among particular means. A n alpha level of 0.05 was chosen to be significant for all analyses. Data were checked for normality and homogeneity of variance. When the assumptions of parametric analysis were not met, data were transformed (e.g. logarithm, square root). However, all data presented here is in the original units. When the transformed data still did not meet the assumptions of a parametric test, non-parametric analyses were performed using Kruskal-Wallis test for independent samples. Pearson correlation was used to determine i f there was a relationship between N concentration (%) and enzyme activity. Linear regression was used to examine whether N concentration reliably predicts the enzyme activity. Statistical analyses were performed using SPSS 10.0.5 (1999 SPSS Inc., Standard Version). 23 3 RESULTS Biomass gains Positive responses of aboveground individual-tree biomass to fertilization were detected at both Kenneth Creek and SCHIRP sites (Figures 3.1 and 3.2, respectively). A t Kenneth Creek, biomass gains following fertilization with N+B and complete fertilizer were relatively small; however, statistically significant compared to non-fertilized plots. There was no significant difference in tree biomass between the two fertilizer treatments (Fig. 3.1). The positive effect of fertilization was more pronounced at S C H I R P (Fig. 3.2). Individual tree biomass responses to N-alone and complete fertilizer were significantly higher than in non-fertilized control plots. Biomass gains in the complete fertilizer treatment were significantly greater than those in the N-alone treatment. A t the stand level (t/ha), increased biomass due to fertilization was apparent (Table 1), but differences between treatments were not significant at either site. Effects of fertilization on other plant species growing at the S C H I R P site (western redcedar (Thuja plicata D.Don) trees, total understory vegetation and salal (Gaultheria shallon Pursh)) were not significant between treatments (Table 3.2). However, there were some changes in the species composition: bunchberry (Cornus canadensis L.) was growing only in the control plots, while fireweed (Epilobium angustifolium L.) was absent in these plots. Although changes in salal abundance were not significant due to high variability, there was more salal in complete-fertilizer plots than in control or N -alone plots. The proportion of total understory vegetation that was salal declined from about 90% in control and complete plots to about half in plots fertilized with N-alone. 24 Stand-level belowground biomass (t/ha) of lodgepole pine and western hemlock did not significantly differ between treatments (Table 3.3). Control NB Treatment COM Figure 3.1 Aboveground-biomass response of lodgepole pine at the Kenneth Creek site to fertilization treatments: Control; N B = nitrogen and boron; C O M = complete, 12 years after trial establishment. Different letters above the error bars indicate significant differences between treatments at the level of p < 0.05. Each value is the mean of 191 trees; error bars indicate standard deviation. 25 100 Control N COM Treatment Figure 3.2 Aboveground-biomass response o f western hemlock at the S C H I R P site to fertilization treatments: Control (n = 70); N = nitrogen (n = 78); C O M = complete (n = 73), 18 years after trial establishment. Different letters above the error bars indicate significant differences between treatments at the level o f p < 0.05. Each value is the mean; error bars indicate standard deviation. Table 3.1 Stand-level aboveground biomass + C content of lodgepole pine and western hemlock to fertilization treatments: Control; N B = nitrogen and boron; N = nitrogen, and C O M = complete. Same letters next to the mean values indicate no significant differences between treatments within each site at the level o f p < 0.05 (n = 3). S D = standard deviation. Site (species) Treatment Tree biomass, t/ha C content, t/ha Mean SD Mean SD Kenneth Creek (lodgepole pine) Control NB COM 100.4 a 5.02 107.4 a 6.13 106.9 a 1.85 50.2 a 2.51 53.7 a 3.07 53.4 a 0.92 SCHIRP (western hemlock) Control N COM 31.0 a 3.44 41.0 a 19.67 60.0 a 8.10 15.5 a 1.72 20.5 a 9.84 30.0 a 4.05 26 Table 3.2 Stand-level aboveground biomass + C content of western redcedar, understory vegetation and salal at the S C H I R P site to fertilization treatments: Control; N = nitrogen, and C O M = complete. Same letters next to the mean values indicate no significant differences between treatments within each plant species/group at the level of p < 0.05 (n = 3). SD = standard deviation. Plant species Treatment B iomass, t/ha C content, t/ha Mean SD Mean SD Western redcedar Control N C O M 156.1a 51.82 124.3 a 81.04 71.4 a 27.25 78.0 a 25.91 62.2 a 40.52 35.7 a 13.62 Understory vegetation * Control N C O M 14.6 a 15.5 27.6 a 18.89 22.9 a 22.86 7.6 a 7.75 13.8 a 9.44 11.5 a 11.43 Salal Control N C O M 13.4 a 10.48 13.0 a 5.16 20.9 a 4.35 6.7 a 5.25 6.5 a 2.58 10.5 a 2.17 Salal has also been included in the understory vegetation biomass. 27 Table 3.3 Estimated belowground-biomass and C content (t/ha) of lodgepole pine and western hemlock at the Kenneth Creek and S C H I R P sites in the fertilization treatments: Control; N B = nitrogen and boron; N = nitrogen, and C O M = complete. Same letters next to the mean values indicate no significant differences between treatments within each root component of each tree species at the level of p < 0.05 (n = 3). SD = standard deviation. Treatment Lodgepole pine Western hemlock (Kenneth Creek) (SCHIRP) Mean SD Mean SD Control 3.7 a 0.05 2.1 a 0.16 Fine-root biomass NB or N 3.7 a 0.05 2.4 a 0.65 C O M 3.7 a 0.02 3.1 a 0.18 Control 1.8 a 0.02 1.1 a 0.08 Fine-root C content NB or N 1.9 a 0.02 1.2 a 0.33 C O M 1.9 a 0.01 1.5 a 0.09 Control 18.6 a 1.07 4.8 a 0.60 Coarse-root biomass NB or N 20.1 a 1.31 6.7 a 3.72 C O M 20.0 a 0.39 10.2 a 1.62 Control 9.3 a 0.53 2.4 a 0.30 Coarse-root C content NB or N 10.1 a 0.66 3.3 a 1.86 C O M 10.0 a 0.20 5.1 a 0.81 Control 22.3 a 1.12 6.9 a 0.76 Total root biomass NB or N 23.9 a 1.36 9.1 a 4.36 C O M 23.7 a 0.41 13.3 a 1.79 Control 11.2 a 0.56 3.4 a 0.38 Total root C content NB or N 11.9a 0.68 4.5 a 2.18 C O M 11.9 a 0.20 6.7 a 0.90 28 Changes in soil C and N content Carbon concentration (%), bulk density (t/m3), dry mass (t/ha), and C content (t/ha) of soils at Kenneth Creek site are summarized in Table 3.4. The complete fertilizer treatment significantly increased C concentration in the forest floor (L/F) layer. The nitrogen + boron treatment increased C concentration in the mineral soil layers, although differences at the 0 - 20-cm depth were not significant. There were no significant differences in forest floor mass and soil bulk density. However, C content (t/ha) within the 20 - 40 cm soil layer was significantly greater in the N+B treatment. Fertilization did not affect the C concentration (%) in any soil layer at the S C H I R P site (Table 3.5). However, the complete fertilizer treatment at S C H I R P had higher forest floor mass in H I (humus formed from litter of the present tree stand) and H2 (residual humus from the previous stand), which resulted in a significantly higher C content (t/ha) in these soil layers (Table 3.5). The N concentration (%) at Kenneth Creek was significantly increased by the complete fertilizer treatment in the forest floor layer and by the N+B treatment in the mineral soil layers (Table 3.6). However, N concentration (%) at S C H I R P was not affected by any treatment within any soil layer (Table 3.6). 29 Table 3.4 Carbon concentration (%), bulk density (t/m3), dry mass (t/ha), and C content (t/ha) of soils at the Kenneth Creek site. Treatments are: Control; N B = nitrogen and boron; and C O M = complete. Different letters next to the mean value indicate significant differences between treatments within each soil layer at the level of p < 0.05 (n = 12). SD = standard deviation. Soil layer Treatment C (%) Bulk density (t/m3) Dry mass (t/ha) C (t/ha) Mean SD Mean SD Mean SD Mean SD L/F Control 44.28 a 1.13 NA NA 13.0 a 6.62 5.7 a 2.90 NB 45.27 ab 1.8 NA NA 13.2 a 5.47 5.9 a 2.45 COM 45.98 b 0.53 NA NA 13.4 a 5.40 6.2 a 2.47 Control 1.20 a 0.31 1.39 0.06 2768.3 127.30 33.3 a 8.69 0 - 20 cm NB 1.50 a 0.42 1.39 0.06 2768.3 127.30 41.4 a 11.63 COM 1.25 a 0.16 1.39 0.06 2768.3 127.30 34.6 a 4.48 Control 0.51 a 0.09 1.49 0.05 2982.6 109.87 15.1 a 2.72 20 - 40 cm NB 0.72 b 0.3 1.49 0.05 2982.6 109.87 21.6 b 8.99 COM 0.50 a 0.12 1.49 0.05 2982.6 109.87 14.9 a 3.48 Table 3.5 Carbon concentration (%),bulk density (t/m 3), dry mass (t/ha), and C content (t/ha) of soils at the S C H I R P site. Treatments are: Control; N = nitrogen; and C O M = complete. Different letters next to the mean value indicate significant differences between treatments within each soil layer at the level of p < 0.05 (n = 3). SD = standard deviation. Soil layer Treatment C (%) Bulk density (t/m3) Dry mass (t/ha) C (t/ha) Mean SD Mean SD Mean SD Mean SD L/F Control 48.13 a 0.38 NA NA 16.4 a 11.54 7.9 a 5.58 N 47.47 a 0.9 NA NA 15.2 a 8.95 7.2 a 4.33 C O M 47.73 a 0.42 NA NA 17.0 a 11.10 8.1 a 5.29 H1 Control 48.43 a 1.17 NA NA 22.7 a 40.41 11.0 a 19.59 N 44.10 a 5.41 NA NA 27.6 a 30.81 12.5 a 14.67 C O M 47.27 a 1.3 NA NA 48.9 b 38.25 23.2 b 18.10 H2 Control 48.20 a 2.19 NA NA 88.5 ab 84.98 43.3 ab 42.19 N 46.37 a 3.01 NA NA 59.3 a 56.05 28.3 a 27.81 C O M 49.28 a 1.45 NA NA 118.1 b 96.52 58.4 b 47.83 Control 9.66 a 0.86 0.55 a 0.17 1106.4 a 344.42 105.3 a 25.17 0 - 20 cm N 9.73 a 1.78 0.48 a 0.23 959.4 a 453.95 97.5 a 61.34 C O M 9.18a 0.35 0.45 a 0.07 905.6 a 134.91 82.9 a 10.49 Control 7.37 a 0.33 0.97 a 0.34 1945.2 a 675.40 142.5 a 46.44 20 - 40 cm N 7.31 a 1.11 0.97 a 0.34 1928.4 a 671.01 136.3 a 27.53 C O M 7.97 a 0.41 1.31 a 0.35 2610.6 a 690.14 209.7 a 66.49 * H I represents humus formed from litter of the present tree stand; ** H 2 represents residual humus from the previous stand. Table 3.6 Nitrogen concentration (%) in different soil layers at the Kenneth Creek and S C H I R P sites. Treatments are: Control; N B = nitrogen and boron; N = nitrogen, and C O M = complete. Different letters next to the mean value indicate significant differences between treatments within each soil layer at the level of p < 0.05 (n = 3). SD = standard deviation. Soil layer Treatment N (%) at Kenneth Creek N (%) at SCHIRP Mean SD Mean SD L/F Control NB or N C O M 0.98 a 0.09 1.06 ab 0.09 1.07 b 0.07 0.95 a 0.06 1.18a 0.14 1.05 a 0.14 H1 Control N C O M 0 NA 0 NA 0 NA 1.04 a 0.10 1.17 a 0.00 1.05 a 0.07 H2 Control N COM 0 NA 0 NA 0 NA 0.90 a 0.06 1.00 a 0.14 0.90 a 0.07 0 - 20 cm Control NB or N C O M 0.06 a 0.01 0.08 b 0.02 0.06 a 0.01 0.25 a 0.02 0.30 a 0.13 0.21 a 0.02 20 - 40 cm Control NB or N C O M 0.04 a 0.00 0.05 b 0.02 0.04 a 0.01 0.20 a 0.03 0.25 a 0.10 0.20 a 0.02 32 E c o s y s t e m response to N addi t ions The total amount of C stored in the stand biomass and soil (to 40 cm) at Kenneth Creek, did not significantly differ among the treatments (Fig. 3.3), although the average amount of C stored in the soil was larger in the N+B plots. There were also no significant differences among treatments at the SCHIRP site, although the complete fertilizer plots had greater average amounts of C in the aboveground biomass + mineral soil (Fig. 3.4). 160 Control NB COM Treatment Figure 3.3 Total C content (t/ha) at the Kenneth Creek site in response to fertilization treatments: Control; N B = nitrogen and boron, and C O M = complete. The included components are: A G B = aboveground tree biomass (lodgepole pine); C R B = coarse root biomass; F R B = fine root biomass; L F = forest floor soil layer; 0-20 cm and 20-40 cm = mineral soil layers. There were no significant differences between treatments at level of p < 0.05 (n = 3). 33 500 Control N C O M Treatment Figure 3.4 Total C content (t/ha) at the SCHIRP site in response to fertilization treatments: Control; N = nitrogen, and C O M = complete. The included components are: AGB - aboveground tree biomass (western hemlock - wh); U V = understory vegetation + western redcedar; CRB = coarse root biomass (wh); FRB = fine root biomass (wh); L F = forest floor soil layer; HI = humus formed from litter of the present tree stand; H2 = residual humus from previous stand; 0-20 cm and 20-40 cm = mineral soil layers. There were no significant differences between treatments at level of p < 0.05 (n = 3). 34 Changes in enzyme activity There was little effect of N fertilization on enzyme activity. Contrary to our expectations, ligninolytic enzymes - phenol oxidase and peroxidase - were not suppressed by N fertilization at Kenneth Creek (Fig. 3.5 and 3.6), or at S C H I R P (Fig. 3.7 and 3.8). Peroxidase activity was significantly higher in the N+B plots in the forest-floor at Kenneth Creek (Fig. 3.6). The activity of P-glucosidase and cellobiohydrolase, responsible for degradation of cellulose, was not significantly affected by any treatment, at Kenneth Creek (Fig. 3.9 and 3.10), or at S C H I R P (Fig. 3.11 and 3.12). Though not statistically significant, the same pattern of decreased cellulolytic activity by the N+B and N-alone treatments was observed in the forest-floor of both sites (Fig. 3.9, 3.10, 3.11 and 3.12). The relationship between enzyme activity and soil N concentration (%) was assessed using Pearson correlation. Peroxidase activity in the forest floor layer at Kenneth Creek site was positively correlated with the N concentration (%) of the same layer (r = 0.612, n = 9, p < 0.05) (Figure 3.13). However, further linear regression analysis indicated that N concentration did not explain a significant amount of the variance in the peroxidase activity (p = 0.08). On the contrary, P-glucosidase activity in the forest-floor at S C H I R P was negatively correlated with the N concentration of H I layer (r = - 0.662, n = 9, p < 0.05). Linear regression analysis showed that N concentration in H I layer accounted for 44% of the variance in P-glucosidase activity (R 2 - 0 . 44 ) (Figure 3.14). 35 - T O 5000 SZ o £ 4 0 0 0 c >. > 3000 T$ CD 5) 2000 CD "O X _ 1000 o c CD Forest floor a Mineral soil 3000 Control NB COM Treatment Control NB Treatment COM Figure 3.5 Phenol oxidase activity in different soil layers at the Kenneth Creek site. Treatments are: Control; NB = nitrogen and boron; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level of p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. Forest floor ° 3 0 0 0 0 c . = 20000 o CD CD </) - § 10000 o I— CD Q . Mineral soil Control NB COM Treatment LU Control NB COM Treatment Figure 3.6 Peroxidase activity in different soil layers at the Kenneth Creek site. Treatments are: Control; NB = nitrogen and boron; and C O M = complete. Different letters above the error bars indicate significant differences between treatments at the level of p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. 36 D ) 2606-O E c > o CD CD W CD "O X o o c CD Forest floor Mineral soil Control N COM Treatment Control N COM Treatment Figure 3.7 Phenol oxidase activity in different soil layers at the S C H I R P site. Treatments are: Control; N = nitrogen; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level o f p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. Forest floor Mineral soil Control N COM Treatment Control N COM Treatment Figure 3.8 Peroxidase activity in different soil layers at the S C H I R P site. Treatments are: Control; N = nitrogen; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level o f p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. 37 o E c > S3 400 o CD CU CO CO 1 200 o o 3 CD Forest floor Mineral soil ao Control NB COM Treatment Control NB COM Treatment Figure 3.9 P-glucosidase activity in different soil layers at the Kenneth Creek site. Treatments are: Control; N B = nitrogen and boron; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level of p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. I300 CD CD co _CD 200 O L . T3 > . - C 100 o la o "53 o O Forest floor Mineral soil Control NB COM Treatment Control NB COM Treatment Figure 3.10 Cellobiohydrolase activity in different soil layers at the Kenneth Creek site. Treatments are: Control; N B = nitrogen and boron; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level of p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. 38 Forest floor Mineral soil D> I 1 , Treatment Treatment Figure 3.11 P-glucosidase activity in different soil layers at the S C H I R P site. Treatments are: Control; N = nitrogen; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level of p < 0.05. Each value is the mean of three samples; error bars indicate standard deviation. Forest floor Mineral soil 1200 1000 800 600 Control N COM Treatment Control N Treatment COM Figure 3.12 Cellobiohydrolase activity in different soil layers at the S C H I R P site. Treatments are: Control; N = nitrogen; and C O M = complete. Same letters above the error bars indicate no significant differences between treatments at the level o f p < 0.05. Each value is the mean o f three samples; error bars indicate standard deviation. 39 CO o <D </> 03 • g X o 24000 20000 16000 A 12000 8000 O 4000 -4 Q _ 0.85 0.90 0.95 1.00 1.05 1.10 1.15 N concentration (%) Figure 3.13 Significant positive correlation between peroxidase activity and N concentration (%) in the forest floor at Kenneth Creek (r = 0.612, n = 9, p < 0.05). CD o E c o CO CD w CD • g w o o _^ (3 1000 800 600 400 200 A 0.90 0.95 1.00 1.05 1.10 1.15 1.20 N concentration (%) Figure 3.14 Significant negative correlation and regression model between glucosidase activity and N concentration in H I layer at S C H I R P (p < 0.05). 40 4 DISCUSSION M y data supported the hypothesis that fertilization would increase the aboveground individual-tree biomass (kg) of both lodgepole pine and western hemlock. Although statistically significant, the lodgepole-pine biomass gains due to fertilization at Kenneth Creek were modest. This finding is consistent with other studies demonstrating poor or variable responses of lodgepole pine to fertilization (Brockley, 1996, Brockley and Simpson, 2004; M i k a et al., 1992). In general, harvest-origin stands and plantations (especially in the Sub-Boreal Spruce biogeoclimatic zone) have responded poorly to N additions in comparison to fire-origin stands response (Brockley, 1996; Blevins et al., 2005). However, the small growth response to fertilization at Kenneth Creek is unusual as other lodgepole-pine installations in the same area have shown better responses to N additions (Brockley and Simpson, 2004). The reason for this is unknown; one of the hypotheses is higher water flow-capacity of lower branches, which may reduce the availability of water to the upper parts of the crown (Amponsah et al., 2004). Another hypothesis is induced deficiency of other nutrients riot included in the fertilizer mix (for example Cu) (Brockley and Simpson, 2004). Although some studies have shown inconsistent response of western hemlock to fertilization (Weetman et al., 1992), growth responses have been significant in many studies (Brown, 2003). The positive aboveground individual-tree biomass response of western hemlock to fertilization at SCHIRP was much more pronounced than the response of lodgepole pine at Kenneth Creek. Our results confirmed the previous positive findings in investigations of the effects of N additions on the productivity of western hemlock on similar sites (Blevins et al, 2006; Bennett et al., 2003; Blevins and Prescott, 41 2002; Prescott and Brown, 1998; Prescott, 1996; Weetman and Thompson, 1992). Blevins et al. (2006) found that the elevated growth response to N-alone was short-lived and the trees did not respond to the second addition of N (10 years later) unless P had been applied at least once. It has been suggested that even a single application of P, in combination with N can promote a much larger long-term response than N-alone (Blevins et al., 2006; Prescott, 1996). This statement was supported by our data showing the highest biomass gains obtained after the complete treatment (containing both N+P and micronutrients). Although the biomass gains after N-alone treatment were not as big as those after the complete treatment, they were also significantly higher than the controls. Therefore it is important to identify on which sites trees are l ikely to be limited by P and/or other nutrients, and to evaluate the economic costs and benefits before deciding which fertilizer treatment to use (Brown, 2003). Stand-level tree biomass gains (t/ha) were not significantly affected by any treatment at either site. This may be due to the small number of samples (n = 3), which increases the variability, and hence increases the percentage of change that is detectable (Yanai et al., 2003). In other words, it is difficult to detect changes between treatments with small number of samples when the true changes are small such as at the Kenneth Creek site. Moreover, the lodgepole pine stand was still young (24 years old in 2005) and it is possible the stand-level fertilization biomass gains may become significant with time (Brockley, 1996). A t S C H I R P , the reason I did not detect significant differences in the stand-level biomass gains between treatments is probably the uneven sample size (i.e., there were different numbers of trees between plots). 42 It is not clear whether the N additions at S C H I R P caused tree mortality in the fertilized plots and hence different number of trees between treatments. Some studies have shown that N additions have negative effects on tree survival. M a g i l l et al. (2004) found increased tree mortality rate after 15 years of N fertilization (15 g/ m 2 / yr). Blevins and Prescott (2002) found lower survival rates of both cedar and hemlock on plots fertilized with N (1000 kg/ha over three years). The amount and frequency of N applications in my study differ from those mentioned above. Moreover, there were also different numbers of trees between treatments at the time of trial establishment (Barker et al, 1987), which makes it difficult to conclude i f N additions w i l l result in significant positive stand-level biomass gains in the western hemlock stands at S C H I R P . M y stand-level root biomass estimations were based on aboveground biomass, and assumed that the belowground stand-level biomass gains followed the same pattern and did not significantly differ between treatments. The effect of fertilization on root biomass is uncertain because the results vary between studies and with fertilizers. Inconsistency has been found mostly in the fine-root biomass gains, while coarse-root and aboveground biomass usually increased by the same proportion after fertilization. For example, Albaugh et al. (1998) found aboveground and coarse-root (diameter (cp) > 2 mm) biomass of loblolly pine (Pinus taeda L.) significantly increased, but fine-root (cp < 2 mm) biomass significantly decreased in all 3 years of fertilization with N + micronutrients. Similarly, 12-year annual additions of N resulted in a decreased biomass of fine ((p < 2 mm) and small (2 < cp < 15 mm) roots of Norway spruce (Picea abies (L.) Karst.) (Iivonen et al., 2006). Smaller positive gains of the fine and small root biomass (only by 50%) compared to the aboveground and coarse-root (cp > 5 mm) biomass gains 43 of radiata pine (Pinus radiata D . Don) have been found 40 years after a single application of P (Zerihun and Montagu, 2004). However, the aboveground to coarse-root biomass ratio remained unaffected by fertilization and both components were closely related to the tree D B H . Contrary, Tingey et al. (1997) did not find a significant effect of N additions (0, 100, and 200 kg/ha/yr) on fine-root occurrence and root length and diameter of ponderosa pine (Pinus ponderosa Laws) seedlings monitored for two years. Similarly, Berch et al. (2006) found no change in the fine-root length of lodgepole pine after annual fertilization with 50-100 kg N / ha, but found decreased fine-root length with 100-200 kg N / ha. However, both Berch et al. (2006) and Tingey et al. (1997) did not present coarse-root and aboveground biomass gains following fertilization and hence we can not account for differences in allocation between above-and belowground biomass. Furthermore, Coyle and Coleman (2005) found little or no effect on allocation to above- and belowground biomass of two eastern cottonwood clones (ST66: Issaquena County, M S ; S7C15: Brazos County, T X ) and American sycamore (open pollinated mixed orchard seed) to irrigation and fertilization, and suggested that root biomass decreases are simply an effect of accelerated development rather than higher resource availability. This hypothesis requires further research. If it is true then the accuracy of my root-biomass estimations depends on how well the equations of L i et al. (2003) used in this study represent the relationship between the below- and aboveground biomass during different tree development stages, rather than soil nutrient availability. Moreover, fine roots are short-lived tissues and would not be considered as a long-term C storage pool, and hence, would not be claimed for C credits. I included these estimates only to provide an indication of overall C distribution in the ecosystem. 44 The understory vegetation biomass was obviously similar among treatments at Kenneth Creek and did not significantly differ among treatments at the S C H I R P site. Results from other studies of effects of N fertilization on understory vegetation biomass have been inconsistent. Canary et al. (2000) did not find significant differences in the C content (Mg/ha) of understory vegetation after fertilization of Douglas-fir (Pseudotsuga menziesii Mirb.) stands with N (896 - 1120 kg/ha over 16 years). Makipaa (1995) also did not find positive effect of N additions (596 - 926 kg/ha over 30 years) on the ground vegetation under a closed canopy of Scots pine (Pinus sylvestris L.) stands. The author concluded that ground vegetation in the boreal forests would not be a C sink under conditions of high N deposition. On the contrary, in another S C H I R P experiment, Bennett et al. (2004) found significantly higher understory vegetation biomass in the N + P treatments (500 kg N/ha + 200 kg P/ha over three years) plots compared to the controls. However, the biomass was significantly reduced in the plots receiving N-alone (1000 kg /ha over three years). Similarly, salal biomass significantly decreased with high levels of N-alone (1000 kg /ha) (Bennett et al, 2004; Blevins and Prescott, 2002), but not when N (1000 kg /ha) was applied together with P or when lower amounts o f N (500 kg /ha) was applied with or without P (Bennett et al, 2004). Prescott et al. (1993) also found reduced salal cover in repeated N-fertilized plots and less pronounced reductions when other nutrients like P and S were applied in addition to N . I did not find significant differences in salal biomass between treatments, but similar trends were apparent (higher biomass only i f P was added in addition to N) . Salal biomass was highly variable between plots of the same treatment, and it was not clear i f it was due to the applied N and/or it was a shading effect of the overstory canopy (some plots have already reached crown-closure 45 and others were close to this stage). So differences in salal biomass may be related to differences in the tree density in the plots. Messier et al. (1989) found a dramatic decrease in understory salal cover and density and also in salal leaf thickness and specific leaf weight with a decrease in the photosynthetic photon flux density and red: far-red ratio. Although total understory vegetation biomass was not significantly affected by fertilization, there were differences in the species composition between treatments. A s already mentioned, bunchberry was found only in the control plots, while fireweed was found in both N-alone and complete fertilizers plots, but not in the control plots. Similarly, Kellner and Marshagen (1991) found changes in the vegetation composition after seven years of irrigation and liquid fertilization (110 - 420 kg N/ha/yr) expressed as decreased dominant moss species and complete loss of lichens. They also found a large number of new vascular species, including fireweed, which increased drastically and dominated the field layer. Nordin et al. (2005) found increased abundance of a relatively fast-growing grass species and decreased abundance of a more slow-growing dwarf shrub following N additions (6 to 50 kg N/ha/yr). They concluded that low N doses applied over a long time period may result in similar vegetation changes as high N doses. On the contrary, He and Barclay (2000) found little changes in the species richness and cover of understory vegetation after 27 years of thinning and fertilization (224 and 448 kg N/ha as urea). These contradicting results indicate that further research is needed to test how different nutrient regimes and/or stand development stages affect growth rate and composition of the understory vegetation. 46 It has been hypothesized that N-fertilized stands w i l l sequester more C in forest floor and mineral soil compared to adjacent non-fertilized stands. Additional accumulation of C could occur due to: 1) incomplete litter decay, and/or 2) increased litter production and hence, increased organic matter input to soils (Johnson, 1992). M y two significant results support this hypothesis - higher forest floor and mineral soil C concentrations (%) in the two fertilizer treatments at Kenneth Creek, and higher humus accumulation (t/ha) in the complete fertilizer treatment at S C H I R P . Humus formation at Kenneth Creek was not sufficiently advanced to look for differences in the organic matter accumulations between treatments. However, i f the response to fertilization maintains the same trend in biomass gains, and i f there are no differences between plots caused by external factors (e.g., fires, diseases), there may eventually be higher humus accumulation in the N-fertilized plots. A t the SCHIRP site, the higher humus accumulation in the complete fertilizer plots can be attributed to the increased tree growth and higher litter production in these plots. The reasons for the higher accumulation of residual humus from previous stand in the complete plots are unknown. The inconsistent results between Kenneth Creek and S C H I R P sites are in keeping with findings from other studies. Mag i l l and Aber (1998) found suppressed decomposition rate of different litter types (red pine (Pinus resinosa Ait . ) and mixed hardwood) by 20-50% and larger original mass remaining in N-fertilized compared to non-fertilized plots. They found suppressed lignin decay and increased total lignin content during the first nine months of N additions. Cellulose decay was also suppressed by N additions, but its content declined immediately and continuously throughout the decay process. In contrast, in a laboratory study of the degradation of hemicellulose, 47 cellulose and lignin in Norway spruce needle litter in relation to N , Sjoberg et al. (2004) found increased cellulose decomposition rate and unaffected lignin and hemicellulose decomposition rates in N-r ich litter compared to control litter. The hypothesis that N additions increase the recalcitrance of soil organic matter was not supported by their study. Nohrstedt et al. (1989) detected a tendency for an increased absolute amount of C per m 2 in all soil horizons in Scots pine plots in Sweden with high N-fertilizer levels, but the differences were not significant. The increased C content could not be explained by increased litter production alone, and reduced microbial activity also appeared to be involved. Makipaa (1995) found a greater amount of C in both humus and mineral soil in N-fertilized Scots pine and Norway spruce stands. On the contrary, Canary et al. (2000) did not find significant differences between control and N-fertilized Douglas-fir stands in any of the sampled soil horizons up to a depth of 85 cm, although there was a significant C increment of the live-tree component after fertilization, h i several field fertilization trials and a microcosm experiment, Prescott (1995) found inconsistent effects of N availability on the rates of litter decomposition. Sewage sludge did not affect the decomposition rate of forest floor material and of mixed-species needle litter during the first 8 to 18 months. However, significantly greater amounts of decaying material remained in the sludged plots of both experiments after 22 to 36 months. In contrast, sewage sludge (alone or mixed with pulp sludge) suppressed the decomposition rate of paper birch (Betula papyrifera Marsh.) only during the first three months but there were no significant differences in the remaining mass decaying between the control and the treated plots thereafter. Moreover, 33 months of incubation of non-fertilized lodgepole pine needles in fertilized and non-fertilized plots did not show a significant effect of N on 48 the litter mass loss at either site, although there was a slightly larger average mass remaining in the fertilized plots. Similarly, the decomposition rate of green and brown needle litter from fertilized plots did not significantly differ from that of green and brown litter from non-fertilized plots, despite having significantly different N contents. In conclusion, the effect of N additions on litter decay has been inconsistent, and the changes in the litter decay rate cannot always be attributed to N availability with certainty, because some other factors could have been changed by N additions (Prescott, 1995). If N additions can affect soil characteristics (e.g. pH) (Nason and Myro ld , 1992), then changes in the enzyme activity could also be expected (Tabatabai, 1994). A s already mentioned, there have been contradicting results in terms of the effect o f N additions on enzyme activity (Keyser et al, 1978; Freer and Detroy, 1982; Leatham and Ki rk , 1983; Fog, 1988; Collins and Dobson, 1997; Carreiro et al, 2000; Miche l and Matzner, 2003; Berg and McClaugherty, 2003; Frey et al., 2004; Waldrop et al, 2004). I found little evidence o f an effect of N fertilization on enzyme activity - the only significant difference was the increased peroxidase activity in the forest floor o f N+B treatment plots at Kenneth Creek. The positive correlation between peroxidase activity and N concentration (%) contradicted my hypothesis of suppressed ligninolytic enzyme activity by N additions. Although there were significant differences in the N concentrations of the mineral soil at Kenneth Creek, and ligninolytic activity tended to be higher in the mineral soil, there were no significant differences in enzyme activity or significant correlations with N concentration in the mineral layer. I also found a significant negative correlation between glucosidase activity and N concentration (%) at S C H I R P . 49 Making conclusions about the effect of N additions on the enzyme activity without knowing the microbial community profile would be incomplete. There is a common misunderstanding that most white-rot fungi follow the same pattern as Phanerochaete chrisosporium which ligninolytic enzyme activity is suppressed and delayed by high N concentrations (Kaal et al, 1995). However, studies have demonstrated that other white-rot fungi (e.g. Pycnoporus cinnabarinus; Bjerkandera spp.; Lentinula edodes; Pleurotus ostreatus) had higher ligninolytic enzyme activity in response to N-r ich media (Collins and Dobson, 1997; Kaal et al., 1995). There is also lack of knowledge as to whether each species produces all ligninolytic enzymes or specialize in producing just some of them. Further research is needed to f i l l in these gaps and to answer further questions, namely: • Which fungal taxa are associated with the increased peroxidase activity at Kenneth Creek? Are these species commonly occurring? • Is the increased peroxidase activity a result of the stimulating effect of N or it is simply due to a higher fungal biomass production? • How can we explain the increased C concentration (%) along with the increased peroxidase activity at Kenneth Creek? Was lignin the major litter fraction responsible for the increased C concentration at this site? 50 5 CONCLUSIONS The key findings of this research are: • N fertilization significantly increased the individual tree biomass (kg), but did not increase the stand-level tree biomass (t/ha) at both Kenneth Creek and S C H I R P sites; • Understory vegetation biomass at S C H I R P was not significantly affected by N additions, but there were some differences in the vegetation composition; • N fertilization increased soil C concentration (%) at Kenneth Creek and soil C content (t/ha) at S C H I R P ; • The total amount of C stored in the stand biomass and soil (to 40 cm) was not significantly affected by N fertilization at either site; • The aboveground tree biomass and mineral soil contained similar amounts of C at Kenneth Creek; at S C H I R P more C was stored in the mineral soil than in aboveground tree biomass. • N fertilization did not significantly affect the enzyme activity, except for the increased peroxidase activity at the Kenneth Creek site; • Further research is needed to determine: o the microorganisms responsible for the increased peroxidase activity; o the major litter fraction responsible for the increased C concentration at Kenneth Creek. 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