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UBC Theses and Dissertations

Gaseous nitrogen transformations in a mature forest ecosystem Cushon, Geoffrey H. 1985

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G A S E O U S N I T R O G E N T R A N S F O R M A T I O N S IN A M A T U R E F O R E S T E C O S Y S T E M by G E O F F R E Y H. C U S H O N A T H E S I S S U B M I T T E D IN P A R T I A L F U L F I L M E N T OF T H E R E Q U I R E M E N T S FOR T H E D E G R E E OF 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 OF G R A D U A T E S T U D I E S D E P A R T M E N T OF F O R E S T R Y We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y OF M A R C H © G E O F F R E Y H. BR IT ISH C O L U M B I A 1985 C U S H O N , 1985 In presenting this thesis in partial fulfi lment of the requirements for an advanced degree at the The University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be al lowed without my written permission. DEPARTMENT OF FORESTRY The University of British Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date: MARCH 1985 Abstract In mature forests, gains and losses of nitrogen may be dominated by the gaseous transformations, asymbiotic nitrogen fixation and biological denitr if ication. Both are reduction reactions and are affected by moisture condit ions, temperature, pH, supply of organic carbon and the availability of mineral nitrogen. Gaseous nitrogen inputs, due to asymbiotic nitrogen f ixation, and outputs, due to biological denitrif ication were quantified for a mature coniferous forest in southwestern British Columbia. Forest f loor material, mineral s o i l , decaying w o o d , foliage and bark were incubated in an atmosphere of 0.1 atm acetylene to al low the simultaneous measurement of N 2 0 production by denitrifying bacteria and acetylene reduction by f ree - l i v ing bacteria and blue-green algae. Forest f loor material .accounted for 80% of a total annual input of 0 .8 ' kg N h a 1 a - 1 . ' Relatively small amounts of nitrogen were fixed in mineral so i l , decaying wood and foliage and no indication of nitrogen fixation activity in bark was detected. Traces of denitrif ication were found, but gaseous output of nitrogen was effect ively 0.0 kg N h a - 1 a - 1 . It is hypothesized that this forest may prevent nitrogen Joss by outcompeting other sinks for mineral nitrogen, thereby allowing a s low accretion of nitrogen by asymbiot ic nitrogen fixation and bulk precipitation input. ii Table of Contents Abstract ii List of Tables v List of Figures vii Acknowledgements viii 1. INTRODUCTION 1 2. LITERATURE REVIEW 5 2.1 Asymbiot ic Nitrogen Fixation 5 2.2 Denitrif ication 6 2.3 Environmental Factors Affect ing ANF and Denitrif ication 8 2.3.1 Soi l Aeration and Moisture 8 2.3.2 Avai labi l i ty of Organic Carbon 10 2.3.3 Temperature 11 • 2.3.4 Soi l Reaction ; 12 2.3.5 Nitrogen 13 2.3.6 Other Mineral Nutrients 15 2.4 Denitrif ication and Asymbiot ic Nitrogen Fixation in Forests 15 2.4.1 Denitrif ication 16 2.4.2 Asymbiot ic Nitrogen Fixation 17 2.5 The Effects of Forest Management Act i v i t y on Asymbiot ic , Nitrogen Fixation and Denitrif ication 19 3. OUTLINE OF RESEARCH 22 4. THE STUDY AREA 23 5. METHODS 31 5.1 Field Procedures 34 5.2 Lab Analys is 36 5.3 Calculations 37 5.4 Site Quantification 38 iii 6. RESULTS AND DISCUSSION 41 6.1 Nitrogen Fixation 41 6.1.1 Forest Floor and Mineral Soi l 41 6.1.2 Decaying Wood 43 6.1.3 Foliage 48 6.1.4 Bark 52 6.2 Denitrif ication ^ . .52 -6.3 Total Flux 56 6.4 Sources of Error 61 7. S U M M A R Y AND CONCLUSIONS 64 LITERATURE CITED ., 67 iv List of Tables Table Page 1 Mean monthly temperature and monthly precipitation in 1984 and the 20 year average (last year 1975) for the UBC Research Forest, Marc Station 26 2 Volume, density and biomass data for the sample strata on the 30m X 30m study site 30 3 Air and soil temperature (12 noon) on sampling days 32 4 Regression equations relating biomass of foliage (Y) to outside bark diameter at breast height (D), //7Y(kg) = a + b/rtD(cm). Source: M.C. Feller (unpublished data) • 39 5 Bimonthly nitrogen fixation rates and total annual fixation for 1984 42 6 Average moisture content (by percent) of sample material 45 7 Nitrogen fixation rates (nmoles N g - 1 day - 1 and total annual flux (kg N h a - 1 a-1) in foliage col lected from mature tree crowns (A) compared with foliage collected at ground level (B) 51 8 Bimonthly denitrif ication rates and total annual denitrif ication for 1984 53 9 Asymbiot i c nitrogen fixation rates in temperate forests 58 v 10 Nutrient losses, nutrient inputs in precipitation and nutrient reserves for logged (A), logged and slash burned (B) and undisturbed (C) watersheds in the UBC Research Forest 60 vi .List of Figures Figure Page 1 The nitrogen cycle 2 2 Access to the UBC Research Forest 24 3 Location of the study site 25 4 Diameter distribution of trees on the 30m X 30m sample plot 28 5 Nitrogen fixation rates in mineral soil and forest f loor for 1984 44 6 Nitrogen fixation rates in decaying wood for 1984 47 7 Nitrogen fixation rates in foliage for 1984 50 8 Total annual nitrogen flux by strata ..57 vii Acknowledgements I wish to express my appreciation to T.M. Ballard, F.B. Hoi I, J.P. Kimmins and especially M.C. Feller for advice and guidance throughout the course of this study. Thanks are also due to Bernice Morosof f for help with drafting work on figures. viii 1. INTRODUCTION "Nitrogen is the principal l imiting factor in the growth of both agronomic and forest crops" (Gordon et al . , 1979). The significance of nitrogen in crop production has generated a huge body of literature' which can be summarized in abstract form by the nitrogen cycle (Figure 1). While this model is complex, it is interesting to note that outside of industrial f ixat ion, there are only two means of entry into the terrestrial nitrogen cycle . These are electrochemical and photochemical fixation of atmospheric nitrogen(N 2), and of far greater s ignif icance, biological fixation of N 2 by microorganisms (Kormondy, 1976). S imi lar ly , there are a limited number of means by which nitrogen is returned to the atmosphere, thus balancing the global nitrogen cycle. By far the most important of these processes is the bacterial reduction of nitrate, or biological denitrif ication (Knowles, 1982a). When nitrogen is the l imiting factor to growth in forest so i l s , nitrogen ferti l izers usually stimulate forest growth (Spurr and Barnes, 1973; Gesse l , 1959). Forest ferti l ization is carried out to increase productivity and also to offset a decline in ferti l ity that may occur as a result of nutrients lost in harvested materials (Marion, 1979; Morrison and Foster, 1979). Intensification of util ization and the shortening of rotation ages in response to increased fibre demand wi l l accentuate the potential for soil impoverishment (Kimmins, 1977). While nitrogen fertil izers are effect ive, they are becoming increasingly expensive in terms of foss i l energy, water pol lut ion, and 'hard cash'. As competit ion for industrially fixed nitrogen develops, it seems clear that forestry wi l l rank a lower priority than agriculture. Biological nitrogen fixation offers two approaches to solving this d i lemma. The possibi l i ty of producing nitrogen fertil izers using cultured bacteria and waste carbon or photosynthesis exists and is being 1 2 Atmospheric nitrogen (N. denitrifying bacteria (NO Nitrogen electrochemical and fixing photochemical organisms Ammonia(NH3) and Nitrate (NO") fixation Nitrate bacteria (NO Industrial fixation birds and fish Shallow marine sediments assimilation and anabolism \ Deep sediments Producers decay £n"d n e r b i v o r y wastes decay and wastes Consumers Amino acids urea, uric acid, organic residues \ Nitrite (NH bacteria Ammonifying bacteria Figure 1. The nitrogen cycle. Adapted from: Kormondy, 1969. 3 investigated (Sprent, 1979). Biological nitrogen fixation may also be used as an ^alternative to chemical ferti l izers. Symbiot ic nitrogen-f ix ing systems, such as those involving plants of the Fabaceae (legumes), have been important in agriculture for a long time (Nutman, 1975). Stewart (1977) tells us that nitrogen fixation by blue-green algae has been the mainstay of sustained rice production throughout the ages. Ecosystem studies have suggested that biological nitrogen fixation may be a major source of nitrogen accumulated in forest biomass during succession (Roskoski, 1980; Todd, Waide and Cornaby, 1975). Much research has focused on the use of nitrogen-f ix ing symbioses to enhance and preserve forest productivity. Less well understood is the significance of nitrogen fixation by f ree - l i v ing microorganisms. Nitrogen accretion by asymbiotic nitrogen fixation (ANF) is generally small when compared to symbiot ic nitrogen f ixation. However, when nitrogen is the l imiting nutrient factor for plant growth, even small inputs of nitrogen may be significant (Granhall and Lindberg, 1980). Evidence also exists that ANF may be important in the wood decay process (Hendrickson and Robinson, 1982). Any discussion of biological nitrogen fixation should also consider the significance of denitrif ication. There is some evidence that the two processes are l inked, responding in kind to the nitrogen status of the site (Knowles, 1978; Granhall, 1981). There is one species of bacteria, Spirillum lipoferum, that has the capacity to effect both transformations, depending on the conditions present (Neyra et al . , 1977). Denitrif ication can be a major source of nitrogen fertilizer loss (Rolston, 1981), resulting in decreased fertilizer eff ic iency. Other reasons for interest in denitrif ication include; 4 1. its potential use in the removal of nitrates from waste water (Schroeder, 1981) and other high-nitrogen waste materials. 2. its contribution of nitrous oxide (N 20) to the atmosphere where it is involved in stratospheric reactions which result in the depletion of ozone (Knowles, 1982a). Our knowledge of these biological nitrogen transformations remains incomplete. The roles of ANF and denitrif ication in forest ecosystems and the effect of management practices such as harvesting, scar i f icat ion, prescribed burning and ferti l ization on them are not wel l understood. This study was undertaken with the main objective of determining the relative significance of ANF and denitrif ication in a mature forest ecosystem. A review of the pertinent literature was undertaken to determine more specif ic objectives and the best methods by .which these objectives might be met. 2. LITERATURE REVIEW 2.1 ASYMBIOTIC NITROGEN FIXATION Biological nitrogen fixation is the reduction of atmospheric nitrogen (N2) to ammonia (NH3). Organisms capable of this transformation are all members of the kingdom Prokaryota (Sprent, 1979), and include species of eubacteria and cyanobacteria (blue-green algae). They occur as f ree - l i v ing organisms (asymbiotical ly) or in symbiotic association with certain species of fungi, bryophytes, cycads (Stewart, 1977) and higher plants. These include members of the Fabaceae and non- legumes such as Alnus sp. Arctostaphylos sp. and Ceanothus sp. (Cromack, 1981). Of interest in this study are f ree - l i v ing bacteria and blue-green algae. Nitrogen fixation by f ree - l i v ing bacteria has been reported in the forest f loor , decaying wood and in the phyllosphere. Free- l iv ing bacteria capable of fixing nitrogen may be aerobic, anaerobic or facultative anaerobic (Hendrickson and Robinson, 1982). Aerobic nitrogen-f ix ing bacteria are thought to be rare or absent from generally acidic, forest soi ls (Jurgensen and Davey, 1971). Anaerobic bacteria that fix nitrogen are generally of the genus Clostridium (Hendrickson and Robinson, 1982). Jurgensen and Davey (1971) found the facultative anaerobe Bacillus polymyxa to be the predominant nitrogen-f ix ing bacterium isolated from a variety of forest soi ls in North Carolina and Washington, and from Alaskan tundra. Other bacteria known to fix nitrogen include members of the genera Bei jerinckia, Enterobacter, Klebsiel la, Pseudomonas, Achromobacter, Spirillum and Micrococcus. Over 20 species of blue-green algae have been reported to fix nitrogen, all of which are members of the order Nostocales (Jurgensen and 5 6 Davey, 1970). Approximately one third of these are found in soil while others are found associated with certain bryophytes, cycads and ferns, and with fungi as lichens (Hendrickson and Robinson, 1982). Jurgensen and Davey (1968) investigated forest soi ls and found low numbers of blue-green algae in some so i l s , but only when pH was above 5.4. They suggest that the contribution of ni t rogen-f ix ing blue-green algae to the nitrogen cycle of established forests is negligible. Of more signif icance in certain ecosystems may be the contribution of ni t rogen-f ix ing lichens (Denison, 1973; Mil lbank, 1978). The reduction of dinitrogen to ammonia, N 2 + 3 H 2 — - 2 N H 3 is an exothermic reaction, but because of the stabil ity of the N 2 molecule, energy is required for the reaction to occur. This energy is provided by adenosine triphosphate (ATP) which is' generated by hydrolysis within the organism. The reaction is catalyzed by a highly oxygen sensit ive system known as the nitrogenase enzyme complex. The reader is referred to Sprent (1979) for a more thorough discussion of nitrogen fixation metabolism and nitrogen fixation in general. 2.2 DENITRIFICATION Denitrif ication may occur abiological ly or biological ly . "Abiological denitrif ication which requires high N 0 2 " and low pH is generally low and insignif icant" (Wollum and Davey, 1975). Biological denitrif ication is the microbial reduction of nitrate ( N 0 3 ) and nitrite (NOf) to the gaseous nitrogen oxides (NO, N 2 0) and atmospheric nitrogen; N 0 3 - — ^ N 0 2 - — - N O — - N 2 0 — - N , 7 Denitrif ication has been reported in soil and aquatic environments where anaerobic conditions prevail. The transformation is effected by essentially aerobic, but facultative bacteria, that utilize nitrogen oxides as terminal electron acceptors in the absence of oxygen (Knowles, 1982a). The reduction is dissimilatory , in that the nitrogen fai ls to enter the cell structure of the organisms involved (Alexander, 1977). Nitrate may also undergo assimilatory and dissimilatory reductions to ammonia which do not result in nitrogen loss and wi l l not be discussed further. Nitrous oxide can also be evolved as a byproduct of the microbial oxidation of ammonia (nitrification) which may be a significant contributor of N 2 0 in certain systems (Aulakh et al. , 1982). The bacterial species that can denitrify come from less than thirty genera (Bryan, 1981). Those from the. genera Pseudomonas and Al call genes are the most commonly isolated and may be of greatest significance (Knowles, 1982a). However, in soil systems, Pseudomonas and Achromobacter may be the most significant (Focht, 1978). Denitrifying bacteria synthesize a series of reductases that enable them to reduce successively more reduced nitrogen oxides (Knowles, 1981). Most possess all the reductases, while some lack the NGy reductase, and are N 0 2 _ dependent. Others lack the N 2 0 reductase and yield N 2 0 as the end product, while sti l l others have only the N 2 0 reductase (Knowles, 1982a). More thorough discussions of denitrif ication may be found in Knowles (1981,1982a) and Delwiche (1981). 8 2.3 ENVIRONMENTAL FACTORS AFFECTING ANF AND DENITR1FICATION Both denitrif ication and ANF are reduction reactions and are similarly affected by a number of environmental factors. These factors include soi l aeration and moisture status, the availability of organic carbon, soi l temperature and soil pH. The effects of soil inorganic nitrogen levels on the two processes wi l l be quite different. Few, if any of these factors are independent of each other making it diff icult to determine a rate-control l ing factor in a particular system. Environmental factors affect not only the overall rate of denitrif ication but frequently exert differential effects on the successive reductases (Knowles, 1981). In this way the relative amounts of the products that accumulate may be affected. 2.3.1 SOIL AERATION AND MOISTURE Soi l aeration and moisture affect ANF and denitrif ication as they affect the presence of oxygen which inhibits both processes. Ni t rogen-f ix ing organisms have developed a number of protective mechanisms against oxygen, including diffusion barriers (as root nodules in symbiot ic nitrogen-f ix ing plants), the formation of heterocysts by blue-green algae and the formation of sl imes in some heterotrophic bacteria (Granhall, 1981). Azotobacter uses high respiratory activity to scavenge 0 2 and keep it away from the nitrogenase. As oxygen is required for the production of ATP , many bacteria fix nitrogen most effect ively under microaerophilic conditions (Silvester et a l , 1982). Other organisms, such as Clostridium sp., can fix nitrogen only under anaerobic conditions. When denitrifying bacteria have access to both nitrate and oxygen, oxygen wil l be reduced rather than nitrate (Bryan, 1981). The / 9 N 2 0 reductase is thought to be the most sensitive to 0 2 and under low concentrations of 0 2 , the overall rate of denitrif ication wi l l decline but the percentage of N 2 0 in the end products wil l increase (Focht, 1974). Denitrif ication has been largely associated with very f ine-textured soils of poor structure, poorly drained s o i l , and normal soi ls of medium or fine texture during periods of excessive rainfall (A l l ison, 1965). The greatest potential for denitrif ication would be in completely water-saturated soil (Rolston, 1981), although this may not be true of forest so i l s , where denitrif ication capacity is greatest while the soil is drying fo l lowing saturation (Binstock, 1984). Evidence that denitrif ication (Fluehler et al. , 1976) and ANF can occur under apparently wel l -aerated conditions, can be explained by the occurence of anaerobic microsites in aggregated soi ls (Currie, 1961; Greenwood, 1961). Gaseous diffusion occurs more readily through the inter-aggregate pores than through the relatively smaller pores within the aggregate (Currie, 1961). Anaerobic conditions may prevail within the aggregate well after the inter-aggregate pores have drained. The 0 2 concentration in soi l depends on the rates of 0 2 consumption (respiration), and 0 2 d i f fusion and the geometry of the di f fusion path (Smith, 1980). The geometry of the diffusion path is complicated by the variation in aggregate size within most aggregated so i ls . Smith (1980) has attempted to model the extent of anaerobic microsites in aggregated so i l s , considering the variation in gaseous dif fusion coeff ic ients , respiration rate and aggregate size. There is a need to extend this type of study to determine the development of anaerobicity in poorly aggregated soi ls (Rolston, 1981). 10 Both ANF and denitrif ication are promoted in soils that remain saturated for long periods of time due to structure, texture and/or precipitation. Any activity that affects these factors wil l indirectly affect ANF and denitrif ication. 2.3.2 AVAILABILITY OF ORGANIC CARBON Photosynthetic ni t rogen-f ix ing organisms, such as the blue-green algae, depend on light quality and intensity for ATP production. Consequently, daily and seasonal fluctuations in growth and nitrogen-f ix ing capacity wi l l occur. Heterotrophic ni t rogen-f ix ing organisms utilize a variety of organic carbon compounds as energy sources, including carbohydrates, alcohols, organic acids and aromatic compounds (Granhall, 1981). ft „ Nitrogen f ixat ion, in general, is most common in environments where organic carbon is abundant and combined nitrogen (NH 4 + , N0 3~) is l imiting. In forests, relatively high rates of ANF have been reported in decaying wood (Todd et al . , 1975; Roskoski , 1980; Granhall and Lindberg, 1980), which has a high carbon to nitrogen ratio. Most denitrifying bacteria are heterotrophs and require organic carbon as a source of energy. Under anaerobic condit ions, denitrif ication is largely controlled by the supply of readily decomposable organic matter (Burford and Bremner, 1975). Denitrif ication capacity has been signif icantly correlated with water -soluble organic carbon (Burford and Bremner, 1975; Bremner and Shaw, 1958) and mineralizable carbon (Burford and Bremner, 1975). Reddy et al . (1982) found denitrif ication rates to be proportional to concentrations of N0 3~ and available carbon and suggested that 11 denitrif ication is influenced by the rate of mineralization of organic carbon. Under some conditions, carbon addition may not affect denitrif ication, indicating that this factor is not rate limiting (Knowles, 1981). Denitrif ication rates wi l l be largely influenced by the amount of carbon in the soi l and its posit ion in the prof i le . Denitrif ication rates are generally higher near the surface (Knowles, 1981) where organic matter deposit ion, mineralization and incorporation occur. ANF rates have been demonstraed to decrease signif icantly with increasing soil depth (Baker and At t iw i l l , 1984). 2.3.3 TEMPERATURE Rates of ANF . and denitrif ication fluctuate diurnally and seasonally in response to temperature. In spite of reports that nitrogen fixation occurs at extremely high and low temperatures, most nitrogen-f ix ing organisms are mesophiles. Evidence exists that nitrogen fixation can be affected more by temperature, than are general growth and photosynthesis (Granhall, 1981). The optimum range for denitrif ication is between 60 and 65°C. (Nommik, 1956; Bremner and Shaw, 1958). Jacobsen and Alexander (1980) found that nitrate was reduced s lowly at 7°C. and the rate increased with increasing temperature. Denitrif ication has been reported at 4°C. in an anaerobic atmosphere (Limmer and Steele , 1982) although Bailey (1976) reported that denitrif ication was completely inhibited at 5°C. The indirect effects of soil temperature on ANF and denitrif ication may be more important than the direct effects . In 12 marine sediments, little variation in denitrif ication rate with temperature has been reported (Knowles, 1982a). Soi l temperature affects the rate of other biological processes such as mineralization and nitrification as wel l as the movement of carbon, water and oxygen in the soil prof i le . Much of the effect of temperature may be related to the development of anaerobiosis (Rolston, 1981) and the availability of carbon and nitrate. 2.3.4 SOIL REACTION Optimum pH for denitrif ication (Nommik, 1956; Delwiche and Bryan, 1976) and nitrogen fixation (Granhall, 1981) is generally around 7. Nitrogen f ixation, although optimum at pH 7, can occur over a broad pH range. Certain asymbiotic bacteria {Bacillus polymyxa and Beijerinckia sp.) and some blue-green algae can fix nitrogen at pH as low as 4 (Granhall, 1981). However, the abundance of nitrogen-f ix ing f ree - l i v ing bacteria is generally lower in soi ls more acid than pH 6. Muller et al . (1980) studied denitrif ication in low pH soi ls (minimum 3.6) and found a highly significant correlation between reaction rate and soil pH. While denitrif ication decreases with decreasing pH, the N 2 0 reductase seems to be more sensitive to low pH than the other reductases. Decreasing pH and increasing 0 2 concentration tend to decrease the overall rate of denitrif ication but to increase the mole fraction of N 2 0 in the final product (Focht, 1974). Interpretation of these results is complicated by the occurence of abiological reactions of nitrite at low pH that yield one or more of NO, N 2 0 , N 2 and C H 3 N 0 2 (Knowles, 1982a). 13 A s with temperature, the indirect effects of soil pH on ANF and denitrif ication may be significant. Soi l pH wi l l affect the availabil i ty of mineral nutrients such as phosphorus and molybdenum which are necessary for nitrogen f ixation. The availabil ity of both these nutrients is optimum at near neutral pH and declines with decreasing pH. Low pH also seems to inhibit nitrif ication (Armson, 1977) which wil l have an important impact on denitrif ication in systems where nitrate is ra te - l imi t ing . 2.3.5 NITROGEN Nitrogen fixation is most common in environments where organic carbon is abundant and combined nitrogen (NH 4 + , N0 3 " ) is l imit ing. It is a sel f - regulated process, promoted or inhibited by changes in the levels of inorganic nitrogen (Granhall, 1981). If supplies of combined N are good, nitrogen-f ix ing organisms are at a competit ive disadvantage for carbon with heterotrophs that can utilize combined nitrogen. Nitrogen fixers must use a considerable portion of the energy available to them to fix nitrogen, leaving less for growth (Sprent, 1979). Denitrif ication rates are often found to be independent of nitrate concentrations as rates of carbon mineralization wi l l be the l imit ing factor (Rolston, 1981). At low concentrations, N0 3~ may exert rate -contro l (Knowles, 1982a). While the forest s o i l , with relatively high levels of soluble organic matter offers an excellent environment for denitrif ication, the latter cannot occur wrthout nitrate and little nitr i f icat ion normally occurs in undisturbed systems (Keeney, 1980). Nitrate concentrations also affect the proportions of the gaseous 14 products evolved. At high nitrate concentrations, the predominant product is N 2 0 (Nommik, 1956) while at lower concentrations, more N 2 is produced. The occurence of common intermediates (N 2 0, N02~) suggests that nitrif ication and denitrif ication may be coupled processes (Knowles, 1978; Focht and Verstraete, 1977). Knowles (1978) suggests two ways by which coupling might occur. Nitr if ication and denitrif ication may occur successively in t ime, in response to alternate wetting and drying cycles that provide the appropriate conditions for each process (Binstock, 1984). The two processes may also occur simultaneously, on opposite sides of an anaerobic-aerobic interface, such as may occur in soil aggregates. Simultaneous nitrif ication and denitrif ication may also occur in soi ls with variable moisture, content. Nitr if ication may occur at the surface of a soil profi le and nitrate then moves downward to an anaerobic zone where denitrif ication can take place (Hendrickson, 1981; Knowles, 1978). Nitr i f ication, and consequently denitrif ication, may be controlled by competit ion for NH 4 + among heterotrophic soil organisms. "When NH 4 + is added the available C/N ratio is drastically reduced and the nitrifiers have a competit ive advantage for NH 4 + supplies"(Johnson and Edwards, 1979). Nitr if ication and denitrif ication may also be coupled with nitrogen f ixation. Nitrogenase has the ability to reduce N 2 0 as well as N 2 . Denitrif ication and nitrogen fixation may occur simultaneously in the same or adjacent microenvironments, or even in the same culture of an organism able to catalyze both processes (Knowles, 1978). 15 2.3.6 OTHER MINERAL NUTRIENTS Lack of phosphorus affects nitrogen fixation before it affects plant growth, because phosphorus is necessary for the synthesis of ATP. The availability of iron and molybdenum is vital for nitrogen fixation as both are components of the nitrogenase enzyme system. Other nutrients thought to stimulate nitrogen fixation include magnesium, boron, cobalt, copper and zinc (Granhall, 1981). Copper, molybdenum and magnesium are all required for denitrif ication. Magnesium is necessary for growth, while molybdenum is an integral part of all the nitrate reductases that have been studied. Copper is involved in the reductase in some organisms while in others it is required for reductase synthesis. Iron and sulphur are both necessary for activity of the denitrif ication enzymes (Bryan, .1981). Sulphur compounds have several effects on denitrif ication. Sulphide appears to inhibit the NO and N 2 0 reductases (Knowles, 1982a). Sulphide can also cause relief of acetylene inhibition (discussed in methods section) of the N 2 0 reductase which may have consequences for the use of this technique in measuring denitrif ication. 2.4 DENITRIFICATION AND ASYMBIOTIC NITROGEN FIXATION IN FORESTS The study of both ANF and denitrif ication has been limited by the lack of ideal methods for quantifying fluxes. The development of the acetylene-reduction assay as an easy, sensitive and relatively inexpensive method of measuring nitrogen fixation has faci l i tated a vast increase in information on nitrogen fixation (Hardy et al . , 1973). While the acetylene-reduction assay has advantages over other techniques, it can be criticized on a number of grounds which wi l l be discussed in Methods. 16 A universal method for the measurement of denitrif ication has proven more elusive (Focht, 1978;"" Rolston, 1981). Denitrif ication has most often been estimated by balance from nitrogen cycle studies (e.g. Bormann and Likens, 1979) and is the subject of much speculation. 2.4.1 DENITRIFICATION Granhall (1981) has suggested that in temperate forests, ANF wi l l more than compensate for generally small losses by denitrif ication, resulting in s low accumulation of combined nitrogen. Todd et al . (1975) provided one of the few studies of denitrif ication in temperate forests. Although their analytical methods al low their results to be considered as rate potentials only, they provided valuable information about the potential rates in different substrates. Highest potential was found in decaying logs on the forest f loor and in the upper 10 cm. of soil where available carbon is at a maximum. Highest total flux of nitrogen gas occurred from the soil because of the greater mass of so i l . Of less signif icance, in terms of denitrif ication capacity, were decaying branches on the forest f loor and the forest f loor itself. Mel i l lo et al . (1983) considered denitrif ication over a successional sequence in organic and mineral soi l horizons. They suggested that denitrif ication potential is greater in young stands fo l lowing clearcutting. Their results indicated the potential for denitrif ication in forest so i l s , in spite of low pH (3.5 to 3.9) and wel l -drained condit ions. The authors speculated that denitrif ication occurred in pulses fo l lowing hydrologic events such as spring runoff and major rainstorms. This agrees with the findings of Binstock (1984) 17 who demonstrated that denitrif ication capacity in forest soils is greatest while the soi l is drying fo l lowing saturation. Mel i I lo et a l . (1983) also found a strong correlation between denitrif ication and nitrate concentration. In forest soi ls where nitrif ication rates are low, nitrate may be the rate - l imit ing factor for denitrif ication. 2.4.2 ASYMBIOTIC NITROGEN FIXATION Considerably more information exists concerning the role of ANF in forests, although the picture is sti l l incomplete. Climax forests in temperate regions have relatively closed nitrogen cycles between vegetation and soi l (Granhall, 1981). Gaseous inputs and outputs may dominate the total gains and losses in the forest nitrogen cycle (Todd et al . , 1975). A survey of the literature indicates that ANF "can account for 1 to 20 kg Nitrogen ha- 1 a - 1 w h i c h ' w i l l compensate for generally small losses of nitrogen caused by leaching and denitrif ication, and may yield some accretion (Granhall, 1981). Paul (1978) suggests that ANF in climax ecosystems may account for 5 to 10 per cent of the amount of nitrogen cycled annually between vegetation and s o i l . While the amounts of nitrogen fixed asymbiotical ly seem relatively smal l , one must consider that 2 kg N h a - 1 a - 1 accrued over a 90 year rotation is almost equal to one 200 kg N h a - 1 application of N ferti l izer. This has the advantage of occurring as s lowly released, easily available nitrogen (Granhall, 1981) in contrast to broadcast fertil izer applications which are subject to potentially high rates of leaching and denitrif ication. Asymbiot ic nitrogen fixation in forests is carried out primarily by heterotrophic bacteria in the soi l and forest f loor . Other potential 18 sources include bacteria in decaying wood and on foliage surfaces, and nitrogen-f ix ing epiphytes living on bark, branches, foliage and the forest f loor . Granhall and Lindberg (1980) present nitrogen fixation data for three coniferous forests in Sweden. Their data indicate the relative nitrogen-f ix ing potentials of different strata. The importance of organic carbon supply was emphasized, as the most imp.ortant nitrogen-f ix ing components were decaying wood , the litter layer, and the rhizosphere and humus layer. Todd et al . (1975) reported that while the highest rates of nitrogen fixation occured in decaying wood and humus, the largest amount of nitrogen fixation occurred in the soil due to the greater mass of so i l . The report by Jones (1970) that n i t rogen-f ix ing bacteria on leaf surfaces of Douglas - f i r could fix considerable quantities of nitrogen, focused attention on coniferous foliage as a potential source of nitrogen f ixation. Subsequent research by Jones (1982) and others (Granhall and Lindberg, 1980; Caldwel l , Hagedorn and Denison, 1979; Todd et al . , 1978) failed to support this f inding, indicating that foliage may not be a significant source of nitrogen f ixation. In a study of forest species in Minnesota and Oregon (Sucoff, 1979), nitrogen fixation on leaf surfaces without epiphytes was considered to be negligible. The presence of epiphytes with a nitrogen-f ix ing blue-green algae component may make the phyllosphere a significant source of nitrogen fixation (Denison, 1973; Millbank, 1978). Roskoski (1980) investigated nitrogen fixation in wood litter in the northeastern United States. Fixation was highest in the oldest and youngest stands studied, as a result of higher acetylene reduction rates and larger quantities of wood litter. The higher acetylene 19 reduction rates were attributed to a greater abundance of large dead logs which provide a more suitable habitat for nitrogen fixation because of their higher moisture content and their ability to retain anaerobic microsites. ANF in the mineral soil in these (Roskoski, 1980) and similiar sites (Tjepkema, 1979) was found to be insignificant. Decomposit ion of woody material in forests is s low, in part because of the high C/N ratio of the material. It has been suggested that nitrogen fixation may help facil itate the decay of woody substrates (Cornaby and Waide, 1973). Jorgensen (1975) postulated that wood decay fungi wil l benefit from added nitrogen while nitrogen-f ix ing bacteria wil l benefit from the carbon-r ich products of wood decay and perhaps from reduced 0 2 levels due to fungal respiration. Research indicates that nitro.gen fixation may be important in the wood decay process, although not as important as other sources of nitrogen, such as crown wash (Larsen et al . , 1982; Si lvester et al . , 1982). 2.5 THE EFFECTS OF FOREST MANAGEMENT ACTIVITY ON ASYMBIOTIC NITROGEN FIXATION AND DENITRIF I CATION The impact of forest management practices on soil biological processes has received considerably less attention than the effects of the same practices on the physical and chemical properties of s o i l . Forest f i res , clearcutting, scarif ication and ferti l ization are examples of treatments that wil l alter chemical and physical soil properties such as moisture content, temperature, aeration, pH, bulk density and available nutrients (Jurgensen et al . , 1979) causing changes in rates of ANF and denitrif ication. 20 Site changes resulting from timber harvesting should tend to promote ANF and denitrif ication. Logging wil l generally increase soil pH and temperature during the growing season, and cause increased soi l saturation due to removal of vegetation. These conditions wi l l favour the anaerobic processes of ANF and denitrif ication. Logging wi l l also promote more rapid mineralization, resulting in a flush of carbon and mineral nitrogen. These conditions wi l l promote denitrif ication, but may conceivably inhibit ANF by putting the organisms at a competitive disadvantage for carbon in the presence of high mineral nitrogen availability (Sprent, 1979). Fire is a natural occurence in many temperate forests and has been used as a management too l . Jorgensen and Wells (1971) reported significant increases in ANF fol lowing prescribed burning in loblol ly pine (P/nus taeda L.) stands in South . Carolina. They attributed this to increases in soi l temperature, soil moisture, pH, available nutrients' and available carbon. These conditions wi l l also promote the activity of denitrifying bacteria (Knowles, 1982a). Denitrif ication wi l l be influenced by any change in soil nitrate levels. Logging and fire may improve conditions for nitr if ication and nitrogen ferti l izers wi l l increase nitrate concentrations in the s o i l , resulting in increased denitrif ication. Rolston (1981) reviewed the literature on nitrogen loss after ferti l ization on cropped soi ls and reported losses of 0 to 75 per cent of applied nitrogen ferti l izer. This wi l l vary considerably with so i l condit ions. Fertilizer applications may potentially inhibit nitrogen f ixation. This should be considered when evaluating the net benefits of fert i l ization. Francis (1982) studied the effects of soil acidity on mineralization, ni t r i f icat ion, nitrogen fixation and denitrif ication in forest so i l . He suggested that increasing acidif ication of forest soi ls by acid precipitation wi l l lead 21 to significant reductions in these microbial processes. The removal of vegetation and the nutrients contained therein, creates an "ecosystem need". This need may be f i l led by the use of chemical fert i l izers. The use of ferti l izers may be unsuitable for economic and ecological reasons. The addition of nitrogen fertil izers may create defiencies in other plant nutrients, and may also cause imbalances in the natural cycl ing of organic matter by changing the C/N ratio. "This could lead to a reduction in total nitrogen and organic matter content, so that long-term soil ferti l ity would be decreased, even if a continuous supply of fertilizer were maintained" (Granhall, 1981). Clearly, a better understanding of ANF and denitrif ication, and their role in forests , is needed. If artificial means for promoting forest productivity are to be used, the long term effects on the nitrogen cycle must be considered. 3. OUTLINE OF RESEARCH In the fall of 1983 a study was begun, with the specif ic objectives of : 1. quantifying the net balance between gaseous N inputs to, and outputs f rom, a mature forest ecosystem and determining the relative signif icance of the processes contributing to this balance. 2. determining where in the ecosystem the gaseous N transformation processes occur and the relative significance of substrate to the rate at which they occur. 3. studying inputs and outputs in relation to season. Based on the preceeding litrature review and some preliminary investigation in the summer of 1983, 11 strata were chosen for study. Foliage and bark .of three tree species were sampled for nitrogen-f ix ing activity and forest f loor , mineral soil and 3 classes of decaying wood were sampled for ANF and denitrif ication. The ensuing report on the results of this study wil l include a description of the study area, a discussion of methods and a discussion of the results and their s ignif icance. 22 4. THE STUDY AREA The University of British Columbia Research Forest is located 7 km. north of Haney, British Columbia, Canada, approximately 40 km. east of Vancouver, B.C. (Figure 2). The 5,151 hectare forest is managed by the Faculty of Forestry, UBC, as an experimental, research and demonstration faci l i ty . It was chosen to be the location of this study because -1. It contained established forest ecosystems that would not be disturbed by management activity during the course of the study. 2. Its proximity to UBC where the analysis was done minimized travel time and handling of sample material. 3. A large amount of mensurational and ecological data exist for various parts of the forest. The site chosen for this study is in the southern portion of the Research Forest approximately 2.5 km. northwest of the main entrance (Figure 3). The Research Forest lies in the foothi l ls which mark the transition from the Fraser Valley to the Coast Mountains. The cl imate, in general, is mesothermal, influenced by the Pacif ic Ocean to the west and modif ied by mountainous relief and the inland location in the lower Fraser Valley. The climate has been classif ied using the Koppen climatic c lass i f icat ion as Cfb (Klinka, 1976), ie equable (marine) mesothermal humid to rainy. This is described as a maritime climate characterized by mild temperatures with common cloudiness and a small range of temperatures, wet and mild winters, cool and relatively dry summers, a long f ros t - f ree period, and heavy precipitation, most of which occurs during the winter. Mean monthly temperature and monthly precipitation for 1984 and 20 year average mean monthly temperature and monthly precipitation are presented in Table 1. The weather in 1984 was slightly warmer and slightly wetter than the 20 year 23 Figure 2. Access to the UBC Research Forest. Figure 3 . L o c a t i o n o f the s tudy s i t e . T a b l e 1 . M e a n m o n t h l y t e m p e r a t u r e a n d m o n t h l y p r e c i p i t a t i o n i n 1 9 8 4 a n d t h e 2 0 y e a r a v e r a g e ( l a s t y e a r 1 9 7 5 ) f o r t h e U B C R e s e a r c h F o r e s t , M a r c S t a t i o n . M o n t h M e a n D a i l y M a x . T . ( " C ) 2 0 y r A v e r a g e 1 9 8 4 M e a n D a i l y M i n . T . ( C ) 2 0 y r A v e r a g e 1 9 8 4 M o n t h l y P r e c i p . ( m m ) A v e r a g e 1 9 8 4 J 3 . 9 7 . 1 - 1 . 6 1 . 9 2 8 9 4 3 2 F 6 . 6 9 2 0 . 2 3 . 8 2 1 9 2 1 7 M 8 . 4 1 2 . 7 0 . 8 3 . 3 2 3 1 2 1 9 A 1 2 . 4 1 3 . 9 3 . 3 3 . 0 1 5 4 1 8 6 M 1 6 . 8 1 5 . 0 6 . 6 5 . 5 1 1 1 2 1 1 J 1 9 . 4 1 8 . 1 9 . 4 9 . 3 9 2 1 3 3 J 2 2 . 6 2 3 . 4 1 0 . 9 1 0 . 9 6 8 2 1 A 2 1 . 9 2 2 . 5 1 0 . 9 1 1 . 0 8 0 6 4 S 1 9 . 2 1 8 . 5 8 . 8 8 . 4 1 2 8 1 1 6 0 1 3 . 2 1 1 . 9 5 . 6 4 . 5 2 4 4 2 5 2 N 8 . 0 7 . 8 1 . 8 2 . 3 2 7 5 3 6 8 D 5 . 3 3 . 2 0 . 0 2 . 6 3 1 5 2 1 6 Y e a r l y T o t a l 2 2 0 6 2 4 3 5 27 average. The southern part of the forest has a submontane physiography, featuring flat to gently rolling terrain with a few granit ic -cored uplands. The entire area is underlain by igneous intrusive rock, predominantly quartzdiorite. Soi ls have evolved from surficial deposits left by pleistocene glaciation, the most recent event occuring some 10,000 years ago. Glacial ti l l and colluvium are the predominant parent materials (Klinka, 1976). The soil underlying the study site is a mini Humo-Ferric Podzol derived from reworked ablation till overlying basal t i l l . It is a sandy loam in texture with 2 0 - 3 0 % coarse fragment content overlain by a 5 cm thick mor humus layer. The study site is located at an elevation of 230 to 235 m asl with a southwest aspect and average slope of 5%. Vegetation in the forest is dominated by temperate marine coniferous forests , with varying mixtures of Douglas - f i r (Pseudotsugau menziesii (Mirb.) Franco var. menziesii), western hemlock (Tsuga heterophylla (Raf.) Sarg.) and western redcedar {Thuja plicata Donn). A synsystematic c lassif icat ion of the UBC Research Forest has been carried out by Klinka (1976). The study site lies in a transition between his Moss(Po/ystichum)-\NRC-\NH and Moss-Mahonia-DF-WH plant associat ions. The vegetation present had developed by natural regeneration fo l lowing a wildf ire in 1868. The overstory was dominated by Douglas - f i r while western hemlock and western redcedar occupied places in the lower tree layer. Figure 4 presents a distribution of tree diameters for the study site. The shrub layer was not well developed and higher shrubs were absent. The lower shrub layer (0 to 2 m.) covered approximately 25% of the area and was dominated by Mahonia nervosa Pursh with lesser amounts of Gaultheria shallon Pursh. Other species present included Vaccinium parvifolium Smith , Acer circinatum 28 JO E H 3 Legend E3 W « s l a r n R«dc«dar •i W . , l . r n H.mlocl< O Douglaa-fir to 30 40 50 60 Diameter Class (cm.) 70 BO 90 Figure 4. Diameter distribution of trees in the 30m X 30m sample plot. 29 Pursh, Tsuga heterophylla and Rosa nutkana Presl . The herb layer was sparse covering 1 to 2% of the ground with Polystichum munitum (Kaulf.)Presl as the major species. Other herb species included Dryopteris assimilis Walker, Trillium ovatum Pursh, Trientalis /at/folia Hook, and Tiarella trifoliata L. Stokesiella oregana (Sull.)Robins. dominated a moss layer covering roughly 30% of the area, with Plagiothecium undulatum (Hedw.)B.S.G. and Rhizomnium glabrescens (Kindb.)Koponen common on decaying woody material. Hylocomium splendens (Hedw.)B.S.G. and Rhytidiadelphus loreus (Hedw.)Warnst. were also present. Biomass data for the 30m. X 30m. study site are presented in Table 2. The phyllosphere was dominated by Douglas-fir(DFF), foliage biomass of this species amounting to 1,140 kg compared to 800 kg for western redcedar(WRCF) and western hemlock(WHF) combined. Two species, Douglas - f i r and western redcedar, comprised the decaying wood on the study site. These occur as deadfall on the forest floor and as standing stumps and snags. Douglas - f i r wood was divided into two classes of decay. The first, referred to as 'white-rot'(WDF), corresponded to an incipient stage of wood decay. The other, referred to as Ved-rot'(RDF), corresponded to an advanced stage of wood decay. The primary decay agent was the brown crumbly rot, Fomitopsis pinicola (Schwartz:Fr.)Karst. The two classes of Douglas - f i r wood accounted for 9,700 kg of b iomass, in roughly equal proportions, while western redcedar(WRCW) accounted for 3,000 kg. Klinka (1976) describes sites such as this one as having medium productivity, but being, wel l suited for forestry use and suggests that they should be intensively managed as commercial forests. T a b l e 2 . V o l u m e , d e n s i t y a n d b i o m a s s d a t a f o r t h e s a m p l e s t r a t a o n t h e 3 0 m X 3 0 m s t u d y s i t e . S T R A T U M ' V O L U M E ( m ' ) D E N S I T Y ( g c m ' ) ' D R Y W E I G H T ( k g ) D R Y W E I G H T / H A ( k g ) L F H S O I L ' 4 5 2 7 0 0 . 2 9 0 . 5 9 1 3 , 0 5 0 1 5 9 . 8 4 0 1 4 5 , 0 0 0 1 . 7 7 6 . 0 0 0 D e c a y e d W o o d R D F W D F W R C W 2 4 1 8 1 2 0 . 1 9 0 . 2 9 0 . 2 6 4 , 5 6 5 5 , 2 8 9 3 . 0 6 5 5 0 , 6 6 8 5 8 , 7 6 8 3 4 . 0 5 0 F o l i a g e D F W R C W H , 1 4 0 4 8 1 3 2 2 1 2 , 6 7 0 5 , 3 3 9 3 . 5 8 0 1 s t r a t u m s y m b o l s a r e e x p l a i n e d o n p a g e 2 9 o f t h e t e x t . 1 d e n s i t y i s r e l a t i v e d e n s i t y f o r w o o d a n d L F H a n d b u l k d e n s i t y f o r s o i l ' s o i l m e a s u r e m e n t s a r e f o r t h e t o p 3 0 c m o f m i n e r a l s o i l . CO o 5. METHODS Gaseous nitrogen fluxes were quantified for a 30m X 30m square plot in the UBC Research Forest. The plot was divided into strata based on ability to fix nitrogen and/or denitrify. The stratif ication was done with reference to the available literature and to some preliminary sampling done in the summer of 1983. Denitrif ication and ANF were measured bimonthly for one year beginning in January,1984 and finishing in November,1984. Air and soil temperatures on representative sampling days are presented in Table 3. Measurement of gaseous nitrogen fluxes was done using the acetylene(C 2H 2) -ethylene(C 2H 4) assay for nitrogen fixation (Hardy et al . , 1973) and the acetylene inhibition method for denitrif ication (Yoshinari et al., 1977). These can be done simultaneously, l imiting the amount of f ield sampling. Nitrogen-f ix ing organisms transform acetylene to ethylene at a rate proportional to that at which N 2 is reduced to NH 3 : N 2 + 3 H 2 — - 2 N H 3 C 2 H 2 + H 2 ~-C 2H 4 This suggests a conversion ratio of 3 :1, but for a number of reasons "empirically determined ratios seldom equal the theoretical" (Roskoski, 1981; Rice and Paul, 1971). These reasons include; 1. C 2 H 2 is roughly 65 times as water -so lub le ' as is N 2 . Hence, it may saturate the nitrogenase system more than would N 2 (Knowles, 1982b). 2. Oxygen, which is required for the production of ATP , may be depleted during long incubations. 3. As ethylene is not involved in metabol ism, nitrogen deficiency may inhibit physiological processes. 31 Table 3. Air and soil temperature (12 noon) on sampling days. Air Temp(°C) Soi l Temp(°C) Jan 25 2 4 Mar 6 ( 12 5 May 8 ' 10 7 Jul 4 17 n Sep 12 11 11 Nov 6 9 6 33 The ef fects of these factors wi l l be most significant when investigating material , such as forest s o i l , with low nitrogen-f ix ing act iv i ty , necessitating relatively long incubation times (Sprent, 1979). In some situations, such as in waterlogged so i l , nitrogen dif fusion may be the limiting factor for nitrogen fixation (Rice and Paul, 1971) and the higher solubil ity of acetylene wi l l cause overestimates of nitrogen fixation rates. This illustrates the importance of calibrating the acetylene reduction assay for specif ic condit ions. Silvester et al . (1982) investigated the relationship between acetylene and nitrogen reduction in decaying coniferous wood , using 1 5 N. The ratio was found to increase with t ime, but averaged 3.5 when samples were incubated less than 7 hours. In this study an incubation time of 8 hours and the stoichiometric conversion ratio of 3:1 were used. The low rates of ANF encountered in this study minimize the potential inaccuracy of this assumption. Ethylene may also be produced by plant tissue as a result of wounding, by contact between terminating acids and rubber stoppers and by microorganisms, part icularly in anaerobic soi ls (Sprent, 1979). Tests for ethylene production in the absence of acetylene are necessary. The measurement of N 2 evolution as a product of denitrif ication is made diff icult by its occlusion into ambient atmospheric nitrogen (Yoshinari et a l . , 1977). The acetylene inhibition method for measuring denitrif ication is based on the principle that acetylene inhibits the reduction of N 2 0 to N 2 (Yoshinari and Knowles, 1976). The evolution of N 2 0 can then be measured using gas chromatography. From measurements of N 2 0 , estimates can be made of denitrif ication rate. A serious problem with this method is the report that acetylene inhibits nitr i f ication, which yields N 2 0 as a byproduct (Aulakh et al . , 1982). In systems where nitrif ication is signif icant, this wi l l 34 cause underestimates of N 2 0 production. Nitrification is also the major source of nitrate which may be rate- l imit ing in certain systems (Knowles, 1982a). However, in a study of northern hardwood forests , Mel i I lo et al . (1983) found that N 2 0 production in the presence and absence of acetylene was not significantly different. This suggests that N 2 was not a significant product of denitrif ication in such systems and that the inhibition of nitrif ication had no major impact on denitrif ication. Alternative methods involve the use of 1 5 N- label led tracer compounds. Methods using 1 5N have been criticized on the basis of the cost of the marking chemical and the cost and relative insensit ivity of mass spectrometric analysis for f ield samples with low activity (Hardy et al . , 1973; Yoshinari et al. , 1977). It has also been demonstrated that denitrifying bacteria discriminate between . naturally occuring 1 4N and isotopic 1 5N (Blackmer and Bremner, 1977). For these reasons, techniques involving the use of acetylene offer the easiest and simplest means of measuring ANF and denitrif ication (Focht, 1978; Hardy et al. , 1973). 5.1 FIELD PROCEDURES The 3 experiments to investigate fol iage, bark and decaying wood were designed as randomised blocks. Each experiment involved 3 treatments (species or decay classif icat ion) and 6 blocks (month). The forest f loor and mineral soi l experiment was designed as a 2X6 randomised block but was not analyzed statist ical ly . At each sampling period, 12 samples of each strata were col lected. For foliage and bark, 8 samples were incubated with acetylene and 4 samples were incubated without acetylene to monitor endogenous ethylene production. For forest f loor, mineral soi l and decaying w o o d , 10 samples were incubated with acetylene and 2 samples without. 35 Sample sizes were halved for the January and March samplings, as no activity was expected. Foliage samples were clipped and then wrapped in wet tissue to maintain turgor pressure. Samples were taken from branches on trees that could be reached from the ground. This sampling scheme raised questions as to whether older trees might have better developed lichen components in their crowns and therefore have a higher potential for nitrogen f ixation. For this reason, an experiment was done in September/1984 using foliage from mature trees collected at a fresh logging area near the study site. Foliage was collected from the top and bottom of crowns of 3 trees of each of the 3 major species. This experiment was designed as a 3 (species) X 2 (top or bottom) randomised block, with variation between trees nested within species. The experiment was done in an incubation chamber, at room temperature under fluorescent .and incandescent lamps, in an attempt to mimic summer condit ions. Bark samples were taken from the dead bark of mature, live trees. Mineral soil and forest f loor samples were taken using trowel and shovel . Wood samples were taken from log sections using chainsaw and axe. Samples were taken randomly from all size classes of woody material. Samples were all placed immediately into 1-L glass mason jars. The jars were equipped with rubber septa to facil itate acetylene amendments and gas sampling. The head space volume of the jar was then amended to 0.1 atm of acetylene using industrial grade acetylene. After al lowing approximately 1 hour for equilibration, gas samples were taken using 5 - m L vacuum tubes to determine initial N 2 0 and C 2 H 4 concentrations. The jars were then left in the f ield under ambient conditions for the length of the incubations. Soi l and wood samples were kept in boxes to prevent 36 exposure to light while foliage and bark samples were left on the forest floor to mimic light conditions within the forest. After 8 hours, vacuum tube samples were taken to measure C 3 H 4 production. Preliminary experimentation produced very low rates of denitrif ication, therefore an incubation time of 24 hours was used. As the goal of this study was to produce information about real rates of gaseous nitrogen flux, incubations were done under conditions that mimicked natural conditions as closely as possible. Other than acetylene, no amendments or additions were made to the samples. Incubations were aerobic, as anaerobic incubations might exaggerate fluxes. Two additional experiments were conducted to validate the low results of the denitrif ication assays. The first involved amending 6 forest floor samples with 60 ml of a 5 mg L*1 nitrate solution to determine if nitrate was rate l imiting. The results were compared to denitrif ication rates for May and July using a f - test to determine if there was a significant effect. To further test the validity of the denitrif ication results, an experiment was done in which material known to denitrify, specif ical ly stream sediments, was incubated. Four samples of stream sediment were placed in mason jars and immersed in water to mimic their natural environment. Other experimental procedures were as previously mentioned. When the incubations were complete, sample wet weight, oven-dry weight, volume and % moisture content were determined. 5.2 LAB A N A L Y S I S Analysis for N 2 0 was done using a Hewlett -Packard 5790A series gas chromatograph equipped with an N i " electron capture detector and using a 1.8 m X 3 mm glass column packed with Porapak Q - 5 (80/100 mesh). 37 Oven temperature was 60 °C, detector temperature was 250 °C and f low rate was 25 ml m i n - 1 . Carrier gas was a 95% Argon, 5% Methane mixture. Calibration was by external standard using N 2 0 in air of 1.5 and 52.5 mg L*1. Response was assumed to be linear based on the investigations of Kaspar and Tiedje (1982). Concentrations of C 2 H 2 and C 2 H 4 were measured using a Hewlett -Packard 5830A series gas chromatograph using two 1.8 m X 3 mm stainless steel columns packed with Porapak N (80/100 mesh) and a flame ionization detector. Nitrogen (N2) carrier gas f lowed through the respective columns at rates of 30 and 37 ml m i n 1 . Oven temperature was 50 °C, injection temperature was 105 °C and detector temperature was 130 °C. Calibration was by external standard with a linear response assumed over the range of expected values. 5.3 CALCULATIONS Gas chromatographic results for denitrif ication were expressed in mg L 1 . This value was multiplied by head space volume and divided by molecular weight and sample dry weight to produce a measure of denitrif ication rate in nmoles N g - 1 day- 1 . This value was then corrected for dissolved N 2 0 as recommended by Moraghan and Buresh (1977). Gas chromatographic results for acetylene reduction were expressed in nmoles ml " 1 . This value was multiplied by head space volume and divided by sample dry weight to produce a measure of acetylene reduction in nmoles g _ 1 per 8 hours. This value was multiplied by 3 to produce a per day value and then divided by 3 to convert to nitrogen f ixation, producing a value in units of nmoles N g _ 1 d a y 1 . 38 Mean rates of nitrogen fixation and denitrif ication in units of nmoles N g - ' day- 1 were calculated and then converted to annual flux rates in units of kg N h a - 1 a - 1 . Multiplying by molecular weight transforms nmoles to units of weight. The number of days of activity per year was calculated from the number of active months in the year - long sample. If 2 out of the 6 monthly samples were active, activity was assumed to occur on 2/6 X 365 days of the year. Multiplying by total biomass h a - 1 then produced a measure of flux in kg N h a - 1 a 1 . 5.4 SITE QUANTIFICATION Foliage biomass was quantified by measuring tree diameters and using regression equations for the UBC research forest relating foliage biomass of tree crowns, to their diameters (Table 4). Bark surface area was not quantified as during the course of the experiment no nitrogen fixation was measured in this stratum. Decayed wood volume was quantified using Smalian's formula, V = (A, + A 2 ) X L / 2 where measurements were made of: A j=top end diameter A 2 =butt end diameter L=sect ion length for each piece of decaying wood present on the study site. Volume was converted to dry weight using densities calculated from selected samples. Density was calculated as M/V where volume was determined by water displacement and mass was the oven-dry (105 °C) weight of the sample. Forest f loor and mineral soi l were quantified by determining volume from horizon depth and area and then converting to dry weight using experimentally calculated densit ies. Bulk density was caculated for mineral Table 4. Regression equations relating biomass of foliage (Y) to outside bark diameter at breast height (D), /nY(kg.) = a + b//?D (cm Source; M.C. Feller (unpublished data). Species a b SE(/n units) r2 n DF -1.216 1.286 .216 .986 10 WRC -3.869 2.100 .408 .930 12 WH -4.130 2.128 .435 .960 18 40 soil and relative density for forest f loor material. Forest f loor volume was determined by water displacement while mineral soil volume was held uniform throughout the experiment. The top 30 cm. of the mineral soil horizon was considered as the area of active microbial act iv i ty , as nitrogen fixation has been shown to decrease significantly with depth (Baker and A t t i w i l l , 1984). Site quantification results and sample densities were presented in Table 2 (page 30). 6. RESULTS AND DISCUSSION 6.1 NITROGEN FIXATION Nitrogen fixation rates and total annual fluxes are presented in Table 5. Fixation rates are indications of potential nitrogenase activity, based on acetylene reduction rates and the stoichiometric conversion ratio of 3:1. Endogenous ethylene production was not encountered during the course of this experiment. 6.1.1 FOREST FLOOR AND MINERAL SOIL The most important substrate for nitrogen fixation was the forest f loor , which accounted for almost 80% of a total measured input due to biological f ixation of 0.8 kg N h a - 1 a - 1 . This finding agrees with other authors (Silvester and Bennett, . 1973; Granhall and Lindberg, 1980; Baker and Att iwi 11, 1984) who found the highest rates of nitrogen fixation in the organic horizons of coniferous forest so i ls . Forest f loor material wi l l have higher nutrient status, higher moisture content and greater available carbon than mineral so i l . Fixation rates in mineral soil were very low. The total input of nitrogen in this substrate is 0.06 kg N h a - 1 a - 1 . This agrees with the reports of Roskoski (1980), Tjepkema (1979), and Granhall and Lindberg (1980) that mineral soil is a relatively insignificant source of nitrogen fixation in northern temperate forests. A n adequate supply of available carbon is often the limiting factor for nitrogen fixation (Sprent, 1979). Forest f loor material wil l have a higher C/N ratio and lower 0 2 tension (ie higher moisture • content) than mineral s o i l . Heterotrophic nitrogen fixation rates have 41 T a b l e 5 , B i m o n t h l y n i t r o g e n f i x a t i o n r a t e s a n d t o t a l a n n u a l f i x a t i o n f o r 1 9 8 4 . S T R A T U M J A N B i m o n t h l y N F i x a t i o n R a t e ( n m o l e s N g - ' d a y 1 ) M A R M A Y J U L S E P N O V T o t a l A n n u a l F i x a t i o n k g N h a - 1 a 1 L F H S O I L n 0 . 0 0 . 0 5 0 . 0 0 . 0 5 1 . 4 3 9 ( 4 7 3 ) 0 . 0 2 ( . 0 1 ) 1 0 0 . 5 5 ( . 1 6 ) 0 . 0 1 0 0 . 2 7 ( . 0 9 ) 0 . 0 1 ( . 0 0 3 ) 1 0 0 . 2 3 ( . 0 5 ) 0 . 0 1 0 0 . 6 1 0 . 0 6 D e c a y e d W o o d R D F W D F W R C n 0 . 0 0 . 0 0 . 0 5 0 . 0 4 ( . 0 5 ) 0 . 0 4 ( . 0 3 ) 0 . 0 3 ( . 0 3 ) 5 0 . 2 7 ( . 0 8 ) 0 . 0 8 ( . 0 3 ) 0 . 0 5 ( . 0 2 ) . 1 0 0 . 0 4 ( . 0 3 ) 0 . 0 7 ( . 0 3 ) 0 . 0 2 ( . 0 1 ) 1 0 0 . 0 5 ( . 0 2 ) 0 . 0 1 ( . 0 1 ) 0 . 1 0 ( . 0 4 ) 1 0 0 . 0 0 . 0 0 . 0 1 0 0 . 0 3 0 . 0 2 0 . 0 1 F o l i a g e D F W R C W H n 0 . 0 0 . 0 0 . 0 5 0 . 1 8 ( . 1 8 ) 0 . 3 4 ( . 2 5 ) 0 . 0 5 0 . 4 7 ( , 2 4 ) 0 . 1 3 ( . 0 9 ) 0 . 2 5 ( . 1 9 ) 1 0 0 . 6 7 ( . 2 9 ) 0 . 2 4 ( , 1 2 ) 0 . 2 9 ( . 1 5 ) 8 0 . 0 0 . 0 0 . 0 8 0 . 0 0 . 0 0 . 0 8 0 . 0 3 0 . 0 1 0 . 0 0 B a r k D F W R C W H n 0 . 0 0 . 0 0 . 0 5 0 . 0 0 . 0 0 . 0 5 0 . 0 0 . 0 0 . 0 1 0 0 . 0 0 . 0 0 . 0 8 0 . 0 0 . 0 0 . 0 8 0 . 0 0 . 0 0 . 0 8 0 . 0 0 . 0 0 . 0 T O T A L 0 . 7 8 - s t a n d a r d e r r o r s i n p a r e n t h e s e s . 43 been correlated with concentrations of organic matter and moisture content and wi l l correspondingly decrease with decreasing soil depth and decreasing soil moisture (Baker and A t t iw i l l , 1984; Granhall and Lindberg, 1980). Coniferous forest soi ls generally have low pH. The availability of molybdenum and phosphorus may limit nitrogen fixation under these condit ions, as the availability of both these nutrients decreases rapidly with decreasing soil pH (Jurgensen and Davey, 1970). Highest nitrogen fixation rates occured in May (Figure 5), when moisture content was highest (Table 6). While nitrogen fixation is posit ively correlated with temperature, high summer temperatures often correspond to periods when moisture content is relatively low. Moisture content has been found to limit nitrogen fixation rates under apparently optimum temperature conditions (Baker and At t iw i l l , 1984). The absence of fixation in November when moisture conditions are favourable may be explained by higher mineral nitrogen availability in November than May. Fall increases in nitrate in streamwater have been observed by Feller and Kimmins (1984). 6.1.2 DECAYING WOOD Nitrogen fixation by f ree - l i v ing bacteria in decaying wood was relatively insignificant (Table 3). Total annual input due to nitrogen fixation in decaying wood was only 0.06 kg N h a - 1 a*1. Because of the large number of inactive samples, the data had a skewed distribution and statistical analysis was invalid. Transformation failed to correct this problem. The data are compared visually in Figure 6. The greatest flux occured in the month of May 44 Legend tZ3 FOREST FLOOR S3 MINERAL SOIL JAN MAR MAY JUL MONTH OF 1984 SEP NOV I STANDARD ERROR Figure 5. Nitrogen fixation rates in mineral soil and forest f loor for 1984. 45 Table 6. Average moisture content (by percent) of sample material. Month of 1984 STRATA J A N MAR M A Y JUL SEP NOV LFH 208 208 259 154 193 247 SOIL 22 20 23 21 18 26 Decayed Wood RDF WDF WRCW 341 340 339 346 264 317 100 113 117 95 61 67 76 89 148 113 151 63 46 when, as with forest f loor , moisture content was high. Highest rates of fixation occurred in Douglas - f i r wood in the advanced stage of decay. Jurgensen et a l . (1984) and Larsen et al . (1978) have reported that nitrogen fixation rates increase as wood decay progresses. Total carbohydrate and soluble sugar have been shown to decrease as wood decay progresses, while total and soluble nitrogen tend to increase, conditions which would tend to inhibit nitrogen fixation (Jurgensen et al . , 1984). The increase in nitrogen fixation activity is probably attributable to increased moisture content in the more decayed wood (Table 4) and consequently reduced 0 2 levels. The fact that decay fungi can readily metabolize woody material, in spite of a high C/N ratio, suggests that .sources of nitrogen input exist. Nitrogen fixation rates from 0.3 to 1.4 kg N h a 1 a - 1 have been reported for decaying wood in northern temperate forests (Roskoski, 1980; Larsen et al . , 1978; Larsen et al., 1982; Jurgensen et al. , 1984). However, these results were achieved using anaerobic incubations, which may exaggerate the nitrogen fixation by anaerobic bacteria. Silvester et a l . (1982) suggested that microaerophilic bacteria may be the most important agent facil itating nitrogen fixation in decaying wood . In an experiment comparing woody material incubated anaerobically and at different 0 2 levels, nitrogen fixation rate was greatest under an atmosphere of 5% 0 2 . S t i l l , Silvester et al . (1982) reported rates of nitrogen fixation in decaying Douglas - f i r wood (4.8 nmoles N g - 1 day- 1 , 1.4 kg N h a - 1 y r 1 ) far greater than rates reported in this study. This discrepancy can, to some extent, be explained by differences in incubation temperature. Incubations in the 47 1.5n 0 •o Ol z « •5 E c < z o X 0.5-JAN j ^ r j , 1%1 r i # U t^ JS MAR MAY JUL SEP Legend eZl REDROT Df O WHITEROT Df CEDAR Y MONTH OF 1984 NOV STANDARD ERROR Figure 6. Nitrogen fixation rates in decaying wood for 1984. 48 Silvester study were done at 22°C, while in this study incubations were done under ambient conditions which never exceeded 17°C and were lower when moisture conditions were most favourable (Tables 3 and 6). Furthermore, Silvester et al . (1982) emphasized the importance of taking samples of large volume to reduce the surface area/volume ratio and prevent drying during the course of the incubation. In the experiment reported here, condensation was observed on the inside of the mason jars during incubations. This could have decreased nitrogen fixation rates by increasing oxygen exposure to nitrogen-f ix ing bacteria. Nitrogen fixation may be less important than other inputs of nitrogen to woody substrates, notably crown wash and litter fa l l . However, nitrogen fixation may have a significant rol.e in the microf loral and faunal succession in decaying wood and warrants careful study (Silvester et al . , 1982). 6.1.3 FOLIAGE Nitrogen fixation associated with foliage is responsible for an input of 0.04 kg N h a 1 a 1 . These low rates are supported by the available literature (page 17). The rates reported here may, in fact, be exaggerated due to the fact that although incubations were done in dayt ime, fixation rates were assumed to be uniform for 24 hours. Stewart (1974) has reported that day - t ime nitrogen fixation rates are usually higher than night - t ime rates. Nitrogenase activity in blue-green algae living in association with sphagnum moss , was reported to respond dramatically to light (Granhall and Lindberg, 1980). 49 Species and time of year were compared in a randomised block design. The preponderance of inactive samples again caused the distribution to be skewed and statistical analysis was invalid. However, it appeared that nitrogen fixation rate was higher in Douglas- f i r than in the other two species (Table 5, Figure 7). Fixation was relatively uniform from May to July. The drop in foliar f ixation from July to September may be related to moisture stress and decreases in nutrient uptake by trees. The presence of epiphytes with a nitrogen-f ix ing blue-green algae component may make the phyllosphere a significant source of nitrogen fixation (Denison, 1973; Millbank, 1978). Foliage samples for this study were collected primarily from younger trees that could be reached from ground level. This sampling scheme raised questions as to whether older trees might have better developed lichen components in their crowns and therefore have a higher potential for nitrogen fixation. An experiment was carried out in which fol iage was collected from mature tree crowns at a fresh logging site. Species and crown posit ion (upper half, lower half) were compared for significant differences (Table 7). The rate of nitrogen fixation in this foliage was similiar to that in the younger fol iage, supporting the claim that nitrogen fixation in foliage was not significant. The only significant difference found in this experiment was between species. Western hemlock was found to have the highest rate of f ixation. In the previous experiment Douglas - f i r was found to have the highest rate of nitrogen f ixation, suggesting that these differences may be attributable to some cause other than an inherent difference between species or to experimental error. 50 P I' m l i n Legend EZJ DF o WRC • WH JAN MAR MAY JUL MONTH OF 1984 SEP NOV I STANDARD ERROR Figure 7. Nitrogen fixation rates in foliage for 1984. 51 Table 7. Nitrogen fixation rates(nmoles N g _ 1 day - 1 ) and total annual flux (kg N h a - 1 a - 1 ) in foliage col lected from mature tree crowns (A) compared with foliage col lected at ground level(B). Species Fixation Rate Annual Flux in Annual Flux in B A DF 0.138(.067) 0.001 0.028 WRC 0.353(.114) 0.001 0.006 WH 1.018(.259) 0.020 0.004 n 12 TOTAL 0.022 0.038 -standard errors in parentheses. 52 6.1.4 BARK Several writers have reported nitrogen fixation on tree boles (Todd et al. , 1978; Granhall and Lindberg, 1980), and Millbank (1978) has confirmed that nitrogen-f ix ing lichens occur on coniferous tree trunks in the Pacif ic Northwest. However, in this study, no nitrogen fixation activity associated with bark was detected. 6.2 DENITRIFICATION Denitrif ication results are summarized in Table 8. While the data suggest that some denitrif ication occured, the total annual output of nitrogen due to denitrif ication was effect ively 0.0 kg N ha- 1 a - 1 . Rates reported for western redcedar wood and mineral soil were the result of only one active sample (n = 10). S imi l iar ly , forest f loor rates were the result of 3 active samples in May and two active samples in July (n = 10, both months). Furthermore, it is unlikely that these measurements are accurate to the 4 decimal places reported. High standard errors support the hypothesis that high spatial variability in denitrif ication rates is typical even in cultivated systems (Robertson and Tiedje, 1984). Robertson and Tiedje (1984) suggested that denitrif ication is a highly variable process both among and within temperate forests. Their results, like the ones presented in this study, often consisted of several active samples and a large proportion of inactive samples which diluted the positive effects of active samples. The reasons for this high spatial variability are not wel l understood, although they may be related to the occurrence of anaerobic microsites and/or nitr i f ication. The large proportion of '0' results raised concern about the validity of the methods used in this study. To test these methods, stream sediments were assayed for denitrif ication potential. Denitrif ication rate in T a b l e 8 . B i m o n t h l y d e n i t r i f i c a t i o n r a t e s a n d t o t a l a n n u a l d e n i t r i f i c a t i o n f o r 1 9 8 4 . B i m o n t h l y D e n i t r i f i c a t i o n R a t e ( n m o l e s N g - ' d a y 1 ) T o t a l A n n u a l S T R A T U M J A N M A R M A Y JUL S E P N O V ( k g N h a ' L F H 0 . 0 0 . 0 0 . 0 0 0 5 ( . 0 0 0 3 ) 0 . 0 0 0 2 ( . 0 0 0 1 ) 0 . 0 0 . 0 0 . 0 0 0 3 S O I L 0 . 0 0 . 0 0 . 0 0 . 0 0 0 2 ( . 0 0 0 2 ) 0 . 0 0 . 0 0 . 0 0 1 0 D e c a y e d W o o d R D F 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 0 0 W D F 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 . 0 0 0 0 W R C W 0 . 0 0 . 0 0 . 0 0 . 0 0 1 3 ( . 0 0 1 3 ) 0 . 0 0 0 2 ( . 0 0 0 2 ) 0 . 0 0 . 0 0 0 1 n 5 5 1 0 1 0 1 0 1 0 T O T A L 0 . 0 0 1 4 s t a n d a r d e r r o r s i n p a r e n t h e s e s . = n u m b e r o f s a m p l e s p e r s t r a t u m . 54 this material was 4.93 nmoles N g - 1 day - 1 (standard error=.46) indicating the validity of the low rates determined for soil and decaying wood. The absence of denitrif ication in decaying wood may be explained by low levels of mineral nitrogen in such environments. That denitrif ication did not occur in mineral soil may be explained by the relatively coarse texture of the s o i l , the lack of available organic carbon and/or the lack of nitrate. Using forest f loor , where both carbon supply and moisture content should be suitable for denitrif ication, an experiment was carried out in which samples (n=6) were amended with 60 ml of a 5 - m g L"1 nitrate solution. This treatment significantly increased denitrif ication rate (p < 0.01) although denitrif ication rate was sti l l relatively low at 0.012 nmoles N g - 1 day- 1 . Assuming that denitrif ication occurs for 122 days a - 1 this would yield a loss of 0.01 kg N h a - 1 a - 1 . The results of this experiment tend to support the suggestion that "nitrate production may be a principle determinant of N 2 0 loss in unfertilized temperate forests" (Robertson and Tiedje, 1984). Very little information about denitrif ication in forests exists. In general, forest soi ls with high levels of soluble organic carbon should provide a favourable environment for denitrif ication. Several recent studies (Meli l lo et al . , 1983; Robertson and Tiedje, 1984) have suggested that nitrate production may be a dominant factor in controlling N 2 0 loss in forests. Mel i l lo et al . (1983) studied denitrif ication potential over a successional sequence of hardwood stands in New Hampshire. In a 50+ year old stand, rates of nitrogen loss were 9 kg N h a - 1 a - - 1 using anaerobic incubation and 1.4 kg N ha- 1 a - 1 using aerobic incubation. Mel i l lo et a l . (1983) suggested that the "true" rate lies somewhere between the two. Denitrif ication potential was highest in a recently logged stand. Robertson and Tiedje (1984) studied denitrif ication potential in a number of sites in Michigan 55 forests and reported highest rates in sites with high potential for nitr if ication and respiration. The most active sites had potential flux rates of 2 to 12 kg N ha- 1 a - 1 , although a number of sites had very low rates of denitrif ication (<0.1 kg N h a - 1 month- 1). In both studies discussed here (Meli l lo et al . , 1983; Robertson and Tiedje, 1984) denitrif ication was found to be highest in spring. In forests, little nitr if ication is thought to occur in unamended systems (Keeney, 1980). Forestry practices such as clearcutting and slash burning wi l l alter the physical and biological environment and may promote denitrif ication by increasing nitrate availabil ity and soil saturation (Vitousek, 1981). Mel i l lo et al . (1983) and Martin (unpublished) have demonstrated that denitrif ication may be more significant fo l lowing clearcutting. Vitousek et al . (1982) suggested a model of nitrate mobil ity in forests where nitrate mobil i ty in forests may be greater fo l lowing clearcutting for' two reasons; 1. mineralization rates are higher, 2. trees are not actively taking up nitrogen. As succession proceeds, competit ion for nitrogen should increase, populations of nitrifying bacteria may be outcompeted and plants may return to a predominantly ammonium-based nutrition (Vitousek et al . , 1982). The implication is that as the site becomes fully occupied, forest trees wi l l take up mineral nitrogen before nitrif ication takes place, thereby preventing denitrif ication losses. If nitr if ication is occurring, the forest may sti l l be able to outcompete denitrifying bacteria for available nitrate. Significant increases in nitrate in soil leachate fo l lowing clearcutting have been demonstrated in the UBC Research Forest (M. C. Fel ler 1 , pers. comm.). A s s i s t a n t Professor , Faculty of Forestry, University of British Columbia, Vancouver, B. C. 56 Martin (unpublished) found increased nitrate fixation by anion absorption resin bags in forest f loor , but not in forest f loor leachate supporting the evidence that nitrate in forest f loor solution was being denitrif ied. Martin (unpublished) has determined denitrif ication rates for a recent clearcut and an old growth forest on Vancouver Island, B.C. Rates in the clearcut (40 kg N h a - 1 a - 1 ) were significantly greater than in the old growth forest (10 kg N h a 1 a 1 ) . However, old growth denitrif ication rates were much higher than the rates measured in this study. While some of this difference can be attributed to differences in methodology, they may also be due to stand dynamics. The old growth stand may become less efficient in taking up available nitrogen, allowing mineral nitrogen to become more accessible to nitr ifying bacteria and consequently denitrifying bacteria. The results presented in this study indicate that denitrif ication is an insignificant factor in the functioning of this ecosystem at its present state of maturity. However, any perturbation such as clearcutting, that might increase nitrate levels, might also increase the potential for denitrif ication. This potential may also be greater at future stages in stand development. 6.3 TOTAL FLUX Figure 8 illustrates the total annual gaseous nitrogen flux for the study site. Denitrif ication rates were effect ively zero (values are magnified 10X) while ANF accounts for an input of 0.8 kg N h a - 1 a - 1 . This is comparable with other published data for northern temperate forests (Table 9). This small input may be significant in balancing potential losses of nitrogen, and may contribute to a slow accumulation of nitrogen in this ecosystem. Feller and Kimmins (1984) reported nitrogen fluxes due to precipitation and streamwater runoff in the UBC Research Forest. Their data 57 YZZZLZZZZ&ZL 0.01 STRATA Legend IZ2 NITROGEN FIXATION •1 DENITRIFICATION Figure 8. Total annual nitrogen flux by strata. 58 Table 9. Asymbiot ic nitrogen fixation rates in temperate forests. Forest type: Estimated kg N fixed h a 1 a 3.8 0.3 Pine/spruce, 160 yrs old (Sweden) Pine, 120 yrs old (Sweden) u oPine, 15-20 yrs old (Sweden) 0.3 (Granhall and Lindberg, 1980) Pine (South Carolina) 1.0 (Jorgensen and Wel ls , 1971) Deciduous (North Carolina) 12.0 (Todd et al. , 1978) Deciduous (Massachusetts) 0.2 (Tjepkema, 1979). Deciduous, 4 yrs old (New Hampshire) 2.0 Deciduous, 57 yrs old (New Hampshire) 0.4 Deciduous, >200 yrs old (New Hampshire) 1.6 (Roskoski , 1980) Mixed conifer (British Columbia) (this study) 0.8 59 suggest a net input of 3 kg N ha1 a~\ Coupled with the input from ANF reported here this yields an annual input of 4 kg N h a 1 a - 1 . Over an 80 year rotation, this amount wi l l more than compensate for losses due to log export. However, losses due to the combined treatment of log export and slash burning may result in a net loss of nitrogen (Feller and Kimmins, 1984, Table 8). The assumption that flux rates are uniform during succession may not be justif ied. Some of the evidence discussed in this thesis al lows us to speculate on changes in gaseous nitrogen flux during secondary forest succession. Similiar successional trends can be suggested for both processes. Flux wil l be greatest fo l lowing clearcutting, decrease as succession proceeds and then increase, as the forest degrades in the later stages of succession. Vitousek and Reiners (1975) have hypothesized that nitrate losses may be controlled by net ecosystem productivity which shows a pattern similiar to that described above. Evidence has been cited for increases in ANF (Roskoski, 1980) and denitrif ication (Melil lo et al . , 1983; Martin, unpublished) fo l lowing clearcutting. Nitrate availabil ity is increased fo l lowing clearcutting due to vegetation removal and/or increased nitr i f ication. As succession proceeds , competit ion for nitrogen wi l l increase and populations of nitr ifying bacteria may be outcompeted for available mineral nitrogen. As the forest grows old and becomes less productive due to senescence and mortal ity, nitrate may again become more readily available for denitrif ication. Gorham et al . (1979) have suggested a simil iar trend for ANF, related to the - availabil ity of phosphorus, an important requirement for biological nitrogen fixation (Granhall, 1981). There is speculation that the availabil ity of phosphorus in forms available to plants decreases as succession 60 Table 10. Nutrient losses, nutrient inputs in precipitation and nutrient reserves for logged (A), logged and slash burned (B) and undisturbed (C) watersheds in the UBC Research Forest. Values are in kg/ha for the two year period 1973-1975. Streamwater export Log export Atmospheric export Total export Forest f loor content Mineral soi l content Total reserve Average annual precipitation input A B C 11 3 1 234 308 - 982 245 1293 1 1632 2180 1490 4566 4647 3924 6198 6827 5414 4 ' 4 4 Source: Feller and Kimmins, 1984. 61 proceeds (Gorham et al . , 1979). Similiar to nitrogen, phosphorus availability wil l be greatest fo l lowing clearcutting. ANF may also be higher after clearcutting and in old growth forests because of an increase in the accumulation of organic matter on the site (Roskoski, 1980). Increases may also be related to net ecosystem productivity as nutrients important for nitrogen fixation may become more readily available as the forest becomes less eff ic ient . Nitrogen fixation after clearcutting may help to offset potentially high nitrogen losses due to denitrif ication and leaching. As the site becomes reestablished, denitrif ication wi l l become less significant and ANF may make a modest contribution to reestablishing the nitrogen pool . 6.4 SOURCES OF ERROR ' During the course of this experiment a number of .potential sources of error were considered. The decision to use the stoichiometric conversion factor of 3:1, for acetylene reduction to nitrogen fixation was based on the work of Silvester et al . (1982). Experimentally-determined ratios generally vary from the theoretical suggesting that comparative C 2 H 2 - 1 5 N fixation studies should be done (Hardy et al . , 1973; Roskoski , 1981). Because of the low fixation rates encountered in this study, it was felt that the use of the theoretical ratio would not create too great an inaccuracy. Using a ratio of 4:1, total nitrogen fixation in this study would be 0.6 kg N ha- 1 a - 1 while using a ratio of 2:1 would result in a total flux of 1.2 kg N ha- 1 a - 1 . Silvester et al . (1982) found the ratio to be 3.5:1 when incubations were kept under 7 hours. Using this ratio, total flux in this study would be 0.7 kg N ha- 1 a 1 . 62 The incubation technique necessitates disturbing samples. Wood samples were cut from larger pieces and fragmented to make them fit into the incubation chambers. Simil iar ly , mineral soi l and forest f loor samples were sampled destructively. These disturbances result in increased surface area/volume and consequently a greater exposure to 0 2 than might occur under natural condit ions. Although aerobic incubations were done to avoid overestimating these reduction processes, aerobic incubation may, in turn, underestimate gaseous fluxes. Si lvester et al . (1982) found acetylene reduction rate to be higher under low 0 2 concentrations than under ambient or anaerobic condit ions. Oxygen dif fusion is strongly affected by moisture content of the material. Increased surface area/volume wi l l make the sample material more susceptible to drying and may cause artifactual ef fects . Future studies of denitrif ication should consider doing incubations with and without acetylene to determine the full effect of inhibiting nitr i f ication. If n i tr i f icat ion/ denitrif ication processes are coupled (Knowles, 1978), inhibition of nitrif ication wi l l uncouple these processes and effect ively halt denitrif ication. In addit ion, nitr if ication may produce significant quantities of N 2 0 in some systems (Aulakh etal., 1982). Both the acetylene reduction assay and the acetylene inhibition method are indications of nitrogen transformation at a specif ic point in t ime. Extrapolating nitrogen fixation rates measured over 8 hours to a per day measurement wi l l cause a decrease in accuracy. Extrapolating flux rates per day to rates per month or year wi l l create an even greater inaccuracy. The high spatial variabil ity reported in this study and elsewhere (Robertson' and Tiedje, 1984) suggest that this source of error should not be over looked. 63 The methods used in this study are inexpensive and easy to use, but are not ideal. Work directed to improving these methods or developing other techniques should be continued. 7. S U M M A R Y AND CONCLUSIONS Evidence has accumulated indicating the importance of biological nitrogen transformations in forest ecosystems (Todd et al . , 1975). Increased demand for wood fibre and fuel wood throughout the world and the realization that supplies of cheap chemical nitrogen ferti l izers are not inexhaustable have kindled interest in biological nitrogen fixation as a means of augmenting nitrogen supplies in biological systems. Ecosystem studies have suggested that biological nitrogen fixation may be a major source of nitrogen accumulated in forest biomass and the forest f loor during succession (Borman et al . , 1977; Todd et al. , 1975). While nitrogen inputs due to asymbiotic nitrogen fixation are generally smal l , coupled with inputs of nitrogen by bulk precipitation, they may be significant in contributing to nitrogen accretion in forests and offsett ing nutrient losses due to log export and/or slash burning (Feller and Kimmins, 1984). Interest exists in denitrif ication because it is a major mechanism of fertilizer nitrogen loss (Rolston, 1981) and it contributes N 2 0 to the atmosphere where it is involved in stratospheric reactions which result in the depletion of ozone (Knowles, 1982a). Denitrif ication in forests is generally thought to be minimal because it does not occur without nitrate and little nitrif ication normally occurs in undisturbed systems (Keeney, 1980). However, clearcutting may signif icantly increase nitrate availability (Feller, pers. comm.) and denitrif ication (Martin, unpublished; Mel i l lo et al., 1983). The objectives of this study were to quantify gaseous nitrogen fluxes due to asymbiotic nitrogen fixation and denitrif ication in a mature coniferous forest. Eleven strata were sampled bimonthly for the year of 64 65 1984. Gaseous nitrogen losses due to denitrif ication were effect ively zero. Forest f loor material was responsible for 80% of a nitrogen input of 0.8 kg N h a - 1 a - 1 . Nitrogen fixation in decaying wood and foliage was relatively smal l . Fixation rates were greater in more decayed w o o d , probably due to its greater moisture holding capacity. Assuming an equal rate of nitrogen fixation over the course of an eighty year rotation, a net input of 64 kg of nitrogen wi l l result. While this amount is small it may contribute to balancing nitrogen losses due to log export, slash burning and other forest management treatments. This input also has the advantage of occuring as a steady accumulation of s lowly released, easily available nitrogen (Granhall and Lindberg, 1980). Future research on ANF and denitrif ication should focus on the effects of site treatments such as clearcutting, slash burning, scarif ication and ferti l ization. ' Changes in rates of ANF and denitrif ication may also occur during the course of forest succession. Research must also focus on improving techniques for measuring both processes. The use of acetylene provides an inexpensive and easy method for measuring ANF and denitrif ication but the potential for inaccuracies exists. Industrial nitrogen fixation has increased the fixed pool of nitrogen in terrestrial environments (Delwiche, 1981). While there is no concern that atmospheric nitrogen wil l be depleted, there is concern regarding the increase of nitrates in food and water and increased N 2 0 concentrations in the atmosphere. Nitrogen fertil izers are becoming increasingly expensive (Beuter, 1979) and are subject to potentially high denitrif ication losses. Biological nitrogen fixation offers an interesting alternative to the forest manager, as a means of maintaining or enhancing forest productivity. 66 Our knowledge of biological nitrogen transformations such as asymbiot ic nitrogen fixation and denitrif ication is incomplete. The role of these processes in forests , their response to treatment and their relationship to each other and other nitrogen transformations warrants further attention. LITERATURE CITED Alexander, M. 1977. Introduction to Soi l Microbio logy. J . Wiley and Sons, Inc. pp 293-308. A l l i son , F. E. 1965. Evaluation of incoming and outgoing processes that affect soi l nitrogen. pp. 573-606 In: Soi l Nitrogen (eds. Bartholomew W. V. and F.E. Clark). Madison: A m . Soc . of Agronomy. Armson , K.A. 1977. Forest So i l s : properties and processes. University of Toronto Press, pp. 100-102. Aulakh, M.S., D.A. Rennie and E.A. Paul. 1982. Gaseous nitrogen losses from cropped and summer - fa l lowed soi ls . Can. J . Soi l S c i . 62:187-196. Bailey, L.D. 1976. Effects of temperature and root on denitrif ication in so i l . Can. J . Soi l Sc i . 56 :79 -87 . Baker, T.G. and P.M. A t t iw i l l . 1984. 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