Earth, Ecological, & Environmental Sciences ELEVATED ATMOSPHERIC NITRATE DEPOSITION IN NORTHERN HARDWOOD FORESTS: IMPACTS ON MICROBIAL MECHANISMS OF PLANT LITTER DECOMPOSITION Jared L. DeForest Earth, Ecological, & Environmental Sciences University of Toledo I welcome everybody and thank you all for attending….

Global rates of atmospheric nitrogen deposition 50.0 20.0 10.0 7.5 5.0 2.5 1.0 0.5 0.3 0.1 On a global scale, human activity has increased the atmospheric input of nitrogen to many ecosystems. The combustion of fossil fuels from industry and automobiles is the primary cause for the anthropogenic release of nitrogen into the atmosphere. A majority of the anthropogenic nitrogen is returned to the earth as nitrate near the point of origin. As you can see from this map, the most industrial regions of the world, Europe, China, United States, and India receive the greatest amounts of nitrogen deposition. kg N ha-1 Galloway & Cowling, (2002)

Total Nitrogen Deposition (2002)

Values in 1012 g; From Schlesinger (1997) Human activities have doubled the amount of available nitrogen Global Nitrogen Cycle 150 This increase in the fixation of nitrogen has altered the global n cycles. Human activities are estimated to now exceed that of the biological fixation of nitrogen by around 10 tera grams. This massive increase in the production of available nitrogen will likely alter nitrogen limited ecosystems. Values in 1012 g; From Schlesinger (1997)

Values in 1012 g; From Schlesinger (1997) Human activities have doubled the amount of available nitrogen Global Nitrogen Cycle 150 This increase in the fixation of nitrogen has altered the global carbon cycles. Human activities are estimated to now exceed that of the biological fixation of nitrogen by around 10 tera grams. This massive increase in the production of available nitrogen will likely alter nitrogen limited ecosystems. Values in 1012 g; From Schlesinger (1997)

The deposition of nitrogen can be in two forms: Nitrate (NO3-) or Ammonium (NH4+) Nitrate represents the majority of total nitrogen deposition in the Midwest Nitrate is rapidly assimilated by the microbial community and through the process of cell death, that nitrogen is released as ammonium Ammonium can represent 75% of extractable total inorganic nitrogen in soil

Human Nitrate Deposition The impact of anthropogenic nitrate deposition on temperate forests is a primary concern, because nitrogen often limits plant growth and therefore has the potential to alter ecosystem function.

Adapted from Schlesinger (1997) The doubling of available nitrogen can be a potent modifier of the carbon cycle Atmosphere 750 Gt C GPP 120 Gt C yr-1 Land Plants 560 Gt C 60 Gt C yr-1 Respiration 60 Gt C yr-1 Decomposition Nitrogen deposition may alter forest ecosystem function by alter the carbon cycles. This can happen in two primary ways, first by increasing plant growth and second by reducing decomposition. Nitrogen, as a plant fertilizer, could cause plants to grow faster and therefore these ecosystem would fix more carbon. However, nitrogen is also know change decomposition rates of plant litter, thus changing the cycling of carbon. The impact atmospheric nitrogen deposition on decomposition will be our focus. Soils 1500 Gt C Adapted from Schlesinger (1997)

Increases in nitrogen deposition can inhibit decomposition because high levels of soil nitrogen can suppress the activity of enzymes that degrade plant litter Lignin degrading enzymes are the most likely to be suppressed by increases in soil ammonium availability

Ligninolytic activity is often inhibited by ammonium (NH4+) Extracellular Ammonium Ligninolytic Activity Ammonium (mM) Ligninolytic Activity The ability of white-rot fungi to degrade lignin is induced by ammonium. Since the late 1970’s, many laboratory experiments have demonstrated that at high concentrations of ammonium, the ability of white-rot fungi to process lignin is limited, if at all. Once ammonium concentrations are reduce, these fungi start producing lignin-degrading enzymes. The production of lignolytic enzymes is independent on the availability of lignin. Culture Age (days) Adapted from Keyser et al., 1978

Basidiomycetes are the primary decomposers of lignin Degrading lignin is a specialized function giving lignin-degrading microorganism access to lignified carbohydrates. A relatively small population of soil bacteria, actinomycete, and fungi have the ability to depolymerize lignin by non-enzymatic and enzymatic means. White-rot fungi are recognized as the primary decomposers of lignin. White-rot fungi are basidiomycetes that can produce an array of enzymes with the ability to fully depolymerize lignin. Degrading lignin is a specialized function giving white-rot fungi access to lignified carbohydrates. White-rot fungi are considered the primary decomposers of lignin because they produce an array of enzymes that can fully degrade lignin.

At least 21 genera are considered white-rot fungi. White-rot fungi are a physiological, rather than a taxonomic, grouping of fungi. At least 21 genera are considered white-rot fungi. Evidence of White-Rot Decomposition

The decomposition of lignin is important because: Lignin is the second most abundant organic molecule Lignin protects plant tissue from decomposition Lignin

The Decomposition of Plant Litter Time  Remaining Mass Lignin Lignified Cellulose Non Lignified Cellulose Labile Compounds The Decomposition of Plant Litter This graph represents the rate of mass loss for four common components in plant litter. The Y-axis is the remaining mass and the X-axis is time. Non lignified carbohydrates are generally the most abundant organic compounds, followed by lignified carbohydrates lignin and soluble carbohydrates. The organic compounds contained within plant litter are decomposed at different rates by microorganisms. The most liable compounds in plant litter are soluble and non lignified carbohydrates, which can be metabolized fairly quickly. These compounds would include sugars, proteins, lipids and cellulose not associated with lignin. Lignified carbohydrates and lignin are more recalcitrant compounds and may take many years or decades to fully depolymerize Adapted from Berg (1986)

Phase regulated by lignin by nutrient level and readily available carbon Phase regulated by lignin decomposition rate Labile Compounds Remaining Mass Non Lignified Cellulose The decomposition of plant litter can be partitioned in two phases. The first phase to decomposition is regulated by nutrient level and readily available carbon. The mass loss of plant litter is typically rapid in this phase of decomposition. A wide array of microorganism are active precipitants at this stage of decomposition Once the liable compounds are utilized, the second stage is regulated by the decomposition of lignin. Lignin is a polyphenolic compound that gives plant cell walls strength and protection against microbial degradation. A relative small group of microorganism are able to decompose lignified plant compounds. Lignified Cellulose Lignin Time  Adapted from Berg (1986)

Mass Loss Time Ambient Nitrogen Elevated Nitrogen Phase regulated by nutrient level and readily available carbon Phase regulated by lignin decomposition rate Ambient Nitrogen Elevated Nitrogen Mass Loss Time Adapted from Fog (1988)

Human Nitrate Deposition Less Lignolytic More Microbial Enzyme Activity Microbial Nitrate Assimilation And Turnover More Available NH4+ Anthropogenic nitrate deposition may initiate a series of events that can eventually reduce the flow of carbon by these ecosystems. Mounting evidence indicates that decreases in plant liter decomposition due to chronic N deposition can be partially explained by the inhibitory effect excess N has on extracellular enzymes that mediate the decomposition of plant litter. Soil microbes, as the primary sink for atmospheric nitrate, rapidly assimilates nitrate and in the process of microbial turnover will release the nitrogen as ammonium. An increase in the amount of ammonium in the soil has the potential to prevent the production of phenol oxidase, which is sensitive to high levels of ammonium. This may diminish the overall capacity of white-rot fungi to degrade lignin. A reduction in lignin decomposition will reduce the decomposition of plant litter at the later stages of decomposition. This, in turn, is likely to reduce the flow of carbon through these ecosystems. Less Litter Decomposition Reduced Carbon Flow Less Lignin Decay

Hypothesis Chronic nitrate additions can suppress the lignin-degrading activity of soil microbial communities I reasoned that chronic nitrate additions can suppress the lignin degrading activity of white-rot fungi. Therefore, nitrate additions may potentially lower their abundance and diminish the overall capacity of microbial communities to decompose plant litter.

Nitrate amended soils will have: Predictions Nitrate amended soils will have: A microbial community composition with less fungi Lower activity of enzymes that degrade lignin and cellulose I predict that nitrate amended soils will have lower activity of enzymes that degrade lignin and cellulose (WHY CELLULOSE)?? In additions, I expect a microbial community composition that has less fungi. (EXPLAIN BEFORE)

Study Sites 7 9 12 12 (kg N ha-1 y-1) To test this hypothesis, I used four sugar maple dominated northern hardwood forests. This type of forest represents a wide geographic range of forested ecosystems in the northern great lake states.

A B C D PLOTS Ambient N Deposition Plus 30 kg N-NO3- ha-1 y-1 Ambient Nitrogen Deposition A B PLOTS C D Each site has 6 plots, three plots served as controls receiving ambient levels of N deposition, whereas the remaining three plots received ambient N deposition plus chronic atmospheric N deposition. From each of the four study sites, within each of the six plot, eight cores were extracted three times a year during spring, summer, and fall. From each core, the top organic portion was separated from the mineral soil to be processed independently. All subsequent analyzes were preformed on these composites litter and soil cores. The nitrate amended plots received 30 kilograms of nitrogen per ha-1 y-1 since 1994. The amount of nitrogen experienced by fertilized plots is similar to nitrogen inputs near industrial regions of northeast United States. Nitrate was used because it is the common form of nitrogen that is atmospherically deposited. The color scheme for ambient N deposition (burgundy) and elevated N deposition (orange) will be used throughout this presentation.

Cell membranes can be used to determine microbial community composition Lipid bilayer Cell membrane Microbial cell Phospholipid

Phospholipid Fatty Acids Unique to fungi Fatty Acids Tails Common to many soil microorganisms The cell membrane in soil microorganisms can be used to determine microbial composition and biomass. The length of fatty acid tails and position of double bonds on the tails can be unique to broad taxonomical groups. Since different microbial groups have different ways they construct cell membranes, with this process, we distinguish four broad microbial groups: bacteria, actinomycetes, fungal, protozoan. This analysis will allow us to determine if nitrate additions have altered soil microbial community composition. The length of fatty acid tails and position of double bonds on the tails can be unique to broad taxonomic groups

Enzyme Analysis Cellulose Lignin Plant Litter Compound b-glucosidase Peroxidase Extracellular Enzymes Cellobiohydrolase Phenol oxidase To determine the potential of the microbial community to degrade cellulose and lignin, I measured the activity of four enzymes. Cellobiohydrolase and beta-glucosidase have the ability to depolymerize cellulose. These cellulytic enzymes were determined by fluormetric analysis. Peroxidase and Phenol oxidase are the two more recognized enzymes involved in lignin depolymerization. These oxidative enzymes were determined by colorimetric analysis. I measured these four enzymes in both the mineral soil and in leaf litter.

Nitrate additions had no noticeable effect on microbial community composition % mol fraction I found that nitrate additions had no noticeable effect on microbial community composition. On this graph, the y-axis represents the percent mole fraction, showing the relative abundance of board microbial groups. The x-axis represents shorthand lipid notation for the four recognized microbial groups. We can see the relative abundance of microbial groups are not significantly influenced by the nitrate treatment. This result is a surprise because I expected to observe a relative reduction in fungal abundance. One possible explanation is that white-rot fungi represent only a small population of fungi in mineral soil. White-rot fungi typically are growing in localized areas like in decaying logs I expect that most fungal PLFAs extracted from mineral soil is derived from mycorrhizae.

Nitrate additions decreased microbial biomass Total PLFA (nmol PLFA mg-1 C) I found that the addition of nitrate has decreased total PLFA by 68%. On this graph, the y-axis is nmols of total PLFA per microgram of soil carbon. Because total PLFA is a indicator for living microbial biomass, this reduction in the amount of soil microorganisms suggests that excess N reduces the microbial community as a whole, not just one functional group like white-rot fungi. This is because microbial community displayed no large shift in composition.

Change in Enzyme Activity Nitrate addition suppressed activity of soil lignin & cellulose degrading enzymes -40% -30% -20% -10% 0% 10% Change in Enzyme Activity Cellobiohydrolase b -glucosidase Peroxidase Phenol Oxidase * * p < 0.05 Within the mineral soil, nitrate additions suppressed the activity of soil lignin and cellulose degrading enzymes. On this graph, the y-axis is the four enzymes measured and the x-axis is the percent change in activity by nitrate additions. Beta-glucosidase and Peroxidase were significantly suppressed by nitrate additions, whereas cellobiohydrolase and phenol oxidase display modest reductions in activity. This results supports my hypothesis that nitrate additions will diminish the overall capacity of microbial communities to decompose plant litter. While excess N has no known direct suppression on beta-glucosidase, this reduction in beta-glucosidase is likely caused by a decrease in the availability of cellulose because it is protected up in lignin. The stage of decomposition in the soil is likely when lignin controls decomposition.

Change in Enzyme Activity Nitrate addition suppressed activity of lignin degrading enzymes in litter -40% -30% -20% -10% 0% 10% Change in Enzyme Activity Cellobiohydrolase b -glucosidase Peroxidase Phenol Oxidase * * p < 0.05 However, in the litter layer I observed the reduction in lignin degrading enzymes, but not in cellulose degrading enzymes. Because the litter layer typically has higher concentrations of N than the soil and nitrate is directly applied to the litter layer, it is reasonable to have such sever reductions in lignolytic activity. The relative unchanged activity of cellulytic enzymes suggests the suppression of lignin-degrading enzyme are having little effect on cellulose decomposition. It is likely that in the litter nutrient and carbon availability control decomposition not lignin decomposition.

No Apparent Change Decrease Decrease Nitrate Additions Microbial Community Composition Lignolytic Activity Total PLFA (Microbial Biomass) The major findings for the first stage of my research was nitrate additions apparently did not change fungal community, but did significantly decrease total PLFA. In addition, lignolytic activity was suppressed in both the litter and soil by the nitrate additions. No Apparent Change Decrease Decrease

Decreases in b-glucosidase activity can help explain lower microbial biomass in nitrate amended soils. Reductions in b-glucosidase activity can diminish the physiological capacity of the microbial community to metabolize cellulose. This reduction could reduce the energy enzymatically derived from cellulose degradation.

Conclusions Anthropogenic nitrate deposition may diminish the physiological capacity of soil microbial communities to degrade plant litter. In conclusion, my results suggest that anthropogenic nitrate deposition may diminish the physiological capacity of soil microbial communities to degrade plant litter.

Does a suppression of lignin & cellulose degrading enzymes indicate a reduction in the flow of carbon from these compounds? Does a suppression of the enzymes responsible for degrading lignin and cellulose result in the flow of carbon from these common plant litter compounds?

Nitrate additions will inhibit the ability of soil microorganisms Hypothesis Nitrate additions will inhibit the ability of soil microorganisms to metabolize and assimilate the products of lignin and cellulose degradation Therefore, I investigated the hypothesis that chronic N additions will inhibit the ability of soil microorganisms to metabolize and assimilate the products of lignin degradation, and thus, will reduce the flow of C through the heterotrophic soil food web.

13C Vanillin Lignin Microbial Assimilation CHO OCH3 OH H To understand changes in the flow of C due to chronic nitrate addition, we used a model compounds enriched with the 13C, which is a stable isotope of carbon that is easily traced. Vanillin was used as the model compound for lignin because it is a simple phenolic that is one, of many, final products of lignin degradation that is metabolized by soil microorganisms. Microbial Assimilation

Microbial Assimilation Cellulose Cellobiose was used as the model compound for cellulose because it is a simple compound that is hydrolyzed by beta-glucosidase and the final products of cellulose degradation, readily available for metabolized by soil microorganisms. By using labeling cellobiose and vanillin composed of the stable isotope carbon 13, we are able to trace the enriched C as it travels through the soil food web in order to determine changes in the metabolism and use of vanillin. Microbial Assimilation 13C Cellobiose

13C Sequential Extractions: Soil was incubated for 48 hours and 13C was traced into respiration, dissolved organic carbon (DOC), microbial carbon, and soil carbon. After labeling with 13C vanillin or cellobiose, the soil was incubated for 48 hours and 13-C was traced into respiration, dissolved organic carbon, microbial carbon, and soil organic carbon. I used an isotope ratio mass spectrometer to trace 13-C into these C pools.

13C PLFA Analysis: Traced the flow of labeled 13C vanillin and cellobiose into cell membranes. Phospholipids fatty acid analysis was used to determine which PLFAs were enriched with C from the 13C labeled vanillin or cellobiose. Using a isotope ratio mass spectrometer allowed us to trace 13C into specific PLFAs. Therefore, I was able to assess which broad members of the microbial community incorporated the C from the labeled compound into cell membranes.

13C PLFA Analysis CHO OCH3 H OH Microbial Extraction & Membrane Separation 13C 13C 13C 13C 13C 13C 13C 13C This diagram illustrates the flow of 13C labeled vanillin into microbial membrane and extracted and separated by PLFA analysis. The 13C is finally detected on the isotope ratio mass spectrometer. 13C 13C Analysis

N additions increased the incorporation of vanillin into PLFAs * Vanillin * * Cellobiose I found that the incorporation of vanillin into PLFAs increased significantly in N amended soils. This result suggests that despite a reduction in lignolytic enzyme activity, more vanillin is used by the microbial community. However, N additions apparently did not effect the incorporation of cellobiose into PLFA.

N additions did not alter the flow of 13C vanillin into carbon pools This graph shows the % recovery of 13C when traced into four carbon pools; microbial respiration, DOC, microbial biomass, and SOC. The percent recovery of 13C vanillin into soil organic carbon, Microbial biomass and respiration, along with dissolved organic carbon was constant among the treatment. The majority of the 13C vanillin was recovered in soil organic carbon, probably incorporated right into soil humus. The amount of 13C vanillin respired and found in microbial biomass was similar. Therefore, it appears that N additions had no noticeable effect on the flow of vanillin into carbon pools.

N additions did not alter the flow of 13C cellobiose into carbon pools This graph is similar to the previous graph, but this is 13C cellobiose. Like vanillin, N addition did is alter the flow of 13C cellobiose into these carbon pools. Unlike vanillin, the majority of cellobiose was incorporated into microbial biomass, followed by soil organic carbon and dissolved organic carbon. It is clear from these results that N additions have little influence in the metabolism and use of vanillin or cellobiose.

N additions increased soil organic carbon Soil Organic Carbon (mg C g-1) However, nitrate additions significantly increased soil organic carbon. I reason that the increase in SOC is caused by, in part, the reduction in phenol oxidase activity preventing the decomposition of lignin and lignified carbohydrates.

UNCHANGED INCREASED Vanillin or Soil Organic Cellobiose into Carbon Excess nitrogen likely inhibits lignocellulose degradation more than vanillin or cellobiose degradation Chronic nitrate additions UNCHANGED Vanillin or Cellobiose into Carbon Pools INCREASED Soil Organic Carbon It is apparent nitrate additions have little effect on the flow of vanillin or cellobiose through the soil foodweb, while it is obvious that carbon is accumulating in N amended soils, thus indicating an increase in soil organic matter formation. Therefore, excess soil N likely inhibits lignocelluloses degradation more then vanillin or cellobiose degradation.

Conclusions Nitrate additions have apparently stemmed the flow of carbon through the soil food web evident by increasing soil organic matter formation through a reduction in lignolytic activity. In conclusion, nitrate additions have apparently stemmed the flow of C through the soil food web by increasing soil organic formation through a reduction in phenol oxidase activity.

Implications Atmospheric CO2 Pools Slower Decomposition Northern Hardwood Forests Atmospheric CO2 Pools The implications of my research suggests that increases in nitrogen deposition have the potential to significantly decrease the decomposition of lignin and lignified carbohydrates contained with plant cell walls. A decrease in plant litter decomposition would slow the cycling of carbon by sugar maple-dominated northern hardwood forests. The capacity for these ecosystems to accumulate carbon could increase through soil organic matter formation. Therefore, anthropogenic N deposition, by slowing the flow of C through the microbial foodweb can be a potent modifier of ecosystem-level patterns of C cycling. Slower Decomposition

Human Nitrogen Deposition If anthropogenic nitrogen deposition can limit the decomposition of lignin and lignified carbohydrates, then how will this alter broad global rates of decomposition?

Global Implication The same mechanism that decreases lignin decomposition could be used to understand the impact nitrogen deposition may have on broad global patterns of decomposition The same mechanism that decreases lignin decomposition could be used to predict the impact anthropogenic N deposition may have on broad global patters of decomposition

Global Controls of Decomposition Environmental Conditions Litter Biochemistry There are two major global controls of decomposition. The most pervasive control of decomposition is environmental conditions followed by the chemical composition of the plant litter. Plant litter decay

Actual Evapotranspiration Environmental Conditions Actual Evapotranspiration (AET) Faster Decomposition Slower Decomposition Environmental conditions can be regarded as the actual evapotranspiration or AET for short which is a function of temperature and precipitation. Moist, warm environments like a tropical rainforest have high AET and faster decomposition then cold or dry environments like a boreal forest or a aired desert. 350-550 mm High > 1000 mm Low < 300 mm

Years Required to Decompose Environmental Conditions Years Required to Decompose 95% of Leaf Litter ~0.5 years ~14 years ~4 years For example, the years it typically takes to decompose 95% of leaf litter is around a half a year in wet tropical environments, four years in temperate deciduous forests and 14 years in boreal forests. Temperate Deciduous Wet Tropical Boreal

Lignin and Nitrogen Concentrations Litter Biochemistry Lignin and Nitrogen Concentrations Faster Decomposition Slower Decomposition Lignin and nitrogen concentration are considered a good broad chemical predictor of decomposition. Litter with low lignin and high nitrogen typically will decompose faster then litter with high lignin or low nitrogen. Lower Lignin Higher Nitrogen Higher Lignin Lower Nitrogen

Human Nitrogen Deposition Increase Litter Nitrogen Concentrations Decrease the Decomposition of Lignin Human nitrate deposition can be a potent modifier of global patterns of decomposition because increases in nitrogen availability can change the chemical predictors of plant litter. Specifically, human nitrate deposition has been shown to increase litter nitrogen concentrations. This in turn may increase decomposition. However, as discussed in the first part of my presentation, nitrate deposition can reduce the ability of lignin-degrading microorganism from degrading lignin. Increase Decomposition Rates Decrease Decomposition Rates

Mass Loss Time Ambient Nitrogen Elevated Nitrogen Phase regulated by nutrient level and readily available carbon Phase regulated by lignin decomposition rate Ambient Nitrogen Elevated Nitrogen Mass Loss Time Adapted from Fog (1988)

Nitrogen deposition impact on decomposition may depend on lignin concentrations Increased Decay Decreased Decay Therefore, nitrogen deposition impact on decomposition may depend on lignin concentrations in plant litter. Litter with relatively low lignin concentrations is likely to increase the decomposition of litter because of the increases in N availability. Lignin in this litter is minor and any suppression of lignin decomposition would have minimal suppression of overall decomposition. Litter with relatively moderate levels of lignin might experience the moderate impact on N deposition. The increase in N concentrations will promote decomposition whereas the later stages of decomposition will be suppressed. Overall, long term decomposition will be suppressed. I reason that N deposition will have the greatest impact on litter with relatively high levels of lignin. “Low” Lignin “High” Lignin

Lignin Control of Decay is Greater at Higher AET As slope decreases, higher lignin concentrations require more energy and moisture to cause decay. Slope = -1.50 Annual Decomposition Rate (%) Slope = -0.75 It is interesting that the control of lignin on plant decomposition is greater at higher AET (CLICK) As we can see, at a modest 15% lignin concentration, environments at 1000 mm AET the annual decomposition rate is reduced by 23%. However, at 500 mm AET, the annual reduction of decomposition is 10%. The reason for this is at higher AET there is less constraints on microbial decomposition because conditions are near optimal and lignin’s control is more pervasive. Decomposition in environments with relatively low AET have more constraints by either low temperature or low moisture. Slope = -0.40 Lignin Concentration (%) Adapted from Meentemeyer (1978)

Anthropogenic nitrogen deposition may have a larger impact on decomposition in wet-tropical environments Less Impact This means that regions that experience high levels of anthropogenic deposition in northern climates may have relatively less impact on decomposition then in tropical areas. More Impact

Summary Nitrogen deposition has the potential to diminish the physiological capacity of lignin-degrading microorganisms to depolymerize lignin. Reductions in lignocellulose-degrading enzymes and microbial biomass suggests a reduction in energy available for microbial metabolism Nitrogen deposition may have a greater impact on decomposition in wet tropical regions than arid or cold regions In summary, nitrogen deposition has the potential to diminish the physiological capacity of lignin-degrading microorganisms to depolymerize lignin. Also, N deposition appears to have little impact on the ability of soil microorganisms to metabolize cellobiose or vanillin. Lastly, anthropogenic nitrogen deposition is likely to have greater impact on decomposition in wet tropical regions than arid or cold regions