Temperature effects on decomposition rates of soil organic ...This experiment measured the effect of...

Temperature effects on decomposition rates of soil

organic matter with differing proportions of labile and

recalcitrant compounds

By Ellen Yeatman

Washington and Lee University

Marine Biological Laboratory, 7 MBL Street, Woods Hole, MA 02543

Advised by Jim Tang and Chris Neill

Collaborators:

Dr. Serita Frey, University of New Hampshire

Shirlie Yang, Grinnell University

Abstract

There is much disagreement regarding the effects of climate change on global soil carbon

stocks. Soil carbon stocks are relatively stable, but fluxes are sensitive to environmental

conditions. This experiment measured the effect of temperature on soil with different

proportions of labile and recalcitrant organic matter by comparing respiration rates of different

aged warmed soils and different soil depths with their controls incubating at 5˚C, 15˚C, and

25˚C. Carbon quality and polyphenol (PPO) enzyme activity, which is a primary enzyme that

breaks down lignin, are measured in order to explain differences in soil sensitivity to

temperature. Most soil carbon cycle models predict that climate warming will stimulate

microbial decomposition of labile soil carbon, producing appositive feedback to rising global

temperatures. Responses of soil carbon to warming by 5˚C conducted at the Harvard Forest 20-

year long term warming experimental plots document that the carbon dioxide loss from soil

declines over time then picks up again after approximately15 years of warming. The long-term

response is found to be a result of changes in microbial physiological properties with increasing

temperature. Warming affects both the soil microbial community carbon use and the carbon

quality of the soil organic matter.

Key Words: global warming, soil microbial respiration, CO2 flux, carbon quality, lignin, labile

carbon, recalcitrant carbon.

Introduction

Soil plays such a large role in our global carbon cycle. Soil is this natural carbon sink

because carbon is sequestered as biomass, such as plant litter, which accumulates as organic

matter. Also, soil can be a carbon source to the atmosphere from the release of CO2 due to

microbial decomposition , which coincides with respiration rates. The ideal carbon compounds

for decomposition is labile soil organic matter, which is an easily decomposable and

energetically favorable energy source. Labile soil organic matter is sugar, starch, and

carbohydrates. An alternative energy source is recalcitrant carbon, which is harder to decompose

soil organic matter such as lignin – a complex compound found in woody biomass. Depending

on the soil organic matter available, the soil microbial community will produce certain sets of

enzymes specifically for degradation of the available energy source (Nabu, 2009). With

increasing global temperatures, it is important to understand how soil properties will change and

thus their role as a carbon sink. This experiment uses forest soils since the world’s forests

account for more than half of the organic carbon stored on land (Melillo et al. 2011). It’s not

clear how this large pool will change in response to temperature.

Numerous warming studies have shown that soil CO2 efflux is initially stimulated by

warming with the effect diminishing overtime (Frey et al. 2008). This attenuation response is

believed to be a result of declines in microbial biomass, such as a decline in the fraction of

assimilated carbon that is allocated to growth, termed carbon-use efficiency, and degradative

enzymes (Alsion et al. 2010). There is little known about how the microbial physiological

properties change over long-term warming and if it is a result of a change in carbon quality or

not. A consensus has yet to emerge on the temperature sensitivity of soil carbon decomposition

mainly over a long-term scale. Unraveling the complex mechanism of soil CO2 flux in response

to temperature is particularly difficult because of the diverse array of soil organic carbon

compounds that exhibit a wide range of kinetic properties (Davidson et al. 2006). More labile

carbon compounds turn over quickly and more recalcitrant carbon compounds may be relatively

stable for years to centuries.

A long-term soil warming experiment at the Harvard Forest research center may show

signs of changes in carbon quality with increased temperature. Initially there is a strong increase

in microbial activity leading to a loss of carbon due to respiration. Here there was a lot of labile

carbon available as quick sources of energy. After 10 years, there is no longer a significant

release of CO2 from the soil. This trend is believed to be due to the depletion of the labile carbon

pool. When the labile carbon pool is depleted, recalcitrant carbon compounds is the primary

energy source. The sensitivity of this more stable soil carbon pool to increased temperature is

much understudied. However, insight by a study by Alison et al. (2010) on the recent trend of an

increased release of CO2 from the Harvard Forest 20 year soil warming plots says that the soil

microbial community adapts to changes in soil carbon quality leading to an upward adjustment

of the efficiency of carbon use, counteracting the decline in microbial biomass and accelerating

soil-carbon loss.

Using soil samples from the 20-year long-term warming experiment and 5-year long-term

warming experiment at the Harvard Forest, this experiment conducts carbon quality analysis,

enzyme activity measurements, and incubates soil subsamples at 5˚C, 15˚C, and 25˚C in order to

analyze changes of carbon use and availability from short-term and long-term warming. The

enzyme measured is polyphenol oxidase (PPO) in order to conclude if the microbial community

has adapted ways to breakdown the recalcitrant carbon pools. PPO oxidizes and breaks down

lignin-like carbon compounds. The carbon quality analysis looks at the proportion of labile

carbon pools versus the recalcitrant carbon pool. Soil-carbon response to climate warming is

strongly dependent on the microbial physiology and the quality of carbon available.

Methods

Field Site

The Harvard Forest is located in Central Massachusetts near the town of Petersham. The

Harvard Forest is an ecological research area of 3,000 acres owned and managed by Harvard

University since 1907. The Harvard Forest is a strong representation of the forests of the New

England Upland Region. There is a research campus on site, which is very similar to the MBL

laboratory. I will be using samples collected from the Prospect Hill 20-year warming site and the

nearby 5-year warming site, which are located near the main campus. Both experiments, led by

Dr. Jerry Melillo and Dr. Serita Frey, have buried self-regulating electrical cables that were

buried around 10cm underground that keep randomly selected plots warmed at 5°C above

ambient temperature. Samples were taken from the disturbed controls plots, which are randomly

selected plots with deactivated electrical cabling.

The Samples

During the week of November 1, soil samples from six designated depths down to 50 cm

depth were collected, sieved, and homogenized at University of New Hampshire by Serita Frey,

the head scientist of the 20-year and 5-year Harvard Forest soil warming projects. Dr. Frey sent

soil samples from five depths (0-10 cm, 10-20 cm, 20-30 cm, 30-40 cm and 40-50 cm) from the

20 yr warming experiment heated and control plots and soil samples from 0-10cm from the 5-

year warming experiment heated and control plots. There are six replicate plots for the 0-10 cm

soil samples and three replicate plots used for the deeper depths of the 20-year warming

experiment heated and control plots. I only received two replicate soil samples from the 20-year

warming experiment control plots at 30-40cm and 40-50cm. These samples allowed me to have a

very dynamic sample pool in terms of variation in carbon quality.

In the Laboratory

The day the soil samples were received, November 12, each 100 g soil sample had 25 g

of soil aliquoted into three subsamples of 25 g. The three 25g subsamples were incubated at 5˚C,

15˚C, and 25˚C. This is noted as Day 0 of incubation. The excess soil per sample were incubated

at 5˚C and used for soil characterization – dry weight, enzyme activity measurements, CHN

analysis, and carbon quality partitioning. The lids of the cup were placed loosely on. See Figure

2 in Appendix for a diagram of the sample set up. A bucket of water is kept in the incubators to

keep the air saturated. Also, soil moisture is monitored by weighing the samples every two to

four days. If mass is lost, then the appropriate amount of water is added to bring the soil weight

back up to 25 grams.

Enyzme assays were conducted using 2 grams of the wet extra soil and following the

protocol found in Appendix Sample C. A 5 mM L-3,4-dihydroxyphenylalanine (L-DOPA)

solution in acetate buffer with a pH of 5 was used as the substrate solution for the PPO assay.

100 µL of L-DOPA was added to every 1 mL of soil sample slurry. After 5 minutes of

incubation at room temperature using a vortex spinner and 4 minutes of centrifugation, the

absorbance of time point one is measured at 460 nm using a spectrophotometer because the

breakdown of L-DOPA releases a fluorescent element at this wavelength. The assay is returned

to incubation for around 90 minutes then the final absorbance is measured. From the control

plots, a background control enzyme assay was measured. The background control soils were

autoclaved for 25 minutes to kill any PPO enzymes that may have been present. The slope

between time point zero and time point one of the control is subtracted from the slope of the soil

samples. The activity is measured as absorbance over time per gram of soil sample.

Approximately 5 grams of soil per treatment plot and depth from the extra soil

subsamples are oven dried for 48 hours at 60˚C. The final dry weight is recorded. The dry soil is

stored in scintillation vials with their caps loosely placed on and stored in the desiccator. This

dry soil is used for CHN analysis carbon quality analysis. A CHN analyzer combusts the soil

(20-30mg) rolled up in tin foil and measures the percent carbon, hydrogen, and nitrogen based on

weight (Foreman, 2010). Approximately 0.5 g of the dry soil samples was used for carbon

quality analysis to measure the amount of labile I, labile II, and recalcitrant carbon pools. I

followed the carbon fractionation protocol by Jim Tang et. al (see appendix Sample D). A DOC

analyzer is used to measure the labile pools and the CHN analyzer is used to measure the

recalcitrant carbon pool.

Respiration measurements were made using a LICOR, an infrared gas analyzer, on Day

1, 8, 18, and 26. The cups were placed in the respirometer and the respiration was autologged for

approximately 5 to 10 minutes each or until the respiration numbers reached a steady state. The

LICOR was set up to record the CO2 flux every 5 seconds and programmed to calculate the

respiration using this equation:

Resp (ug CO2/g sample/min) =

(CO2ref – CO2sample)*(1000-H20ref)/(1000/H20sample)*-2640.588*Flow*(10^-7/Sample

Mass)

(Equation 1)

The files can be opened in excel. In excel, the mass of the dry weight of the soil sample must be

entered. The respiration rate of the sample is the average of the respiration rates at steady state.

Analysis

The second story of respiration is how time played a role in change of soil activity over

the short incubation period at the various temperatures. This is analyzed using the Q10 value,

which is a simple indicator commonly used to quantify the temperature sensitivity. Q10 equals

the increase in respiration rate when temperature increases by 10˚C. For each treatment plot, the

replicates at each temperature are plotted on a graph of temperature versus CO2 flux. An

exponential trendline is plotted and the equation

(Equation 2)

is used to calculated the Q10 value.

The values of the labile and recalcitrant carbon pools must be converted to g C/g sample.

The labile carbon pools that had to be diluted 10 to 1 before being measured in the DOC

machine, the measured values were multiplied by 10. The values must be corrected from the

standard curve. The standard curve was made using potassium hydrogen phthalate (KHP) at

1,5,10,20,50, and 100 ppm. The DOC values are corrected using Equation 3.

Standard correction value (mg C/L) = (Measured Value – Standard Curve Y-intercept)/Standard

Curve Slope

(Equation 3)

Then the values are converted to g C/g sample by multiplying by the liters of liquid of the labile

carbon pools (0.02 L for Labile I carbon pool and 0.27 L for Labile II carbon pool), dividing by

1000, and multiplying by 2.

Results

Soil from Four Treatment Plots at 0-10cm

The average respiration for the two warming plots at 0-10cm depth and their two

disturbance control plots at 0-10cm (Figures 1-4) increases at all three incubation temperatures

with each round of respiration measurements (Day 8, 18, and 26). The difference between 5˚C

and 15˚C average respiration rates is more pronounced than between 15˚C and 25˚C, at least for

the respiration measurements made on day 8 and day 26, for all treatment plots except for the 20

year heated plot (Figure 4). Day 1 respiration measurements are not shown because they were

measured using a different version of the LICOR, the 6200. The numbers were an order of

magnitude lower than Day 8. The control plots seem to have a little bit higher respiration rate

overall than the heated plots. The control plot average respiration rates were highest at15˚C

(Figures 1 and 3). The average respiration rates for the control plots on Day 8 did not have a

clear trend as there is no significant difference of soil incubating at 15˚C and 25˚C incubations.

The average respiration rates of the five year heated plot have a larger increase in the

average respiration rate from 5˚C to 15˚C compared to the 20 year heated plot throughout the

incubation period and this difference increases as the incubation period goes on (Figures 2).The

average respiration rates of the five year heated plot do not decrease from 15˚C to 25˚C as the

five year control plot respiration rates do (Figure 2 and 1). There is no significant difference

between the 15˚C and 25˚C respiration rates (Figure 2). The 20 year heated plot increases in

average respiration rate with temperature and with time (Figure 4). The average respiration

values at 25˚C of the 20 year heated plot are higher than the other treatment plots throughout the

incubation period.

The temperature sensitivity varied over the 26 incubation period for all four treatment

plots at 0-10cm (Figure 5). The five year warming plots’ Q10 values were not significantly

different than the five year control plots’ Q10 values. Both the five year heated and control plots

showed a decreasing trend in temperature sensitivity, but not as a significant decreasing trend as

the 20 year heated plot. The 20 year heated plots showed a decreasing trend in temperature

sensitivity over time where the 20 year control plots did not significantly change over the

incubation period. The 20 year warming experiment heated plots had significantly higher Q10

values than the 20 year control plots.(Figure 5)

In Figure 6, the results of the enzyme assay for polyphenol oxidase activity show that the

20 year warming experiment heated plot has the highest activity by 0.00273 nm/min/2 g soil

sample. The next highest enzyme activity was found in the five year warming experiment heated

plot. The control plots had similar levels of enzyme activity. (Figure 6) The total carbon of each

four treatment plot shows the opposite trend with the heated plots having less average total

carbon than their control counterpart plots (Figure 7). The 20 year warming experiment heated

plot had the least amount of total carbon overall (Figure 7). Also, the difference between the 20

year warming experiment heated and control plots is more significant than the difference

between the five year heated and control plots (Figure 7). The 20 year warming experiment

heated plots had the least amount of recalcitrant (Figure 8). The heated plots both had less

average recalcitrant carbon per gram of soil than their control plot counterparts (Figure 8). There

is not a significant difference in the average recalcitrant carbon pools between the 5 year control

plots, 5 year heated plots, and 20 year control plots (Figure 8). There is no significant difference

between the labile I and labile II carbon pools between the four treatment plots except for the 20

year heated plot has less labile I carbon than its control counterpart plots (Figures 10 and 11).

The percentage of the carbon pools versus the total of the three carbon pools is shown in Table 1

and there is no distinct trend. There is more of labile II carbon across the board, but the 20 year

heated plot shows to have the highest amount of labile II carbon percentage (Table 1). Also, the

5 year warming experiment heated plot has the highest average percent of recalcitrant carbon and

the 20 year warming experiment heated plots have the lowest average percent of recalcitrant

carbon (Table 1).

Soil from 20 year warming experiment heated and control plots at depth

The respiration rates of the 20 year warming experiment heated and control plots at depth

are very variable. There is an overall increase in respiration rates with incubation time in each

incubation temperature (Figures 13 to 15) identical to the trend of the four treatment plots at 0-

10cm depth. At 20cm to 50cm depths, the control plots did generally have lower respiration rates

(Figures 13 to 15).

There were clear trends in the enzyme activity at depth with an overall decrease in

enzyme activity with depth. The 20 year warming experiment heated plots’ microbial

communities have developed significantly higher levels of lignase than the control counterpart

plots to 30 cm depth over their long-term warming lifetime (Figure 16). At 30-40cm depth, the

heated plot did have a higher measured value of lignase activity than the control counterpart plot,

but it is not a significant difference (Figure 16). The most significant difference between the 20

year heated and control plots is at 20-30cm depth.

The total carbon at the five depths of the 20 year warming experiment heated and control

plots decreases with depth (Figure 17). At depths 0 to 30cm, the control plots have more total

carbon than the heated counterpart plots and, with depth, the average total carbon is less than half

of the previous depth average total carbon (Figure 17). The soil samples from 10-20cm depth

from the heated plots have half the total carbon compared to the soil from 0-10cm depth from the

heated plots (Figure 17). The average recalcitrant carbon pools of the20 year warming heated

and control plots steadily decreases with depth with a large drop in recalcitrant levels between

10-20cm soil and 20-30cm soil (Figure 18). From 0-20cm depth, the heated plots have less

recalcitrant carbon than the control plots (Figure 18). At 20-30cm depth, the heated plots have a

slightly higher average recalcitrant carbon pool (Figure 18).

The average labile I carbon pool mass of the 20 year heated plots decreases steadily from

0-10cm depth to 20-30cm and is significantly less than the labile I carbon pool mass at 30cm to

50cm depths (Figure 19). The most significant difference of the average labile I carbon pool

between the heated and control plots is at 20-30cm depth in which the labile I carbon pool of the

control plot is significantly higher. Then this carbon pool of the control plots decreases with

depth. (Figure 19) The average labile II carbon pool mass of the 20 year warming experiment

heated plot is significantly lower at 0cm to 20cm soil layers, but then jumps up from 0.005 g C/g

sample to 0.025 g C/g sample at 20-30cm depth. The heated plots have less labile II carbon pool

than the control plots at 30-50cm depth. The average labile II carbon pool mass at 0-20cm depth

is significantly less than the subsequent depths measured. (Figure 19)

Discussion

The 20 year heated plots are the most temperature sensitive and have the most

pronounced and steady increase in average respiration rates with temperature. This may be

because the 20 year heated soils are more quickly adapting to warmer temperatures so activity is

not hindered by the higher temperature as it clearly is in 20 year warming experiment control

plots and the 5 year warming experiment control plots. The five year warming experiment

heated plots’ soil is not as adapted to warmer temperature as the 20 year warming experiment

heated plots’ soil as represented by the slight increase, but not significant, in average respiration

rates on Day 8 and Day 18 and there is no real change in the average respiration rate between

15˚C and 25˚C soil incubations on Day 26.

Since each sample took a different amount of time for the LICOR to read a steady rate in

the CO2 flux, it was difficult to find consistency in calculating the average respiration value from

the raw data for each sample. If the experiment could be redone, I would have more soil per cup

and I would begin recording the respiration as soon as the cup is enclosed in the respirometer and

AutoLog for a longer period of time (around 15 minutes instead of 5 to 8 minutes). Time ended

up being a huge limitation for this project. Also, I would use the LICOR 6400 from the

beginning as it more accurately measures small CO2 fluxes. Another example of the time

limitation being an obstacle is that I was unable to measure enzyme activity and CHN analysis at

the end of the incubation period. A comparison of the initial total carbon to the final total carbon

would be useful in explaining the respiration trends. The increase in overall average respiration

rates over the incubation period is hard to explain without a final CHN analysis or final enzyme

activity measurements.

The temperature sensitivity of the 20 year warming experiment heated plot is defended

by literature, which states that more recalcitrant soil is more temperature sensitive (Davidson et

al. 2006). The decrease in sensitivity over time with respiration measurements showing

increasing activity could lead to the conclusion that over 26 days the 20 yr warmed soil was

breaking down recalcitrant soil, but a final total carbon measurement of the recalcitrant pool was

not measured to prove this theory due to time constraints.

The five year control plots had unusually high temperature sensitivity on day 1, which

may be due to differences in how the LICOR 6200 measured respiration on day 1 versus the

LICOR 6400 measurements made on days 8, 18, and 26. It is surprising that the five year

warming experiment heated plots did not stay consistently higher than the five year warming

experiment control plots as the twenty year warming experiment heated plot stayed so much

higher than its control counterpart. Again this may be due to the way respiration rates were

calculated. The average respiration rates trend for the 5 year warming experiment control plots

where there is a sharp decrease in average respiration rate between 15˚C and 25˚C soil

incubations is not explained by the temperature sensitivity trend where the temperature

sensitivity is lowest at 25˚C. The decrease in average respiration rates at 25˚C for the control

plots must not be a temperature sensitivity issue, but a total carbon issue. The control plots’ soil

may burn through their labile carbon very fast at the 25˚C incubation so activity is hindered from

the beginning practically. A final total carbon measurement was not taken to be able to prove this

theory. The control plots’ have not developed PPO enzymes as the 20 years warming experiment

heated plots and the 5 year experiment heated plots, which keep the soil activity, as represented

by respiration, up at the high 25˚C temperature. From the differences seen in enzyme activity

one can conclude that there is a change in the microbial community over both five year and 20

year warming experiment’s heated plots, which explains why these soil communities react

different to the short-term temperature changes of their environment in this experiment.

The total carbon of soil samples that were not incubated show that the heated plots

already have less average total carbon than their control counterparts due to the long-term

warming having stimulated much more soil activity relative to a soil community in their normal

environment. The overall lower amount of carbon coincides with the overall slightly lower

respiration rates seen in the heated plots compared to the control plots. The total carbon analysis

makes it obvious that long-term heading leads to a more rapid depletion of carbon stored in soil.

The total carbon as calculated by the CHN analyzer did match up perfectly to the total carbon as

calculated by adding the labile and recalcitrant pools. I should have done both of these analyses

as soon as I received the soil, when the soil is as close as possible to its natural state, and at the

very end after incubation. The total carbon numbers did show that the long-term heated plots

have had an overall depletion of carbon from the soil.

The analysis of labile I, labile II, and recalcitrant carbon pools helps tell the story of what

mechanism is being used by the soil microbial community to be more active under the long-term

higher temperature. Unfortunately, there is not a significant difference in the labile carbon pools

between the four treatment plots, but I would still like to point out the interesting trends. The 20

year warming experiment heated plot had the least amount of labile I carbon, but the most

amount of the labile II carbon pool. This coincides with a theory proposed about changes in the

carbon pools of the 20 year warming experiment that long-term warming stimulates an overall

increase in nitrification due to warming-enhanced decay of soil organic matter and warming-

enhanced plant growth (Melillo et al. 2011). As carbon levels are proportional to nitrogen

concentrations, maybe the high amount of labile II carbon of the 20 year warming plots are

sustained or reproduced over long-term warming due to this increased nitrification. A more

precise method needs to be developed to look at labile carbon pools so significant differences

can be found as my experiment hints at that are possible. However, the carbon quality analysis

did prove useful in providing further evidence along with the enzyme assay that the 20 year

heating plot has developed a different microbial community in response to warming because

there is significantly less recalcitrant carbon in the 20 year heated plots than the 20 year control

plots. The heated plots have adapted ability via lignase to breakdown this recalcitrant carbon

compounds as an energy source.

For the respiration measurements of the 20 year warming experiment soil from depths

past 10cm, there is no clear trend. It is very surprising that the heated plots soil at 10-20cm depth

did not follow the same trend as the 0-10cm soil from the heated plot as the electrical wires that

warm the soil are located at 10cm, which is right in between these soil layers. The heated plots

could be seen to be more adaptive to the short change in temperature as the heated plots at 20 to

50cm depth have generally higher average respiration rates than the control counterpart plots.

This could be reflective of a change in the microbial community at depth as well as the changes

that are evident at 0-10cm soil layer.

Soils at depth are expected to have less labile and more recalcitrant carbon, thus one

would expect the soils to increase in temperature sensitivity with depth, but the results did not

reflect such a pattern. The fact that respiration rates increased over the incubation time period at

all depths is very unexpected as one would think the since the soil at depths have much less

carbon compared to the 0 to 20cm depths that respiration rates would slow with time as there is

not enough carbon to provide enough energy. Also, the very low enzyme activity at 20 to 50cm

depth could insinuate that there is an overall small microbial community population at these

depths so one would expect for the respiration rates to decrease over time.

There are signs of lignase being adapted by the soil microbial communities at depth over

time as visible in the enzyme activity data and the average recalcitrant carbon pool

measurements. The high amounts of lignase activity at 0-20cm depth has worked through a lot of

the recalcitrant soil already as there is a lot less recalcitrant soil in the heated plots than the

control plots at these depths. Now, it looks as if the soil at 20-30cm depth has recently adapted

the lignase enzyme as there is the largest difference in enzyme activity between the heated and

control plots at this depth, but the level of the average recalcitrant carbon pool is the same as the

average recalcitrant pool as the control plot soil at that depth. It would be interesting to measure

the recalcitrant carbon pool for the next few years and watch for a decrease in the amount of

recalcitrant carbon in the heated plot relative to the control plot at depth, especially this depth –

20 to 30cm. The labile carbon pools are not as telling. The labile pools are significantly less in

the 20 year heated plots at 0-20cm than the deeper soil layers, but this also applies to the control

plots so not much can be deduced from these results. Less of the labile II carbon pool in the 20

year heated plots would hint at possible biological adaptations by the microbial community to be

able breakdown the harder to decompose carbon relative to the labile I carbon pool.

Conclusions

Both short-term and long-term warming of soil cause a change in the carbon composition

of the soil due to changes in the microbial community. There are clear sign of microbial

adaptation of lignase with 20 year warming experiment heated plots and the initial development

of lignase in the five year warming experiment heated plots. The fact that more recalcitrant soil

can be confirmed to be more temperature sensitive has major implications of the consequences of

global warming on soil carbon stocks. As the global temperature continues to rise, the soil will

become more temperature sensitive. Adaptations to recalcitrant carbon as a primary energy

source will lead to the further release of CO2 from the soil with long-term warming, which

stimulates a positive feedback to global warming with the release of such a strong greenhouse

gas. The role of terrestrial soil as a major carbon sink and the overall balance of the global

carbon cycle will be hampered by these changes in global temperature.

The magnitude of acclimation of soil respiration in response to warming and its

mechanism is still unclear. The mechanisms are very complex. This experiment did not consider

other biogeochemical cycles that are crucial to soil health, such as the nitrogen cycle or root

respiration and nutrient uptake. To take this experiment one step further, soil and aboveground

vegetation would be kept intact and simultaneous respiration measurements would be made.

Also, it would be interesting to measure the rate of mineralization and the percent of carbon from

the soil that is being redistributed from the soil to woody tissue in aboveground vegetation both

in response to warming.

Acknowledgements

Thank you so much Jim Tang for being a great and patient advisor and mentor. Special

thanks for Chris Neill for helping to figure out the logistics of this experiment. I would like to

thank the Marine Biological Laboratory, the Semester in Environmental Science faculty and

students, Dr. Serita Frey and her University of New Hampshire Harvard Forest Research group

for providing me with this unique opportunity to examine the precious soil of the Prospect Hill

and UNH Soil Warming LTER I also would like to particularly thank Stef Strebel, Rich

McHorney, Laura Van der Pool, and Carrie Harris for being such amazing lab teaching

assistants.

Literature Cited

Alison, Steven et al. 2010. Soil-carbon response to warming dependent on microbial physiology.

Nature Geoscience 3: 335-340.

Conant, Richard, T. et al. 2008. Experimental warming shows that decomposition temperature

sensitivity increases with soil organic matter recalcitrance. Ecology 89(9): 2384-2391.

Davidson, E.A., and I.A. Janssens. 2006. Temperature sensitivity of soil carbon decomposition

and feedbacks to climate change. Nature 440: 165-173.

Foreman, Ken. 2010. Preparing, Packing, and Organizing CHN samples.

Frey, S.D. et al. 2008. Microbial biomass, functional capacity, and community structure after 12

years of soil warming. Soil Biology & Biochemistry 40: 2904-2907.

Melillo, J. et. al. 2011. Soil Warming, carbon-nitrogen interactions, and forest carbon budgets.

PNAS Early Addition: 1-5.

Nabu, Masaru. 2009. Soil warming activates soil fungal ligninolytic secondary metabolism

through carbohydrate exhaustion at the Harvard Forest soil warming LTER: an enzymatic

approach. MBL: Semester in Environmental Science.

Figures and Tables

Figure 1: The five-year warming experiment control plot average respiration rate as measured by the

LICOR 6400 versus temperature over the 26 day incubation period.

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25CO

2 f

lux

(ug

C/g

sam

ple

/min

)

Temperature (degrees Celsius)

5 yr C Average Respiration Rate versus Temperature

Day 26

Day 18

Day 8

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

5 15 25

CO

2 f

lux

(ug

C/g

sam

ple

/min

)


20 yr C Average Respiration Rate versus Temperature

Day 26

Day 18

Day 8

Figure 2: The five-year warming experiment heated plot average respiration rate as measured by the


Figure 3: The twenty-year warming experiment control plot average respiration rate as measured by the


Figure 4: The twenty-year warming experiment heated plot average respiration rate as measured by the


0

0.1

0.2

0.3

5 15 25

CO

2 f

lux

(ug

C/g

sam

ple

/min

)


5 yr H Average Respiration Rate versus Temperature Day 26

Day 18

Day 8

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

CO

2 f

lux

(ug

C/g

sam

ple

/min

)


20 yr H Average Respiration Rate versus Temperature Day 26

Day 18

Day 8

Figure 5: Temperature Sensitivity over time as measured by Q10 values from each day of respiration

measurements of the 20 year warming experiment heated and control plot and the five year warming

experiment heated and control plot.

Figure 6: Relative average Polyphenol Oxidase enzyme activity over 50 to 100 minutes incubation time

with L-DOPA enzyme substrate added to two grams of soil sample from the four treatment plots’

replicate samples done at room temperature.

0.8

1

1.2

1.4

1.6

1.8

2

2.2

2.4

0 30

Q1

0 V

alu

e

Time (days)

Temperature Sensitivity of Four Treatments at 0-10cm over Time

20 yr Control

5 yr Heated

5 yr Control

20 yr Heated

0.000

0.003

0.006

0.009

0.012

0.015

5 yr C 5 yr H 20 yr C 20 yr H

Act

ivit

y (A

bso

rban

ce/T

ime/

2 g

sam

ple

)

Four Treatments Average Lignase Enzyme Activity

1 8 18 26

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0.08


g C

to

tal/

g so

il

Total Carbon of Four Treatment Plots

Figure 7: Average total carbon of the four treatment plots as measured by CHN analysis.

Figure 8: Average recalcitrant carbon pool of four treatment plots as calculated by CHN analysis.

Figure 9: Average labile pool one and labile pool two of four treatment plots as calculated by a DOC

analyzer.

0

0.005

0.01

0.015

0.02

0.025

0.03


g C

arb

on

/g s

oil

sam

ple

Recalcitrant Carbon of Four Treatments

0

0.005

0.01

0.015

0.02

0.025

0.03


g C

arb

on

/g s

amp

le

Labile I and Labile II Carbon Pools of Four Treatments

Labile II

Labile I

Figure 10: Average labile I Carbon pool of four treatment plots as measured by the DOC analyzer.

Figure 11: Average labile II Carbon pool of four treatment plots as measured by the DOC analyzer.

0.0015

0.0016

0.0017

0.0018

0.0019

0.002

0.0021

0.0022

0.0023

0.0024


g C

/g s

amp

le

Average Labile I Carbon pool of Four Treatments

0.0062

0.00622

0.00624

0.00626

0.00628

0.0063

0.00632

0.00634

0.00636


g C

/g s

amp

le

Average Labile II Carbon pool of Four Treatments

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)

20 yr Control @ 10-20cm Average Respiration versus Temperature over Time

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

20 yr Heated @ 10-20cm Average Respiration versus Temperature over Time

Day26Day18Day 8

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)


Day 26

Day 18

Day 8

Day 1

Table 1: Percent averages of total labile I, labile II, and recalcitrant carbon pools in total carbon per gram

of soil sample of four treatment plots measured from initial soil subsamples.

Treatment Total C/g soil Labile I (%) Labile II (%) Percent

Recalcitrant (5%)

5 yr Control 0.0335 11 18 66

5 yr Heated 0.0294 8 17 75

20 yr Control 0.0290 7 22 72

20 yr Heated 0.0173 9 24 65

Figure 12: Average respiration rates of soil from 10-20cm depth from the 20 year warming experiment

heated and control plots (the same units for x- and y-axis and legend at right).



Temperature (˚C)

Temperature (˚C)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)


Day 26

Day 18

Day 8

Day 1

00.05

0.10.15

0.20.25

0.30.35

5 15 25

CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)


0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

5 15 25

CO

2 f

lux

(ug

CO

2/g

sam

ple

/min

)


Day 26

Day 18

Day 8

Day 1

0

0.002

0.004

0.006

0.008

0.01

0-10 10-20 20-30 30-40 40-50

Act

ivit

y (A

bso

rban

ce/T

ime/

2g

sam

ple

))

Depth (cm)

Average Lignase enzyme Activity: Depth Profile of 20 yr H and C





Temperature (˚C)

Temperature (˚C)

0

0.005

0.01

0.015

0.02

0.025

0-10 10-20 20-30 30-40 40-50

g C

arb

on

/g s

amp

le

Depth (cm)

Depth Profile of Average Recalcitrant Carbon of 20 yr Plots

Recalcitrant 20 yr Control

Recalcitrant 20 yr Heated

Figure 16: Relative average Polyphenol Oxidase enzyme activity at room temperature incubation over 50

to 100 minutes with L-DOPA enzyme substrate added to two grams of soil sample from the five depths of

the 20 year warming experiment heated and control plots done at room temperature.

Figure 17: Average total carbon of the four treatment plots 20 year warming experiment heated and

control plots at depth as measured by CHN analysis.

Figure 18: Average recalcitrant carbon pool of 20 year warming experiment heated and control plots at

depth as calculated by CHN analysis.

0

0.01

0.02

0.03

0.04

0.05

0.06

0.07

0-10 10-20 20-30 30-40 40-50

g C

arb

on

/g s

amp

le

Depth (cm)

Total Carbon at Depth

20 yr Control

20 yr Heated

0

0.001

0.002

0.003

0.004

0.005

0.006

0-10 10-20 20-30 30-40 40-50

g C

/g s

amp

le

Depth (cm)

Labile I Carbon Pool of 20 yr H and C Plot at Depth

Labile I 20 yr Control

Labile I 20 yr Heated

0

0.005

0.01

0.015

0.02

0.025

0.03

0-10 10-20 20-30 30-40 40-50

g C

/g s

amp

le

Depth (cm)

Labile II Carbon Pool of 20 yr H and C Plots at Depth

Labile II 20 year Control

Labile II 20 year Heated

Figure 19: Average labile I Carbon pool of 20 year warming experiment heated and control plots as

measured by the DOC analyzer.

Figure 20: Average labile II Carbon pool of 20 year warming experiment heated and control plots as

measured by the DOC analyzer.

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30

Q1

0 V

alu

e

Time (days)

Temperature Sensitivity of 20 yr Control Plot at Depth over Time

0-10cm

10-20cm

20-30cm

30-40cm

40-50cm

Table 2: Percent averages of total labile I, labile II, and recalcitrant carbon pools in total carbon per gram

of soil sample of 20 year warming experiment heated and control plots at depth as measured from initial

soil subsamples.

Treatment Total C/g soil Labile I (%) Labile II (%) Percent

Recalcitrant (5%)

20 yr Control

0-10cm

0.0290 6 22 72

20 yr Heated

0-10cm

0.0195 9 33 58

20 yr Control

10-20cm

0.0231 5 21 74

20 yr Heated

10-20cm

0.0204 6 26 68

20 yr Control

20-30cm

0.0322 19 63 18

20 yr Heated

20-30cm

0.0333 2 78 19

20 yr Control

30-40cm

0.0192 19 60 21

20 yr Heated

30-40cm

0.0277 8 66 16

20 yr Control

40-50cm

0.0122 22 57 22

20 yr Heated

40-50cm

1 17 61 22


measurements of the 20 year warming experiment control plots at depth.

1 8 18 26

0

0.5

1

1.5

2

2.5

0 30

Q1

0 V

alu

e

Time (days)

Temperature Sensitivity of 20 yr Heated Plot at Depth over Time

0-10cm

10-20cm

20-30cm

30-40cm

40-50cm


measurements of the 20 year warming experiment heated plots at depth.

Appendix

Figure A –Prospect Hill Tract of Harvard Forest where sampling locations located (see soil

warming).

1 8 18 26

Treatment Layer (cm) 5˚C 15˚C 25˚C

20 year warming 0-10 6 replicates 6 replicates 6 replicates

20 year control 0-10 6 replicates 6 replicates 6 replicates


20-30 3 replicates 3 replicates 3 replicates









TOTAL 46 46 46

Figure B – Sample set-up in the incubators.

Sample C: Polyphenol Enzyme Assay Protocol

Polyphenol Oxidase Enzyme Assay Protocol (by Ellen Yeatman)

Organic carbon can be classified into two main categories: labile and recalcitrant. Labile carbon

pools are easily decomposable sources of energy for microbes while recalcitrant carbon pools are

more difficult for microbial decomposition. The purpose of this enzyme assay is to determine if

soils of various proportions of recalcitrant and labile carbon pools adapt the ability to breakdown

the more recalcitrant carbon compounds when there is not enough labile carbon to meet

microbial energy demands. Soil polyphenol oxidases (PPO) are enzymes that catalyze the

oxidation of recalcitrant aromatic compounds, such as lignin, into more readily available

substrates. This protocol is a simple method to assay PPO activity directly by a

spectrophotometric test using 5 mM L-3,4-dihydroxyphenylalanine (L-DOPA) as the substrate

and using an acetate buffer with a pH of 5.

PREPARATION

Preparing Buffer (Acetate)

The acetate buffer is a general purpose buffer used to assay a suite of soil enzyme activity

(Sinsabaugh, 1994). The pH is made to be 5 as presumed to most closely mimic realistic

environmental conditions.

Mix 357 mL of 0.1 M acetic acid and 643 mL of 0.1 M sodium acetate then adjust the pH to five

using a pH meter (De Lloyd, 2000). Acetic acid is 17.4 molarity. Mix 5.747 ml of acetic acid in

1000 mL of distilled water to create 0.1 M acetic acid. Using the molecular weight, it was

determined that 13.6 g of sodium acetate in 1000 mL of distilled water results in 0.1 M sodium

acetate.

Preparing Enzyme Substrate (5 mM L-DOPA)

The molecular weight of L-DOPA is 197.19 g/mol and multiplied by 0.005 mol/L tells you that

you need 0.98595 g L-DOPA per liter of solution. For every mL of solution, 100 uL of L-DOPA

is needed (Sinsabaugh, 2000).

To make 50 mL of enzyme substrate, add 0.0492975 g L-DOPA (50 mL multiplied by

0.00098595 g/mL L-DOPA) to 50 mL of the acetate buffer solution.

L-DOPA is light sensitive so keep shielded from light as much as possible even during the assay

process.

Preparing Samples

Weigh two grams of sample soil into a 15 mL falcon tube by taring the falcon tube first and

adding soil directly to the tube. Depending on the number of time points wanted to measure, add

7-15 mL of acetate buffer. The spectrophotometer uses 3 mL of sample. For each desired time

point, supply 3.5 mL of acetate buffer. For this experiment two time points were measured so 7

mL (or 8 mL for extra precaution) of acetate buffer added to the falcon tube with the two grams

of sample soil.

The background control is 2 grams of control soil sample that has been autoclaved for 25

minutes to kill any enzymes that may be present and add 8 mL of acetate buffer.

Methods

1. Once falcon tubes prepared with 2 g soil sample and 8 mL acetate buffer, add 800 uL of L-

DOPA to initiate the assay (100 uL L-DOPA per mL of acetate buffer). Note exact time of

addition of substrate; this is time zero.

2. Use shaker or vortex for an hour to two hours at temperature soil has been stored at (or at

room temperature) in a dark place because L-DOPA is light-sensitive.

3. Centrifuge sample at 2000 rpm for 4 minutes or more if necessary. If spin at too high of an

rpm, the soil gets stuck at the bottom of the falcon tube.

4. Aliquot 3 mL of supernatant from the falcon tube to a glass tube.

5. Measure the absorbance of the supernatant at 460 nm using a spectrophotometer and note

readings. Remember to measure the background control sample also.

6. Compute activity as absorbance (nanometers) per minute per gram of soil sample as follows

using the slope and subtracting the background control activity (the slope).

Bibliography

De Lloyd, Dhanlal. 2000. Preparation of pH buffer solutions. AnalChem Resources. University

of the West Indies: Chemistry Department. http://delloyd.50megs.com/moreinfo/

buffers2.html

Flock, C., E. Alarcon-Gutierrez, and S. Criquet. 2007. ABTS assay of phenol oxidase activity in

soil. Journal of Microbiological Methods 71: 319-324

Sinsabaugh, Robert L. 2000. Phenol Oxidase and Peroxidase Assays. Center for Dead Plant

Studies. University of New Mexico: Biology Department.

http://enzymes.nrel.colostate.edu/enzymes/protocols/PhenOx-

Perox_Enzyme_Assays_Protocol_09.15.2000.pdf

Sample D: Carbon Quality Analysis Protocol – Fractionating Carbon samples.

http://delloyd.50megs.com/moreinfo/%20buffers2.html

http://delloyd.50megs.com/moreinfo/%20buffers2.html

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