Semester in Environmental Science, Independent Project, 2011

Effects of warming on soil respiration

measured by CO2 flux and extracellular

enzyme activity

Xiao Yang (Shirlie)

Grinnell College

1115 8th

Ave, Grinnell, IA 50112

Mentor: Jim Tang

Marine Biological Laboratory

7 MBL Street, Woods Hole, MA 02543

Abstract

Soil respiration plays a significant role on global carbon cycle as the primary path that releases carbon into the atmosphere CO2. Since global warming is the general trend on climate change, this study focuses on the effects of warming on soil respiration on three different types of soil. Soil from Harvard Forest LTER control plot, agricultural corn field in Deerfield, MA and grassland in Woods Hole, MA were collected and incubated under three different temperatures: 10°C, 20°C and 30°C. Measurements including CO2 flux, extracellular enzyme assay and soil characters. From our results, we concluded that the heating increase the enzyme activity rate on cellulose as well as the soil respiration rate, but the sensitivity to temperature decreases through the incubation period. Also, soil contains more organic matter, such as forest soil, has higher respiration rate than other types of soil. Therefore, forest ecosystem will be more sensitive to global warming.

Key words: Soil respiration; soil warming; CO2 flux; extracellular enzyme assay; cellulose, lignin; Harvard forest; agricultural; grassland; incubation.

Introduction

Soil respiration, which includes the respiration of plant roots, the rhizoshpere, microbes and fanua, is the primary path for the CO2 fixed by land plants returns back to the atmosphere. Every year, approximately 75-77 Pg C/yr (Raich and Potter. 1995) is released to the atmosphere by soil respiration. Global soil also contains a large amount of carbon store, which is up to 3150 Pg C/yr and is more than four times the carbon containing in the atmosphere. (Sabine et al. 2007) Therefore, a small change in soil respiration can seriously alter the balance of atmosphere CO2 concentration and the soil carbon stores. In addition, soil respiration rate in specific ecosystem also close related to its net primary production (NPP). One good example is the tropic area usually has the greatest respiration rate, in response, the vegetation in tropic area growth luxuriant and the conditions are ideal for decomposers. According to past researches, several factors will significantly affect the soil respiration rate, which includes temperature, nutrient content and human activity. When the global temperature increases every 2°C, the additional 10 Pg Carbon will be released from soil respiration (Friedlingstein, 2003). Also, human activities changes land use by fertilization and cultivation. Different soil nutrient content because of vegetation and geography history results in changing the microbial metabolic rate, which consequently affects the soil respiration rate. During decomposing process, organic matter is divided into two groups based on the difficulty to break down. One group is relatively easily decomposed, such as sugars, starch, non-lignified carbohydrates, and another group is much more difficult to decompose, such as lignin and lignified carbohydrates (Melillo, 2011). The enzyme assay on the enzymes that break down these two groups of organic matter shows the microbial activity during the soil respiration, indicating the microbe metabolic rate, which alters the soil respiration rate.

Although most ecosystem models predict that climate warming will stimulate microbial decomposition of soil carbon and therefore producing a positive feedback on soil respiration to rising temperature (Lloyd and Taylor, 1994), some researchers mentioned the different

response to changing climate between short-term and long-term treatment. A study on soil warming shows that the soil respiration is strongly and positively related to temperature but it acclimates after a long-term period (Bradford et.al, 2008). Also the reduced carbon-use efficiency limits the biomass of microbial decomposer and decreases the loss of carbon within a few years (Allison et al. 2010). Therefore, it will be interesting to explore more about how soil respiration rate responds to warming along with the incubation period.

In this project, I was looking at how the temperature and soil type affect the soil respiration rate and extracellular enzyme activities. This study therefore sought to address the following questions: 1) How does the CO2 flux respond to the changing temperature? 2) Does the CO2

flux respond differently according to different soil types? 3) How does the soil sensitivity to temperature change during the incubation peirod? 4) How does these changes explained by extracellular enzyme assay and soil characters?

Soil samples from Harvard forest, grassland and corn field are collected and incubated under temperature at 10°C, 20°C and 30°C for 25 days. My hypothesis is that the heating increases the soil respiration rate as well as the high nutrient content. Enzyme activities on cellulose shows the same trend as soil respiration rate but the enzyme activities on ligninase shows the opposite trend. Soil respiration rate decreases slightly during the incubation period.

Methods

Soil sample collection and field measurement: Soil samples were collected from the surface soil layer in Harvard forest LETR control plot, an University of Massachusetts’s agricultural experimental site at south Deerfield, MA and grassland in Peterson Farm at Woods Hole, MA. Soil was homogenized and separated into 27 standard 5 gallon plastic buckets, 9 buckets for each soil type. Soil from grassland and corn field was 40 pounds per buckets and soil from Harvard forest was 25 pounds per buckets. CO2 flux of soil respiration was measured by LiCor 6400 in these plots. Soil temperature and moisture level were also measured in the field. Bulk density CHN content and the PH of these 3 types of soil were measured in the lab.

Soil respiration measurement during incubation period: Soil buckets were incubated for 25 days in 3 different temperature control room that were set at 10°C, 20°C and 30°C. There were 3 replicates for each treatment, meaning that under each temperature, there were 3 buckets of each type of soil. CO2 flux is measured constantly on day 5, day 15, day 23 and day 25 by LiCor 7000 on the Mobile GHG measurement system during the incubation period. The measurement for one sample took 5 minutes (Between chamber on and off). The CO2 flux was measured per 2 seconds during this 5 minutes and were transferred to respiration rate based on the equation:

A

V

RT

P

dt

dCF v ,where C is mole concentration (μmol m-3), V is volume (m3), A is area (m2), P is air

pressure (Pa), T is soil absolute temperature (K), and R is universal gas constant (8.3144 J mol-1 K-1) (Tang,2011).

Enzyme Assay: The enzyme activities of cellulose and ligninase were measured by the enzyme assays. Enzyme assay samples were collected in day 2, day 16 and day 25 during incubation period. For cellulose, 1 gram of soil was collected from each soil buckets and was mixed with 5.5 ml acetate buffer (0.1M) and 550 μl enzyme substrate MUF-β-D-glucoside (M-3633) in the 15 ml falcon tubes. After centrifuged 2 minutes with 3000 rpm, 2.5ml of enzyme-sample solution was transferred into test tubes and added into 2.5ml glycine buffer (0.1M, PH 10.5). The fluorescence was read by fluorometer. The sample-enzyme mixture was incubated under the same temperature as the soil samples for 25 minutes. During the incubation, the falcon tubes were shaked constantly to completely mix the soil and enzyme substrate. After 25 minutes, the enzyme-sample solutions were measured again by fluorometer again after killed by glycine buffer. To calculate the enzyme activity, plot MUF concentration (nM) versus time and use linear regression to calculate enzyme activity in units of nmolel-1h-1. Method for ligninase was basically same as cellulose. But ligninase used L-3,4-dihydroxyphenylalanie (L-DOPA) as the enzyme substrate and incubation time is 2 hours. Also, UV sepectrophotometer set wavelength at 460nm was used for the reading.

Results

According to the data, soil respiration rate on all three type of soil increases significantly when temperature increases from 10°C to 30°C (Fig. 1a, 1b, 1c). However, the increase on soil respiration rate did not increase proportionally along this the increasing temperature. The respiration rate increase from 10°C to 20°C was relatively higher than it from 20°C to 30°C, showing that very high temperature did not benefit soil respiration as well under warm treatment. Also, comparing with the soil respiration on corn field and grassland soil, Harvard Forest soil has much higher respiration rate under all three temperature, but especially in the warmer environment such as 20°C and 30°C (Fig. 2a, 2b, 2c). Soil respiration rate on grassland and corn field did not have significantly difference from each other when they were under same treatment (Fig.3).

Enzyme activity on cellulose for soil under most of the treatments, the enzyme activity increased rapidly from the beginning of the incubation to day 15 and decreased back to low activity rate by the end of incubation under all three different temperature (Fig.4a, 4b, 4c). Under 10°C and 20°C, forest soil had much higher maximum enzyme activity rate than grassland and corn field and overall the enzyme activity rate of all types of soil were the highest under 20°C and the lowest under 30°C. Enzyme activity on ligninase does not show significant change on different type of soil or temperature and the activity rates were very close to zero, indicating the very few enzyme activity on peroxidase (PER), which is the ligninase we measured.

Among all three types of soil, Harvard Forest soil has the lowest bulk density (Fig.5) and the highest carbon content (Fig.6), indicating that Harvard forest soil contains much more organic matter than grassland and corn field soil. Furthermore, measurement on soil respiration rate in the corn field (0.554 μmol•s-1•m-2) was close to the soil respiration rate measured during the incubation period under similar temperature (0.501 μmol•s-1•m-2).

Discussion

Comparing with the respiration rate measured in corn field, our incubated corn field soil under similar temperature treatment had very close soil respiration rate, proving that the incubation environment in the temperature room successfully modeled the field environment. Therefore our data can also be compared with the field measurement in related research. Previous study indicated that increasing temperature effectively increases the soil respiration rate, which was also supported by our experimental results. Soil respiration rate was positively correlated with the temperature in Harvard Forest, grassland and corn field. Among all three types of soil, Harvard forest soil had the highest soil respiration rate under all three different incubation temperatures. Also, our soil respiration rate results shows no significant difference between grassland and corn field soil, where soil has been disturbed by human activities and have less organic matter accumulation. Therefore, both temperature and soil type were significantly alter the soil respiration. Furthermore, soil respiration showed less sensitivity to changing temperature along with the incubation period, especially on samples incubated under 30°C. Soil respiration rate decreased slightly during the incubation period, suppored Bradford et.al’s research result, indicating that soil respiration rate declined during a long-term heating. Although our incubation period was only 25 days, because of the lack of organic matter source, soil sample were under the similar situation as the soil under long-term heating treatment in the field and shows the same trend as field experimental soil respiration. Therefore, the length of period of soil warming also affects the soil respiration rate significantly.

To explore why the soil respiration rate changed under different temperature, we tested the enzyme activity on both cellulose and ligninase at the beginning (day 2), middle (day 15) and the end (day 25) of the entire incubation period. Enzyme activity on cellulose showed the interesting trend that the enzyme activity increased rapidly from the beginning to the middle of the incubation period then decrease again by the ending point. This trend was shown on all three type of soil under different temperature treatment. As we discussed above, our soil respiration rate showed the same trend, which was that the soil respiration rate decreased through the entire incubation period. One possible explanation on the changes on enzyme activities was that the microbial community in soil decomposed all the cellulose in the soil sample at about day 15 during the incubation and began to decompose other organic matter. Although our enzyme assay on ligninase did not show a significantly increase after day 15 on all of our samples. Since we were only measured the activity of peroxidase (PER), which is one of the major ligninases, there may more enzyme activities on other ligninases, such as polyphenol oxidase (PPO). In addition, different methods on enzyme assay results different results, especially the time long of incubating soil sample during enzyme assay. In our project, the incubation time is around 2 hours, which may be too short for enzyme substrate to fully active. Another important finding from the enzyme assay on cellulose was that enzymes had the highest activity rate under 20°C and the lowest was under 30°C, indicating that the microbial enzyme community did not adapted the higher temperature as well as the normal(20°C) or lower temperature (10°C). Therefore, warming may effectively alter the soil respiration by effect the microbial community activity. Furthermore, similar as soil respiration rate, under

10°C and 20°C temperature treatment, the enzyme activity in Harvard forest soil was relatively much higher than grassland and corn field soil. Combing with the temperature’s effect, the extracellular enzyme activity and soil respiration rate are close correlated with each other and will be seriously affected by both the change on temperature and soil type.

In the measurements on soil characters, including bulk density and carbon content, Harvard Forest soil had the lowest bulk density and highest carbon content, presenting that Harvard forest soil contained most organic matter. The more organic matter the soil has, the higher enzyme activity rate on cellulose it will be and then further increase the soil respiration rate. Our hypothesis was fully supported by the soil respiration, enzyme activity rate on cellulose and soil character measurement results during the incubation period. Overall, warming increase soil respiration rate and extracellular enzyme activity but the sensitivity to changing temperature will decrease if no new organic matted added into the soil. Soil that contains more organic matter will have much higher respiration rate than the soil with less organic matter. Therefore, soil respiration in terrestrial ecosystem such as forest will be more sensitive to the global warming comparing with other ecosystems. In the future experiment, it will be helpful to incubation soil samples longer and measure the enzyme activities more frequency with better method. Also, it will be interesting to compare the field data and incubation data for both long-term and short-time incubation in order to better explore the effects of climate change on soil respiration. Soil samples from different ecosystem and more replicates also help us to better understand how disturbance, including agricultural behavior and fertilization affect soil characters and furthermore affect soil respiration.

Acknowledgement

Thanks a lot for my metor, Jim Tang, for his patient advice and resources support during the entire project period. Also thanks Joseph Vallino for his great help on enzyme assay and Jerry Melillo for helping me set up the project.

Thanks Richard McHorney, Stefanie Strebel and Carrie Harris for all the lab work. Especially thanks Tim Savas for guiding me using the equipment and analyzing data. Also thanks a lot to everyone in the soil group for all the field work.

Thanks again for the Marine Biological Laboratory and Grinnell College for giving me such a wonderful opportunity and strong resource support for this project.

Reference

Allison,S.D., Wallensterin, M.D.,and Bradford, M.A. 2010. Soil-carbon response to varming

dependent on microbial physiology.Vol 3, page 336-340 in Nature Geoscience.

Bradford, M.A. et al.2008. Thermal adaptation of soil microbial respiration to elevated

temperature. vol 11, Issue 12,pp 1316-1327. Ecology letters. Blackwell Publishing Ltd.

Frederick.S .1992. A New Assay for Lignin-Type Peroxidases Employing the Dye Azure B. Applied

and Environmental Microbilogy.Vol.58.,No.9.pp.3110-3116. American Society for Microbiology.

Lloyd, J., and Taylor,J.A. 1994. On the temperature dependence of soil respiration .Funct,

Ecol,8, pp315-324. Functional Ecology. British Ecology Society.

Nobu, M. 2008. Soil warming activates soil fungal ligninolytic secondary metabolism through

carbohydrate exhaustion at the Harvard Forest soil warming LTER: an enzymatic approach. SES

Raich J, and Potter C. 1995. Global patterns of carbon dioxide emissions from soils. Global

Biogeochemical Cycles. vol.9, page 23-36.

Sabine C et al. 2003. Current status and past trends of the carbon cycle. Toward CO2

Stabilization: Issues, strategies, and consequences. Island Press. Washington DC.

Vares.T et al.1995. Lignin Preoxidases, Manganese Peroxidases, and Other Ligninolytic Enzymes

Produced by Phlebia radiate during Solid-State Fermentation of Wheat Straw. Applied and

Environmental Microbilogy.Vol.61.,No.10.pp.3515-3520. American Society for Microbiology.

Forman,K. 2011. Preparing, Packing & Organizing CHN Samples. SES

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Tang, J. 2011. Class lecture. SES

Graph 1. Map of soil sample plot

Fig. 1a Cornfield soil respiration rate under three different temperature during the incubation period

Fig. 1b Harvard Forest soil respiration rate under three different temperature during the incubation

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Fig. 2b Soil respiration under 20°C

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Fig. 3 Soil respiration for all types of soil under different temperature though the incubation period.

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Fig. 4a Enzyme activity rate on cellulose under 10°C

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Fig. 4b Enzyme activity rate on cellulose under 20°C

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Fig. 5 Carbon content percentage on Harvard Forest, grassland and corn field soil.

Fig. 6 Bulk density of Harvard Forest, grassland and corn field soil.

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