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Massachusetts Institute of Technology : Undergraduate Research Opportunities Program

Understanding the Hydrogen Soil Sink Harvard Forest Long Term Ecological Research Site

WRITTEN BY DEEPA RAO
Direct Advisor: Laura Meredith - predawn@mit.edu Faculty Supervisor: Professor Ron Prinn - rprinn@mit.edu

Fall 2010 - Spring 2012

d r a o @ m i t . e d u • 2 2 9 Va s s a r S t . R o o m 1 0 7 3 S i m m o n s H a l l , C a m b r i d g e , M A 0 2 1 3 9 • 602.410.6576

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Atmospheric H2 is a secondary greenhouse gas (GHG) and is vital to the removal of methane (CH4) from the atmosphere. Natural and anthropogenic outputs are nearly equally responsible as sources of H2 and human output is expected to increase in the future. Soil microorganisms are responsible for about 80% of the Earth’s atmospheric H2 sink and are the least understood term in the overall H2 budget. It is speculated that Streptomyces, a ubiquitous genus of soil microbe, is a large contributor to H2 deposition through its enzyme: hydrogenase. Specifically, Streptomyces has a unique high-affinity, low-threshold hydrogenase. How Streptomyces and other hydrogenasecontaining microbes respond to changes in the climate can help us predict if and how the H2 cycle can potentially change. We will examine soil uptake of H2 by isolating and varying the environment of Streptomyces from the Harvard Forest (HF) and by measuring soil’s H2 flux in a set of plots that cover a wide range of environmental conditions (soil moisture, temperature, increased and decreased levels of H2, etc.). So far, we have optimized a protocol for Streptomyces isolation from forest soil, extraction of DNA, and amplification of the hydrogenase DNA sequence through polymerase chain reaction (PCR). We plan on testing our isolated strains to see if their H2 uptake drastically alters under different environmental conditions. The second part of our project deals with measuring H2 uptake by using flux chambers in various long-term plots around the HF (nitrogen amendment, heated, litter removal. plots, etc.) With these two methods combined, we will gain a better understanding about the variability of the Earth’s largest H2 sink and how it may respond to climate change.

U n d e r s t a n d i n g t h e E a r t h ’ s H y d r o g e n S i n k!

MIT

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Understanding the Microbial Control of Earth’s Hydrogen Soil Sink through the Isolation of Streptomyces
N A T U R E O F R E S E A R C H

As our global community explores new and exciting energy options, we must understand the consequences of these technologies on our biosphere. Along with new energy solutions come changes in the emissions of direct and indirect greenhouse gases (GHGs). These molecules can dramatically affect the chemistry of the atmosphere, oceans, and soils, leading to potential changes in the radiative budget of the atmosphere. Before radical shifts in energy use occur, we must thoroughly examine the potential unintended consequences of these new energy technologies on the concentrations of their associated direct and indirect GHGs. To replace fossil fuels, one of many solutions includes using H2 as an energy carrier. Whether H2 technologies are used as mobile or stationary energy carriers, there will be leaks associated with its storage. If these technologies are increasingly employed, the anthropogenic input of H2 into the atmosphere will increase, and could potentially alter the oxidative environment of our atmosphere. Atmospheric H2 is an important indirect greenhouse gas; it attenuates the removal of methane (CH4) and other GHGs by reacting with the atmosphere’s cleansing hydroxyl radical (· OH). · OH is generated by photochemical reactions and has a very short lifetime and fairly low concentrations. It acts as an atmospheric cleansing radical by breaking down other atmospheric gases. A pertinent example to climate change models is of· OH’s ‘cleansing’ the atmosphere of the strong GHG methane (CH4). Methane as a GHG is ~25 times stronger per ton than CO2. · OH is a major sink for methane by the following reactions:     (1) CH4 + · OH → · CH3 + H2O

It is also the minor sink for H2 ( approximately 20%): (2)        H2 + · OH → · H + H2O (reaction 4)

The reason H2 is an indirect greenhouse gas is that H2 is competing for · OH with CH4. So, if there are more H2 emissions, more· OH will be taken by H2 and less will be available to react with CH4. This will allow CH4 to remain in the atmosphere longer and exert more of a greenhouse effect. There are other greenhouse gases like CH4 that can also remain longer because· OH is their main sink (e.g., hydrofluorocarbons widely used as refrigerants and solvents). The largest sink for H2 is through consumption in the soil by microbial-mediated soil uptake. Microbial uptake accounts for ~80% of all H2 that is taken out of our troposphere. UnfortuU n d e r s t a n d i n g t h e E a r t h ’ s H y d r o g e n S i n k! MIT

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nately (or fortunately for the sake of our research), these H2-metabolizing microorganisms are poorly understood. The response of these microorganisms to an increase in atmospheric hydrogen will dictate the rate at which H2 is taken out of our atmosphere. This information will be highly important if we are exuding enough H2 to fundamentally change biogeochemical cycles. One large accomplishment of my research as a UROP this sumer was that I isolated several strains of microorganisms that contain hydrogenase. We are currently sequencing the 16S rRNA of these organisms and will be able to compare them to known strains in the near future. Once we know more about their genomes, we intend on then testing them under different environmental conditions. We are planning on growing them under zero, low, normal, and high H2 concentrations. We explore how these organisms respond to heat, increased nutrients in their media, and other factors that simulate environmental change.
L O C A T I O N S A N D P E O P L E

This fall I will be working with Laura Meredith, my direct supervisor, under the guidance of Professor Ron Prinn, my faculty advisor. The bulk of this semester’s microbiological work will be conducted in Tanja Bosak’s lab and we will be taking gas chromatography measurements in Shuhei Ono’s lab, which are both in room E25-650B at MIT. We are also planning on taking syringe samples of H2 from the Harvard Forest (HF) Long Term Ecological Research Site in Petersham,s  MA (http://harvardforest.fas.harvard.edu/research/lter_new.html). The HF contains well-studied field sites and refurbished laboratories that I worked in over the course of the 2011 Summer REU Program and will be working in for the remainder of my thesis work.
P E R S O N A L D U T I E S & T E N TA T I V E W O R K P L A N

I will be working most of the time at MIT this term for my UROP. Over the summer at the HF, we successfully isolated strains of organisms from HF soils that contain hydrogenase. Since we have these strains, we can test them with the above mentioned methods. The timeline of the full project is until May 2012, when I must complete my senior thesis for EAPS.  
M E T H O D S

In order to understand the variability of soil H2 uptake, we must learn more about the specific microorganisms that remove H2 from the atmosphere. Herein lies the dual goal of our project: Firstly, we will take various measurements to determine how H2 uptake varies within different soil plots (heated, increased nitrogen, control soils, etc). Secondly, we will isolate novel strains of soil microorganisms that can utilize H2 in order to understand 1) the biodiversity of this subset of microorganisms and 2) to see how their H2 metabolism changes under various environmental conditions that simulate future climate change. With this knowledge, we will extrapolate on how these microorganisms will react on a global scale and also their effect on the H2 cycle.
U n d e r s t a n d i n g t h e E a r t h ’ s H y d r o g e n S i n k! MIT

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For the microbiological work: there is one published genus of microorganisms--Streptomyces sp. -that has been isolated with the ability to metabolize H2 (at our current low atmospheric concentrations) with an enzyme called hydrogenase. There is available genomic data suggesting that there are other species with the same ability to metabolize H2; however, they have not yet been isolated. Our goal for the past summer was to isolate organisms that contain hydrogenase enzyme. Given that there are over 1 billion microorganisms per gram of soil, we had to optimise our methods for finding hydrogenase-producing organisms from fresh HF soil. Over the course of the summer, we successfully isolated 4 strains with the relevant hydrogenase. Isolations aid our understanding of the diversity of microbes responsible for the uptake of atmospheric H2. We have successfully sequenced the genetic 16S rRNA and Hydrogenase genes of these four organisms and have determined that they are indeed in the genus of Streptomyces. After genetic analysis of these strains, we will compare their phylogenetic and hydrogenase genes to known strains in available databases and to metagenomic data from Harvard Forest soils. This approach will give us greater insight into the diversity, prevalence and ecological distribution of these organisms. Next, we will test the isolated strains under different simulated environmental conditions; for instance, we could test how temperature affects their H2 uptake. With so are many potential questions to address, research on these isolates could be continued into the following Spring. Through this work we will better understand the microbes and potentially their role in the community of these microorganisms as a whole. Flux chambers are a traditional method used to measure gas exchange between the soil and the atmosphere. It consists of a volume of air trapped above the soil within a chamber resting on a collar in the soil; trapped air will undergo compositional changes as trace gases exchange between the trapped air and the soil. We can measure the flux of H2 from the trapped air to the soils inside the chamber to assess H2 uptake. We will have some definitive and some exploratory soil sites, which will change after we assess whether the plots are informative or not. We will isolate microbes and characterize them. Further microbial experiments will depend on which species we can isolate. I will also be doing some computer-based research on HF sites about soil type, hydrology, and past microbial community data.

U n d e r s t a n d i n g t h e E a r t h ’ s H y d r o g e n S i n k!

MIT

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M O T I V A T I O N S

F O R

R E S E A R C H

First, I want to express my thanks to the MIT UROP Program, the Harvard Forest REU Program, and the Lord Foundation for funding my research. I had an invaluable experience at the Harvard Forest Summer Research Program and learned an immense deal from the advisor, fellow students, and staff at the forest. I look forward to expanding on my current research through the next year. I am interested in this UROP because I have finally found a project that combines my passion for understanding changing ecology in the context of climate change. I am majoring in Earth, Atmospheric, and Planetary Sciences with a focus in Environmental Biology. Through my interdisciplinary coursework, I have learnt the fundamentals of ecology but this will be the first time that I will conduct research in the field. My scientific perspective has always been through the lens of how our biosphere operates, evolves, and regulates itself. We are constantly playing a tugof-war with the Earth’s carrying capacity (technology vs resource depletion) and toying with our planet’s homeostasis. With current population growth expected to reach 9 billion people by 2040, our combined anthropogenic effect is also always growing. Understanding natural cycles gives us great insight into our anthropogenic forcing. Through our research, we are interested in better understanding the natural biogeochemical cycle of H2 and therefore how humans are perturbing its natural balance. This vital information can help us to better regulate our GHGs and one day mitigate our negative effects on our global climate. Through this UROP I will get the chance to exercise my biological, chemical, and earth science education, and my chemical and microbiological laboratory skills. This summer REU and UROP are also now the basis of my senior thesis. We have made much progress thus far and will continue this research through the end of my time as an undergraduate here at the Massachusetts Institute of Technology.

U n d e r s t a n d i n g t h e E a r t h ’ s H y d r o g e n S i n k!

MIT

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