Investigating the role of microbial composition in soil formation within the Critical Zone Background and Project Description:

The Critical Zone (CZ) is the thin, near surface environment that extends from the top of vegetation down to groundwater. The CZ hosts complex interactions involving rock, soil, water, air and living organisms that regulate the natural habitat. This layer determines the availability of life-sustaining resources such as food production and water quality (NRC, 2001). Critical Zone Observatories (CZO) have been established globally to form a network that aims to quantify the interactions between rocks and life in a consistent and comparable manner to help sustainably manage our valuable natural resources. Representing a thin section within the CZ is soil. Soil loss is a major issue facing humanity as it is essential for food production and other immensely important resources (Banwart, 2011). In addition, global temperatures are anticipated to rise in future years, therefore studying the influence of climate on soils will help us predict how climate change will impact the CZ.

To investigate the influence of climate on soil properties, study sites in North America, Puerto Rico and Wales were established and added to the Susquehanna Shale Hills CZO in central Pennsylvania (Dere et al., 2013) (Fig. 1). Study sites vary in mean annual temperature (MAT) and precipitation (MAP) from cold and wet in the north to warm and wet in the south. All sites are underlain by shale, which is an abundant sedimentary rock on Earth. Soil cores were collected from the surface to bedrock, or as deep as possible, on the ridgetops of each site. The cores were evaluated for geochemistry, mineralogy and physical properties and compared across the sites. Initial findings show that soils are deeper and more weathered in warmer and wetter climates (Dere et al., 2013; Dere et al., 2016). Further data would improve our understanding of the role climate plays in soil formation and weathering processes, particularly from a variety of climates, and at a global scale.

Another significant component that would help us understand the influence of climate on soil properties would be to investigate microbial composition. Microbes are vital for driving a variety of weathering processes either directly or indirectly through their activities. For example, microbes can directly change iron oxide minerals by oxidizing iron or indirectly accelerate rock weathering by decomposing organic carbon to produce carbon dioxide and organic acids, causing soil acidification and then mineral dissolution (Amundson et al., 2003). The composition and function of microbial communities is known to change with different environmental variables (Fierer et al., 2006; Lozupone et al., 2007; Rousk et al., 2010), which can be expected to change across soil horizons and with depth. However, most studies have focused only on the upper 0-10 cm of soil (Eilers et al., 2012) and have neglected to evaluate the changes in microbial community composition with depth and how this may affect their function. Here, I propose to examine samples collected across a shale weathering gradient (resulting in soils of different depth) to identify microbial community composition with depth and identify potential changes in microbial community function in response to resource availability (such as organic carbon or iron).

Methods: In August of 2016, I was part of a team led by Dr. Dere and Dr. Santini from the University of Queensland that traveled to the North American Shale Transect sites to collect soil samples from the ridgetop experiment sites. Since samples from these sites have already been evaluated for geochemistry,

Figure 1. Map showing the North American shale sampling sites. DNA from soil samples collected from PA, VA, TN and AL this past summer will be extracted and analyzed for microbial composition.

mineralogy and physical properties (Dere et al., 2013; Dere et al., 2016), the main objective of the new samples is to analyze microbial composition and function with depth. Our goal is to determine the microbial composition, including archaeal, bacterial, and fungal species present, in shale derived soils across the climosequence of the Shale Transect. The work involved digging up to 1.5 m deep pits and collecting samples in each horizon while maintaining a clean and sterile environment by wearing nitrile gloves and sterilizing all tools and sampling tubes with ethanol. The samples are preserved with LifeGuard! Soil Preservation Solution and have been kept frozen until we are able to begin analysis. The next step is to extract the DNA from the samples using PowerSoil DNA Isolation Kits and send the samples for DNA sequence analysis (16S rRNA amplicon sequencing at the Australian Centre for Ecogenomics, University of Queensland).

Dr. Santini is a soil scientist and geomicrobiologist at the University of Queensland, Australia. She will provide her expertise in DNA extraction, sequencing, and bioinformatics techniques (Santini et al., 2016). Dr. Santini leads the Biogeosciences Research Group (BiGS) at the university where they have the equipment to perform microbial analysis. Australia also presents an exciting opportunity to collect new samples at a shale site near Mt. Barney, approximately 100 km southwest of Brisbane (Fig. 2). Shale here is geochemically similar to the previously studied shale in North America, but presents a subtropical environment in the southern hemisphere. The timing of the new sample collection (February 2017) is late summer in Australia, similar to the seasonal timing in which the North American shale microbial samples were collected. This will help ensure seasonal differences in microbial composition are avoided. The process to collect new soil samples near Mt. Barney will be identical to the previously collected North American samples. We will dig a pit 1.5 m deep and collect samples from each horizon while maintaining a sterile environment. The new samples from Australia will also undergo DNA extraction using PowerSoil! DNA Isolation Kits followed by sequencing. In addition, all soil samples from the North American and Australian study will be ground and sent for analysis at the Pennsylvania State University Materials Characterization Lab. Geochemical data will allow us to identify the drivers of microbial community composition with depth and climate across the North American and Australian shale sites. Ultimately, this study will help us understand how different climates are impacting shale soil formation and how microbial composition at depth may enhance or decrease weathering rates. Project Timeline:!February 2017 Travel to Brisbane, Australia; collect new samples, extract DNA March 2017 Send soil samples for geochemical analysis April 2017 – Aug. 2017 Analyze DNA and geochemical data Sep. 2017 – March 2018 Submit abstract, FUSE report and present at Student Research Fair

Student/Faculty Mentor Roles:

I have worked closely with Dr. Dere since starting school at UNO and am very interested in CZ research, especially the process of sampling for microbial composition. I have experience collecting these samples at three of the U.S. CZ Observatories this past summer, and will be able to follow the process further by testing and analyzing them with help from Dr. Dere and Dr. Santini. Dr. Santini will provide me with geomicrobiology knowledge and training for DNA extraction and sequencing along with how to interpret the results. Dr. Dere will provide support for writing and presenting results and help interpret the geochemical data.

Figure 2. Map showing the new shale sampling location 100 km southwest of Brisbane, Australia.

Budget Justification: Budget Item Cost International Travel Airfare roundtrip Omaha, NE to Brisbane, Australia February 11-26 $1975 Visa $25 Lodging (provided by Dr. Santini, Univ. Queensland) $0 Local Transport (provided by Dr. Santini, Univ. Queensland) $0

Total $2000 Project PowerSoil® DNA Isolation Kit $515 Lifeguard® Soil Preservation Solution $210 Geochemical Analysis ($33/sample; 35 samples total) $1155 Organic Carbon Analysis ($7/sample; 35 samples total) $245 Stipend $375

Total $2500 Total amount requested $4500

I am requesting a total of $4,500 to investigate the role of microbial community composition in

soil formation within the Critical Zone with Dr. Dere at the University of Nebraska at Omaha and Dr. Santini at the University of Queensland in Brisbane, Australia. $2,000 of the funds will support travel to Brisbane, Australia in February 2017 where I will meet Dr. Santini at the University of Queensland. Dr. Santini has generously offered transport for the collection of new samples near Mt. Barney and DNA extractions of all samples at the university labs. She has also graciously offered that I could stay at her home during the trip, meaning no funds are requested for lodging and transportation expenses. I have also requested a stipend of $375 to cover the cost of meals, estimated at approximately $23/day for sixteen days. To facilitate the completion of the research objectives, funds have also been requested to purchase PowerSoil! DNA Isolation Kits ($515) to analyze microbial composition and Lifeguard! Soil Preservation Solution ($210), to preserve new samples collected near Mt. Barney. Soil chemistry analysis at $33/sample for 35 samples is requested for total elemental geochemical analysis which will to be completed at the Pennsylvania State University Soil Characterization Lab (State College, Pennsylvania). Finally, $7/sample for 35 samples is requested for soil organic carbon analysis at Ward Laboratories (Kearney, Nebraska). Funding requested here would enable both travel and completion of the initial research objectives outlined in the project proposal.

References: Amundson R., Guo Y., and Gong P. (2003) Soil diversity and land use in the United States. Ecosystems

6: 470-482. Banwart, S.A. (2001) Save our soils. Nature 474:151-152. Dere, A.L., White T. S., April R. H., and Brantley S. L. (2016) Mineralogical transformations and soil development in shale across a latitudinal climosequence. Soil Sci. Soc. Amer. J. 80:623-636. Dere, A. L., White T. S., April R.H., Reynolds B., Miller T. E., Knapp E. P., McKay L. D. and Brantley S. L. (2013) Climate dependence of feldspar weathering along a latitudinal gradient. Geochim.

Cosmochim. Acta. 122:101-126. doi: 10.1016/j.gca.2013.08.001 Fierer N., Jackson R. B. (2006) The diversity of biogeography of soil bacterial communities. Proc. Natl.

Acad. Sci. 103:626-631. Eilers, K. G., Debenport S, Anderson S, and Fierer N. (2012) Digging deeper of fine unique microbial communities: the strong effect of depth on the structure and bacterial and archaeal communities in soil. Soil Biol. Biochem. 50:58-65. Lozupone C., and Knight R. (2007) Global patterns in bacterial diversity. Proc. Natl. Acad. Sci.

104:11436-11440. National Research Council (NRC) (2001) Basic Research Opportunities in Earth Science. National Academy Press, Washington, 154 p. Rousk J., Baath E., Brookes P. C., Lauber C. L., Lozupone C., Caporaso J. G., Knight R., and Fierer N.

(2010) Soil bacterial and fungal communities across a pH gradient in an arable soil. ISME J. 4:1340-1351.

Santini T. C., Malcolm L. I., Tyson G. W., Warren, L. A. (2016) pH and organic carbon dose rates control

microbially driven bioremediation efficacy in alkaline bauxite residue. Environ. Sci. Tech. doi: 10.1021/acs.est.6b01973

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!!!! October 6, 2016

Dear FUSE Proposal Committee,

I am writing to express my support of Sara Parcher’s FUSE proposal. Sara is currently a second year geology student in the Department of Geography/Geology at UNO. I first had the pleasure of working with Sara in my Fall 2015 semester Introductory Physical Geology course and she was an outstanding student in the classroom. She displayed a great aptitude for learning and completed all assignments on schedule and with high quality. Despite completing only one geology course, Sara wasted no time in applying for, and obtaining, an Iowa EPSCoR summer research internship working at the University of Iowa on soil hydrology research. She thrived in the research environment, completing and presenting a research project investigating slope aspect differences in shallow soils under agricultural land management. Following the completion of the summer internship, Sara joined my research team for a week of intensive field work in the Appalachian Mountains. Field conditions were some of the more difficult I have experienced, with high heat and humidity, intermittent rain, labor intensive soil pit excavation, and hiking with sample- and gear-laden packs. Sara did not complain and was up to the challenge each day, providing a much needed boost in morale. She also took advantage of every opportunity to learn a new technique or ask questions about the research methods or interpretations. Sara is eager to continue the research she began this summer and has taken the initiative to develop and submit this FUSE proposal.

The construction of this FUSE proposal has been highly collaborative, with the initial ideas germinating from discussions while conducting fieldwork this past summer. Sara is especially interested in helping complete the microbial analysis of soils she collected with the research team this summer but she is also proposing to collect additional samples in Australia that would expand the dataset on comparable shale soils in the southern hemisphere. The proposed project is related to my research activity and would greatly complement my studies on soil formation in various climates. I do not, however, have the expertise to guide her in microbial research efforts so she will work with Dr. Talitha Santini, from the University of Queensland in Australia, to learn additional techniques necessary to complete the project. Dr. Santini has the research facilities and expertise to mentor Sara in the laboratory while my role will focus more on helping her write up and present her findings when she returns from Australia. The proposed work would fill a knowledge gap in our understanding of how microbial communities differ with depth and as a function of climate and contribute data to a larger international effort to understand how life and rock interact in the Critical Zone.

I am confident that Sara has the skills to succeed in this project. She is self-motivated and driven by an intense curiosity to understand earth systems but is also detail oriented, careful and deliberate, all of which instill confidence in her ability to conduct high quality research. Sara is an excellent student and promising researcher. She is still new to research and geology, but has shown incredible ambition and a desire to learn. I am excited about her proposed work and willing to provide the mentoring and support to ensure she is successful in this endeavor. I thank you for considering her proposal and please do not hesitate to contact me if you have any further questions. Sincerely,

Ashlee Dere Assistant Professor of Geology Department of Geography/Geology University of Nebraska – Omaha Omaha, NE 68182 (402) 554 – 3317 adere@unomaha.edu

School of Geography, Planning and Environmental Management Level 4, Chamberlain Building (35) Campbell Drive St.Lucia

The University of Queensland Brisbane QLD 4072 Australia

T +61 7 3365-6455 F + 61 7 3365 6899

E gpem@uq.edu.au W www.gpem.uq.edu.au CRICOS PROVIDER NUMBER 00025B

School of Geography, Planning and Environmental Management Dr Talitha Santini, PhD, BSc (Hons), BA Senior Lecturer, Environmental Management CRICOS PROVIDER NUMBER 00025B

9 October 2016 To whom it may concern, RE: Support for FUSE grant application, Sara Parcher I would like to express my strong support for the research project proposed by Sara Parcher, supervised by Dr Ashlee Dere, to be completed within the School of Geography, Planning, and Environmental Management at the University of Queensland (UQ), Australia in 2017. This proposal has been developed through a research collaboration between myself and Dr Dere, for which we recently completed a week-long field trip along the Appalachian Mountains with four UNO students including Ms Parcher. I was impressed by Sara’s interest and knowledge of soil weathering and development during this trip, and I believe that Sara shows substantial promise as a researcher. Sara’s proposed work would fill in a crucial knowledge gap in our understanding of shale weathering across an international climate gradient, by providing the first insights into the composition and potential roles of microbial communities in controlling weathering rates. Sara would be travelling to my laboratory at UQ to acquire skills in microbial analysis and analyse recently collected samples plus another set to be collected from our Australian site (Main Range Critical Zone Observatory, south-east Queensland) during her visit. Our School will provide access to our field equipment, office, laboratory facilities as an in-kind contribution to this project. I will personally train and supervise Sara during her visit, and facilitate and assist with field work. Sara will also have the opportunity to network with other research students and staff across Schools and research Institutes at UQ including the School of Earth Sciences, the School of Agriculture and Food Sciences, the School of Chemical and Molecular Biosciences, the Centre for Microscopy and Microanalysis, and the Sustainable Minerals Institute. I would be delighted to host Sara Parcher’s research visit, and I look forward to building our fruitful research collaboration with the University of Nebraska at Omaha. Sincerely,

Talitha Santini Senior Lecturer (Associate Professor) School of Geography, Planning, and Environmental Management The University of Queensland