Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720
Performing Department
Environ Sci, Policy and Mgmt
Non Technical Summary
The proposed research addresses the current issue of identifying agricultural practices to bolster soil fertility. Arable soil is a finite and dwindling resource under increased demand for agricultural productivity as the global population increases and consumes higher-input foods. Meeting this challenge in the coming decades will require technologies and management strategies that both address loss of soil fertility to erosion and increase the productivity of agricultural soils. Polysaccharides are known to constitute a major component of natural soil organic carbon, and soil polysaccharides have been shown to improve soil aggregation leading to reduced erosion loss, as well as increase soil water holding capacity and nutrient availability to crops. Here we propose to use cutting edge approaches to study microbial ecology in order to identify organisms most contributing to soil polysaccharide production and their responses to varying soil moisture and sources of plant carbon.To accomplish these objectives, we propose to use stable-isotope labeling techniques to trace carbon from atmospheric CO2, through microbial communities in soil, and into the polysaccharides those microorganisms produce. We have completed an experiment in which plants were grown in an atmosphere with CO2 made up of a heavy isotope of carbon, 13C, which is distinguishable from the naturally-abundant form 12C. This enabled us to incorporate 13C carbon into plant biomass. We then incubated soil either with 13C-labeled root litter from, with living plants growing in a 13C atmosphere, or reciprocal combinations of both. Each of these plant-carbon treatments was also conducted at both high and low moisture levels. We propose to implement a technique known as metagenomic quantitative stable isotope probing (metagenomic qSIP) which enables us to reconstruct the genomes of organisms living in the soil and quantify how much carbon each organism used from the labeled source (13C-labeled living-root exudates, or 13C-labled root detritus). The structure of the experiment enables us additionally to measure how soil organisms that consume plant carbon respond to the source of carbon and to drought. Genomic reconstruction enables inference of the genomic capacity of these organisms to metabolize various substrates and produce various compounds, including extracellular polysaccharides. Polysaccharides were extracted from soil for all treatments. The research proposed under this fellowship includes the metagenomic sequencing of stable-isotope probing samples and the development of a computational pipeline to predict the structure of polysaccharides produced by a microorganism given its genome. The fellow will then apply this computational pipeline to predict polysaccharides produced by organisms in the experiment, and then use isotope-aware spectroscopic methods to quantify polysaccharides in soil produced by 13C-consuming microorganisms, thus enabling us to identify microorganisms responsible for increasing soil polysaccharides under varying conditions (plant-carbon source and moisture level).The ultimate goal of the proposed research is to generate tools for farmers to improve soil structure, water retention capacity, and fertility through microbial polysaccharide production. Biotechnology has primarily generated products farmers can use to combat agricultural pests. To date, the promises of molecular and cellular biology have not been brought to bear to improve the fertility of farmer's soils. Few tools exist for farmers to modulate soil properties in a way that is economical and scalable. This fellowship represents on attempt at applying modern technologies in biology--which have enabled advances in breeding and pest management--to the problem of managing soil productivity directly. One tangible outcome towards this objective is the nomination of soil microorganisms that can be developed into commercially available inoculants that farmers can apply to crops to modulate soil properties. This will require identifying microorganisms that contribute substantially to soil structure and water retention through polysaccharide production, and an understanding of what conditions influence the organisms' polysaccharide biosynthesis. These are the knowledge gaps that the proposed work seeks to fill. Additional outcomes will include at least two peer-reviewed scientific publications, including for the computational pipeline to predict polysaccharide structure, and for the quantification of population-specific polysaccharide production in the experiment described above, as well as the computational tool itself, which can enable discovery of novel polysaccharides with industrially-useful material properties.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
(N/A)
Goals / Objectives
The overarching goal of this project is to contribute towards development of agricultural practices and technologies for farmers to directly improve soil fertility. Our framework in this pursuit is that extracellular polysaccharides (EPS: complex sugar molecules produced by plants and microorganisms) improve soil structure and agronomic soil properties. The goals of the project in illuminating this process are 1.) Quantify contributions to soil polysaccharides of different populations of soil microbes. 2.) Characterize responses of EPS producing microbial populations to soil moisture and carbon source. 3.) Identify candidate taxa / strains for improving soil properties through (EPS) production. 4.) Contribute to mentoring future generations of agricultural scientists.Towards these goals, we have the following objectives:1.) Quantify contributions to soil polysaccharides of different populations of soil microbesa. Develop bioinformatic pipelines to predict EPS structures produced by an organismb. Culture EPS producing strainsc. Verify eps structuresd. Publish EPS prediction pipelinee. Predict EPS structures produced by organisms consuming plant carbon in previous experiment2.) Characterize response of EPS-producing microbial populations to soil moisture and carbon sourcea. Quantify plant-biomass consumption by soil microbesb. Predict EPS structures produced by soil microbesc. Quantify predicted EPS molecules in experimental samplesd. Multivariate analyses to determine effects of drought and source of plant carbon on EPS productione. Publish results of experimental quantification of population-specific EPS production in soil3.) Identify candidate taxa / strains for improving soil properties through EPS productiona. Secure additional funding for targeted culturing of microbial populations that contribute most soil EPS4.) Contribute to undergraduate mentorship and gain mentoring experiencea. Work with programs at UC Berkeley offering undergraduate research opportunities to students from backgrounds underrepresented in science to recruit traineesb. Student mentees will engage with proposed research primarily on objectives 1a and 1b
Project Methods
The overarching goals of the project are to identify soil microorganisms that can improve soil fertility by quantifying polysaccharides produced in soil by specific microbial populations in response to variable plant carbon inputs and drought. To accomplish this we will use cutting edge techniques in microbial ecology to trace plant carbon from live and dead roots through soil microbial communities and into specific polysaccharide molecules produced in soil under varied moisture conditions. Specifically, we have grown plants in a CO2 atmosphere of the stable heavy isotope of carbon, 13C, and extracted DNA from soil microbial communities associated with living roots, with root detritus, and with the two combined. We propose to determine the identity and genomic capacity of organisms consuming each source of plant carbon through metagenome quantitative stable isotope probing (qSIP), whereby genomes from 13C-consuming organisms are differentiated based on density. qSIP is a well-developed technique in microbial ecology, but applying metagenomics (whole-genome reconstruction from environmental DNA) to qSIP is a novel approach that the fellow has developed so far during his postdoctoral appointment. Metagenomic qSIP will enable prediction of polysaccharide structures from microbes consuming both living and dead plant material under varying moisture conditions. Polysaccharide structural predictions will be carried out by a bioinformatic pipeline which will be developed for this fellowship. We predict that microbial extracellular polysaccharide structures can be predicted from genomes because known microbial polysaccharides are synthesized by genes organized in operons (clusters of genes in the genome serving a related function), the main genetic components of which are conserved enough to identify in novel genomes. We propose to apply machine learning algorithms to extract genetic features that are important for determining polysaccharide structures in known systems in order to develop predictive rules for uncharacterized polysaccharides. Polysaccharide predictions will be validated by extracting polysaccharides from microbial cultures previously isolated from the experimental soils and solving polysaccharide structures through targeted digestion and HPLC, as well as liquid-state NMR. Polysaccharides were extracted from the same samples as DNA for metagenomic qSIP. Predicted polysaccharides from 13C-consuming microbial genomes will be quantified under each treatment by selecting enzymes that cleave expected glycosidic linkages from genomic predictions, and quantifying resulting monosaccharides and oligosaccharides and their isotopic composition (representing whether the constituent polysaccharide was derived from 13C-labled plant carbon or not in each treatment) through high-performance liquid chromatography (HPLC).These research efforts will result in a state change in knowledge of polysaccharide production in soil, and the identification of organisms that can play a role in boosting agricultural soil fertility through polysaccharide production by microbial inoculants. This scientific knowledge will be disseminated through publication of at least two manuscripts in peer reviewed journals (and submission to preprint servers), deposition of metagenomic data on relevant public databases (e.g. NCBI), and availability of computer code and bioinformatic pipelines on github. Additionally, the research will be presented at both scientific and agricultural industry-focused conferences.The success of the research will be evaluated primarily based on the timeline of manuscript submission for review and publication. A manuscript describing the polysaccharide prediction computational tools will be submitted for publication by the end of the first year of the fellowship. A manuscript describing the results of the 13C-labeling experiment will be submitted by the end of the second year. The fellow has a well-developed mentorship plan with both postdoctoral advisors, professors Mary Firestone and Jill Banfield, which includes regular meeting and presentation of new results.