Performing Department
(N/A)
Non Technical Summary
Current agricultural practices often lead to reductions in soil C content and soil health. Soil management strategies that aim to improve soil organic matter content use long-term practices such as no till and organic manure additions. There is a great need to improve soil management practices with the goal to increase soil C content: a benefit for farmers as well as for society by sequestering large amount of CO2 from the atmosphere.Modern techniques such as analyses of genomes and transcripts from microbes reveal a very complex behavior of microbes in soil communities. Since soil microbes are important for soil organic matter formation and degradation, we will try to manipulate microbes in intact soil communities to stimulate microbes that eat plant materials, but discourage microbes that eat other microbes. To differentiate between these groups of microbes, very precise and frequent measurements of microbes in soil communities are required. Using modern techniques, we are able, for the first time, to achieve detailed view of microbial activities at very high temporal resolutions. This information is foundational to developing new soil management systems.We will conduct experiments with soil from Texas cotton production systems. These soils have very low C contents and are extremely vulnerable for wind and water erosion. If we are able to improve soil organic matter content of these soils, we will reduce wind and water erosion, improve soil health, and stimulate cotton crop productivity.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
Our long-term research goal is to develop techniques and approaches to manage short-term microbial processes to improve long-term soil organic matter formation. We are specifically focused on how short-term microbial processes of primary and secondary consumers result in a net transfer of C to the soil organic matter pool or, as CO2, to the atmosphere. The research is focused on C-poor soils from cotton production systems in NW Texas. We have the following three research objectives:to study how soil moisture, dry-wet transitions, N addition, and litter quality affect microbial physiological processes and growth rates of primary and secondary consumers in response to an exudate or litter addition.to analyze how changes in activity of primary and secondary consumers affect C use efficiency, microbial turnover, soil organic matter formation and C retention.to test whether inoculation with cultures of primary consumers alters the balance between primary and secondary consumers and leads to increases in soil organic matter sequestration.
Project Methods
Two methods will be used to determine how microbes respond to changes in their environment. 1 - DNA and RNA extraction and shotgun sequencing: Shotgun sequencing of DNA and RNA extracted from the soil at several times during the incubation. We will extract DNA and RNA using established laboratory protocols. Nucleic acid concentrations will be quantified with a Qubit fluorometer and purity will be assessed with a NanoDrop photospectrometer. High-quality samples will be sent to a commercial sequencing facility for shotgun sequencing after ribo-depletion.Once shotgun sequencing is complete, sequencing reads will be quality-trimmed and adapters removed using Trimmomatic. Corresponding metagenomes and metatranscriptomes will be coassembled using MEGAHIT and reads mapped to contigs using Bowtie2. Contigs will be binned for prokaryotic metagenome-assembled genomes (MAGs) using Metabat2 and Concoct via a combination of subassembly and co-assembly methods, curated/dereplicated using DasTool, and completeness quantified using CheckM259. Remaining bins will be annotated for eukaryotic genes (BUSCO database), and completeness assessed using EukCC . Remaining contigs will be used for viral binning using Virsorter2 and CheckV. Viral and prokaryotic binned contigs will be annotated using DRAM/DRAMv and eukaryotic bins using NCBI-nr and KEGG databases. MAGs and metagenome annotations will be used to map metatranscriptomes for differential expression analyses.Metagenomes and metatranscriptomes will be normalized using Deseq2 and statistically analyzed with a likelihood ratio test and Wald test. Further statistical analyses include various multivariate analysis of variance methods (PERMANOVA, SIMPER, PCA test, etc) using R scripts. Transcripts for enzymes will be grouped into physiological functions using existing R scripts.2- Quantitative Stable Isotope probing: We will use qSIP to measure taxon-specific growth and death rates following published protocols. In short, microbial DNA is labeled during soil incubation in the presence of 13C-glucose or litter and compared to DNA from natural abundance glucose and litter incubations. DNA is then extracted, purified and density-separated using isopycnic centrifugation on a CsCl gradient. After centrifugation, ~10-14 fractions will be collected. By comparing the density (position on the density gradient) of the 13C-labeled DNA with that of the natural abundance DNA, a density shift can be calculated per taxon. Growth rates are calculated from the density shift. Information from abundance and density shift is then used to calculate taxon-specific growth rates and mortality. rRNA genes are sequenced on an Illumina MiSeq with 515f/806r (bacteria, archaea) and 1380F/1510R (V4 region; fungi and protists) primers. Gene sequences will be processed using QIIME software package. Generated data will be analyzed using Usearch and ASVs classified against SILVA, UNITE, and PR2 databases.Three techniques will be used to evaluate soil C losses and gains in soil organic matter. 1- Loss of (13)C from added litter: Respiration and CO2 isotope composition will be measured using an LI-6262 CO2 analyzer connected to a sample loop and a Picarro CO2/CH4 isotope spectrometer2- 13C incorporation into microbial biomass, Biochemical and C use efficiency, and microbial turnover: Soil (8g) will be extracted with 1 M KCl solution on melting ice, shaken and the extract filtered. Nitrate and ammonium concentrations will be analyzed on a SmartChem 200 discrete analyzer (Westco Scientific Instruments, Brookfield, CT, USA). Microbial biomass will be measured using an extraction-fumigation-extraction protocol. This protocol is optimal for soil with high soluble C and N concentrations. Soil (10 g) will be extracted with 0.05 M K2SO4 solution on melting ice, shaken and filtered. Chloroform (2 ml) will be added to remaining soil, and incubated for 24h. After chloroform evaporation (under vacuum), soil will be extracted with 0.05 M K2SO4 solution as before. The first extraction provides an estimate of the K2SO4-extractable organic C and N, while the second extraction will be used as a measurement of microbial biomass C (MBC) and N (MBN). Extracts will be dried at 60 °C in a ventilated drying oven, and, after grinding, combusted using an Elemental Analyzer and analyzed on an IRMS. The 13C incorporation into MBC combined with measurement of 13C respiration is used to calculate CUE. We will measure microbial turnover as the decline in CUE over time (24 and 72 h after glucose addition).We will use metabolic flux analysis and loss of position-specific isotope labels as CO2 as an indication of Biochemical Efficiency and a second measure of microbial turnover. The calculation of the Biochemical Efficiency is done using standardized and published methods in metabolic flux analysis. In short, we will add 2 ml of a 3.6 mmol L-1 position-specific isotopologues of glucose (1, 2, 3, 4, 5, 6, U-13C) to soil (20 g), and measure the position-specific 13CO2 production over one hour. The patterns of position-specific CO2 production are very sensitive to a change in efficiency and are used to calculate BE. For upland soils, C-positions 2,3,5,6 of glucose are incorporated into biomass. The microbial turnover is estimated as the CO2 release from C2, C3, C5, and C6 over time (24 to 72 h).3- 13C Content in Soil and Density Fractionation: Soil will be dried at 60°C in a ventilated drying oven. Dry soil will be ground to a fine powder and analyzed on an Elemental Analyzer connected to a Mass Spectrometer. For soil density fractionation, we will separate heavy and light soil fractions using sodium polytungstate and measure 13C incorporation in light and heavy mineral-bound SOM. We will measure %C and 13C using an elemental analyzer coupled to an isotope ratio mass spectrometer (IRMS) at the isotope lab at NAU.We will use one approach to isolation and culture of primary consumersWe will use culture and inoculation techniques to alter the balance between primary and secondary consumers and determine what the outcome is on soil organic matter production.Using general culture techniques, we will isolate cultivable species by extracting soil with a phospho-saline buffer and, using a dilution series, inoculate the extract on media in petri dishes. Three different media will used: 10% Tryptic Soy Agar and defined minimal media with glucose or plant litter as a sole C source. We will include antibiotics that target bacteria when isolating fungal strains. Pure cultures of bacteria and fungi will be identified through Sanger sequencing of 16S rRNA genes or ITS2 sequences. Strains will be identified as primary consumers by comparing the 16S rRNA or ITS2 gene sequences with those in our amplicon and metatranscriptome sequencing libraries from the first 24h after glucose or litter addition. Once cultures have been transferred to fresh media several times, they will be grown in a liquid medium, identical to the original growth medium with the omission of agar. The batch culture will be grown into the stationary phase, which will be documented through consistent optical density readings. The behavior of the strain, for example, sporulation or death, during the stationary phase will be recorded. Cells will be precipitated by centrifugation, washed and used as live or heat-killed inoculum.The inoculum (dead and live) will then be added to a control soil and soil with 13C labeled glucose or litter as a substrate (0.7 mg C g-1 soil).