Improving soil health using beneficial microbiomes in urban agriculture

Progress report for GNE21-269

Project Type: Graduate Student
Funds awarded in 2021: $14,991.00
Projected End Date: 11/30/2023
Grant Recipient: Cornell University
Region: Northeast
State: New York
Graduate Student:
Faculty Advisor:
Jenny Kao-Kniffin
Cornell University
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Project Information

Project Objectives:

            The overall aim of this research is to improve the physical and biological health of soils in urban tomato farms by increasing soil aggregation, accumulation of SOC, and P cycling. The specific research objectives include:

 

            1) To discover how different combinations of AMF and PSB impact the production of the soil-aggregating glomalin-related soil protein (GRSP), SOC accumulation, different species of P in soils, and P uptake by tomatoes, consequently impacting the growth and yield of tomatoes in urban agriculture.

 

            Reasoning: 

            This objective is designed to measure how AMF and PSB contribute to SOC accumulation, soil P cycling, and plant P uptake and growth. AMF are known to increase SOC accumulation by producing a sticky and hydrophobic glue-like glycoprotein, GRSP, which can bind adjacent small-sized aggregates and enlarge them. Hypothetically, increased organic carbon (C) in soils becomes an energy source for PSB to grow, thereby facilitating P cycling and P supply for plant uptake22. The growth and yield of tomatoes can also be affected by increased SOC and P availability in soils.

 

            2) To identify the genotype and phylogeny of soil microbiota that are abundant within different sizes of soil aggregates in the rhizosphere of tomatoes in urban agriculture. 

 

            Reasoning:

            At the spatial level, soils are composed of different sizes of aggregates: microaggregates (< 0.25 mm) which helps protect soil C from erosion and macroaggregates (0.25 to 2 mm) which limit the influx of water and oxygen into soil particles. Different microbial communities can inhabit two different soil matrices, influencing soil metabolic functions and structural stability within aggregates.  However, little is known about the biological and genetic characteristics of different sizes of aggregates within compost-type soils used in urban agriculture.  Thus, this objective will allow us to compare the genotypes of the abundant microbial residents between microaggregates and macroaggregates and reveal their biological potential for establishing aggregate structure.

 

            3) To compare microbial community compositions and metabolic functions using integrated omics studies on the soils of urban farms growing tomatoes under the treatment of AMF and PSB.

 

            Reasoning:

            This objective aims to elucidate how AMF and PSB treatments affect soil biochemical metabolisms and soil nutrient enrichment by examining the protein expressions for N, C, and P cycling in urban agricultural soils. By integrating shotgun metagenomics with metaproteomics26, we can identify how the inoculation of AMF and PSB creates a shift in soil protein expressions in relation to SOC accumulation and soil nutrient metabolisms, and how this can relate to tomatoes’ nutrient uptake and growth27,28.

Introduction:

            The purpose of this project is to tackle chronic issues in urban agriculture such as poor soil structure and low nutrient availability using microbial resources to help produce higher economic returns to urban farmers. It is expected that more than 60% of the world’s population will inhabit urban areas by 20301. Urban agriculture holds great promise to feed such rising urban populations; in fact, urban agriculture is meeting from 2% to 150% of vegetables and fruit demands in urban areas2. Urban agriculture is also critical for food security by providing cheaper and more nutritional foods to the urban poor.

            However, many urban farmers face challenges in generating high yields and profits, because their soils lack good physical structure2. They grow crops in raised beds using Technosol soils, which are comprised of mixtures of natural soil, compost, and soil-less media (e.g., perlite). These constructed raised beds often fail to develop sufficient soil aggregates where soil organic carbon (SOC) accumulates as aggregated structures that act as a reservoir for nutrients critical for plant uptake3,4. Lacking SOC results in low soil fertility with low yields in urban agriculture, driving urban farmers frequently to add composts or fertilizers in an attempt to maximize their yields. Nonetheless, their attempts quite often lead to over-supply of nutrients in the form of highly mobile nitrogen (N) and phosphorus (P), which are subject to losses through waterways contributing to eutrophication5. Salomon et al.6 surveyed 12 urban farms and found that their soils had P greatly exceeding the recommended level for horticultural production, concluding that improper nutrient management in urban agriculture could hamper plant growth and increase environmental risks.

           In this proposal, we describe a study to enhance SOC accumulation via soil aggregation and biological P cycling using two soil microorganisms: arbuscular mycorrhizal fungi (AMF) and polyphosphate solubilizing bacteria (PSB) to improve plant growth in urban soils that minimize environmental pollution. AMF can help develop soil structure by increasing SOC stocks7, while PSB could assist in soil P enrichment8. Both soil parameters are important to maximize crop yields and farm net income3, so AMF and PSB would promote the overall economic stability for urban farmers. Moreover, increasing nutrient cycling and soil structure would result in a reduction of nutrient loading to stormwater via runoff which will enhance environmental sustainability in urban agriculture2,5. Our research is novel and will advance knowledge in agricultural use of microbes in urban agriculture and the broader field crops systems.

           Our work will also demonstrate the possible deployment of PSB as P biofertilizers. PSB are commercially available and affordable beneficial bacteria that can be easily used by farmers. P fertilizers derived from nonrenewable sources are rapidly decreasing and are predicted to be exhausted within 50‒100 years, therefore new approaches to P recovery and re-use are urgently needed to sustain a rise in the world population9,10. We will address this need by examining the use of PSB as biological enhancers that can reduce the overdependence on limited reserves of rock phosphate-based P fertilizers and improve agricultural sustainability.

Cooperators

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Research

Materials and methods:

 

Figure 1. An overview of the experimental design and research workflow.

 

            Our research will be based on three urban farms in New York City (NYC) that grow tomatoes in raised bed soils. The research will have four different microbial treatments—a control that does not contain either PSB or AMF; AMF only; PSB only; and AMF + PSB (Figure 1). I will use commercial microbial inoculums because they are affordable and easily accessible to farmers. A commercial AM fungal product (DYNOMYCO®) will be applied to the soils as inoculums of Glomus intraradices and Glomus mosseae. Another commercial product, Mammoth P, will be used as a source of PSB which include Enterobacter cloacae, Citrobacter freundii, Pseudomonas putida, and Comamonas testosteroni. Each treatment will be applied to a pot that contains different soil media that each farm has created, and each treatment will have five replications per farm (4 × 5 = 20 separate 18-inch pots will be used). Prior to growing tomatoes, individual soils will be collected and submitted to Cornell Soil Health Laboratory for extensive soil analysis such as soil pH, organic matter, labile P, potassium, micronutrients, soil texture, and SOC. Tomatoes will be planted in late spring and grown for three months (April 2022 ‒ June 2022). Root and shoot will be harvested at full maturity. The soil samples will be collected at 20 cm depth, and stored in three different ways; 1) dried at room temperature for SOC quantification; 2) stored at 4 ℃ for GRSP extraction, soil P analysis, and flow cytometry; 3) stored at ‒20℃ for DNA extraction and metaproteomics.

 

Objective 1: To discover interactions between AMF and PSB for GRSP and SOC production, soil P cycling, and the growth and development of tomatoes.

 

            In order to achieve this objective, we plan to investigate multifaceted aspects of SOC and P interplay between AMF and PSB via GRSP production. This aspect of the research will address our main question: whether a combination of AMF and PSB will produce the largest amount of GRSP, organic C, and P in soils compared to individual treatments of AMF, PSB, or neither. Soil nutrients such as N, P, and SOC will be measured and correlated with Tomato’s biometry parameters to show how the soil nutrients impact the growth and development of tomatoes.

 

1) GRSP extraction and UV spectroscopy

 

            Response variable: Concentration of easily extractable GRSP (EE-GRSP) and difficultly extractable GRSP (DE-GRSP)

            Replication number: Five replicates per AMF+PSB, PSB-only, AMF-only, control

 

            The two fractions of GRSP (easily extractable GRSP (EE-GRSP) and difficultly extractable GRSP (DE-GRSP)) will be extracted from the soil using autoclaving with citric acid in five replications per treatment27. EE-GRSP is a labile fraction of GRSP, which is chemically unstable so easily accessible to microbial consumption. DE-GRSP is a recalcitrant fraction of GRSP, sustaining aggregate structure and contributing to the accumulation of SOC7. Extracted GRSP will be quantified with a modified Lowry assay using a Total Protein Kit, Micro Lowry, Onishi & Barr Modification (Sigma-Aldrich, USA) and spectrophotometry at 590 nm27.

 

2) Soil chemical analysis

 

            Response variable: SOC, organic matter, N, P, Ca, Mg, K, and Na

            Replication number: Five replicates per AMF+PSB, PSB-only, AMF-only, control

 

            Collected dried soil samples will be ground to < 250 μm particle size. SOC will be measured after removing inorganic C, such as carbonates, using a vacuum desiccator along with a 150 ml beaker with 100 ml of 12M HCl for 24 hours28. This HCl fumigation is effective as a pretreatment because no water-soluble C will be lost from the soil during removal of inorganic C; otherwise lost in the HCl dissolution method29. Then, soils will be completely oxidized using high-temperature combustion (1100°C) at Cornell Nutrient Analysis Laboratory, producing CO2 gases which will be calibrated to SOC content. Other nutrients such as N, P, Ca, Mg, K, and Na in soils, as well as soil organic matter, will be measured at Cornell Nutrient Analysis Laboratory.

 

3) Plant tissue analysis and biometry measurements

 

            Response variable: The cellular nutrients in tomatoes’ root cells and tomatoes’ biometry data

            Replication number: Five replicates per AMF+PSB, PSB-only, AMF-only, control

 

            Plant root samples will be air-dried and ground, and then submitted to Cornell Nutrient Analysis Laboratory for the element analysis for N, P, Ca, Mg, K, and Na. Biometry data of tomatoes, such as root and shoot height, shoot dry weight, and fruit dry biomass, will be measured in 5 replications in all different treatments.

 

Objective 2: The genotype and phylogeny of  microbiota abundantly present in different sizes of aggregates in the rhizosphere of tomatoes

 

            Little is known about bacterial communities inhabiting different sizes of aggregates and associating with AMF. Our research will be the first to discover the identity genetic and biological functions of two groups of microbiota associated with microaggregates and macroaggregates that inhabit the soils of tomatoes in urban agriculture and interact with AMF.

 

1) Flow cytometry for quantifying abundance of soil microorganisms

 

            Response variable: Relative abundance of bacterial cells in soils

            Replication number: Five replicates per AMF+PSB, PSB-only, AMF-only, control

 

            Flow cytometry (FCM) will be used to compare physical characteristics and relative abundances of diverse species of PSB in different treatments. For FCM analysis, soil samples will be suspended in phosphate-buffered saline (PBS) according to established protocols for soils31. Total PSB will be quantified in all conditions using flow cytometry using a dual staining method (using SYBR Green II and Tc). The quantification of total cells will be performed using a BD LSR II available in the Institute of Biotechnology at Cornell University. Excitation at 488 nm will generate forward scatter and side scatter of each cell, which will be used to characterize different phenotypes of PSB and calculate relative abundance. All cytometric measurements will be performed in five replicates.

 

2) Soil fractionation and 16S rRNA sequencing

            The collected soils will be prepared for soil aggregate fractionation using dry-sieving to minimize disturbance on microbial communities and their activities from wet sieving procedure. Soils will be dried to approximately 10% gravimetric water content at 4°C and then sieved and separated into 0.25-2 mm macroaggregates and 0.053-0.25 mm microaggregates. DNA from each aggregate will be extracted from frozen soil samples using the DNeasy PowerSoil Pro Kit (Qiagen N.V., Hilden, Germany. The DNA samples of different sizes of aggregates will be processed and submitted for 16S rRNA Illumina sequencing to the Cornell Life Sciences Sequencing Core.

 

Objective 3: Shotgun metagenomics and metaproteomics analysis of the soil microbiome

 

            We aim to discover how the treatment of AMF and PSB alters compositions of soil bacterial communities and how this change affects soil microbiome functions.

 

1) Shotgun metagenomics

 

            Response variable: Metagenome data

            Replication number: Four replicates per AMF+PSB and control

 

            We will perform shotgun metagenomic sequencing (i) to identify signals of specificity in the taxonomic and functional composition of the soil microbiota and (ii) to generate a reference library for the metaproteomic analysis. We will generate metagenome-assembled genomes (MAGs) to assess the functional potential of the soil microbiota and to generate a protein sequence database for comparative metaproteomics across the two different treatments (AMF+PSB and control) from using methods that Dr. Kleiner (our collaborator) has successfully employed previously34. In order to sequence these samples deeply enough for assembly and binning of MAGs, we will sequence these eight samples to a total read depth of 150bp paired-end reads (one full Illumina NovaSeq S4 flow cell). We will assess read quality withFastQC and trim them using BBdduk to remove adapter sequences and low-quality regions. We will assemble reads from individual or combined samples, comparing the output of the three metagenomic assemblers IDBA-UD35, MEGAHIT36and metaSPAdes37. We will determine read coverage profiles for all contigs >2 KB across all 8 metagenomic samples using BBmap38and use an ensemble of binning tools (ABAWCA, ABAWACA2, MaxBin2, CONCOCT, and MetaBAT) to generate MAGs. We will assess the completeness and contamination of our MAGs using CheckM39. For the purpose of generating a reference database for metaproteomic analysis, we will retain all bins with <10% contamination, and also include unbinned scaffolds, provided they contain a reliable phylogenetic marker gene34.

 

2) Metaproteomics

 

            Response variable: Soil metaproteome data

            Replication number: Four replicates per AMF+PSB and control

 

            For metaproteomics, we will prepare protein extracts from the soils of AMF+PSB and control groups in four replications, followed by peptide preparation for liquid chromatography (LC) mass spectrometry (MS)/MS analyses following the filter-aided sample preparation protocol40. We will use one dimensional (1D) or two dimensional (2D) LC and analysis of eluting peptides in a QExactive HF mass spectrometer as Dr. Kleiner has done previously41. Mass spectrometric raw data will be processed for protein identification using the ProteomeDiscoverer software (Thermo Scientific) or MaxQuan42. Differentially abundant proteins will be identified by using t-tests that are corrected for multiple comparisons with permutation based q-values and false discovery rates (5% cutoff) using the Perseus Platform43. We will use a protein sequence database derived from our MAGs and unbinned contigs for peptide and protein identification.

 

 

Statistical analysis

 

            Statistical analysis will include one-way ANOVA (difference between different combinations of PSB and AMF), correlations (the relationship between soil P, GRSP, SOC, other soil nutrients, and plant biometry data), and non-metric multidimensional scaling (NMDS) and other ordination tools to show variations in the bacterial metagenome and soil proteome. Heatmaps will be used to visualize and compare protein expression at taxonomic and functional levels between AMF+PSB and control treatment.

Participation Summary

Education & Outreach Activities and Participation Summary

Participation Summary:

Education/outreach description:

            The outreach plan for this research has three components: 1) working closely with farm staff and youth interns, supporting youth in sharing research results and applications with local NYC farmers and networks, 2) sharing findings and applications with regional farmer and Extension networks, and 3) sharing detailed analytical results with national and international academic audiences.

 

            This research will build upon and extend many of the goals the large Cornell Agricultural Soils of the City project (https://blogs.cornell.edu/nyurbansoils/) to better understand and support the sustainability and productivity of urban agriculture. Specifically, we will work with three NYC farms that have been part of this project: East New York Farms! (ENYF), BK ROT, and Red Hook Farms. Each of these farms facilitates dynamic youth farmer training and internships, and the youth and staff at each site will be instrumental in implementing the research. Youth and farm staff are being consulted in the planning of the research and will help establish plots, tend to crops, assist with sampling and harvest, and will be engaged in a series of iterative dialogues interpreting the data. Youth will be supported in co-presenting findings at local conferences, namely GreenThumb’s GrowTogether, Brooklyn Botanic Garden’s Making Brooklyn Bloom, and The Urban Farmer to Farmer Summit. These events are widely attended by NYC farmers and organizations and will be ideal for encouraging adoption of AMF and PSB applications.

            Regional urban agriculture endeavors are also expanding, and this research will be shared with these audiences at conferences including the annual meeting of the Northeast Organic Farming Association, as well as Cornell Cooperative Extension’s Ag-In Service Conference. Collaborating youth will also be encouraged to co-present at these conferences, to support their agriculture-, STEM- and career-skills, as well as to ensure that the findings are presented in comprehensible ways. We anticipate that urban farmers and organizations in other cities will not only be interested in the impacts of AMF and PSB, but will also be interested in enhancing soil structure, SOC, yields, and profits. We will write a series of relevant Extension documents that can be shared with these networks.

            Finally, the results of this research will be submitted to peer-reviewed journals such as Frontiers in Ecology and the Environment. The results presented in this manuscript will be addressed towards academic researchers, who we anticipate will be interested in the novel approaches that we use both in terms of investigating PSB in agriculture and in the range of analytical methodologies that we employ. These results will also be shared at academic conferences including the annual meeting of the Soil Science Society of America as well as the international Soils of Urban Industrial, Traffic, and Mining Areas conference.  

Project Outcomes

Knowledge Gained:

I have been working on a pilot study using polyphosphate accumulating organisms (PAOs) and arbuscular mycorrhizal fungi (AMF) via a growth chamber study that grew sorghums. I found a very interesting result which waits for publication. However, we realized that PAOs may not be suitable inocula to be easily used by farmers. It is because PAOs need some activation process to trigger their metabolism for polyphosphate accumulation which includes several hours of repetitive anoxic and oxic conditions. We believe most farmers will not process within their usual farm setting and we are afraid that the research outcome would not be helpful for farmers to use it actively. As the SARE grant wants more practice-focused research, my supervisor and I decided to use commercially available and affordable beneficial bacteria (phosphate solubilizing bacteria:PSB) instead of non-commercially available poly-P bacteria, PAOs, because it would result in more practical outcome instead of simply research that only leads to a publication. There is little known about a combination of PSB and AMF in a compost type of urban soils, and little study is available for their biological and functional properties in terms of soil aggregation within urban soils. Thus, we believe using PSB instead of PAOs will not only provide a novel discovery, such as their roles in establishing soil structure in urban farms in association with AMF, but also produce more practical outcomes considering the affordability and accessibility of commercially available microbial products to farmers.

 

Any opinions, findings, conclusions, or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the view of the U.S. Department of Agriculture or SARE.