Progress 07/01/24 to 06/30/25
Outputs
Target Audience:Our target audiences include farmers, landowners, the general public, extension professionals, researchers, foresters, government organizations, and non-government organizations. As you can see from our other products section, our extension efforts target a broad range of individuals. We target farmers and landowners in an effort to educate those directly able to implement woodchip bioreactors on the land they farm or own. We target extension professionals, government organizations, and non-government organizations to increase knowledge the woodchip bioreactor conservation practice and to encourage adoption when interacting with farmers and landowners who express interest in implementing a conservation practices. We engage foresters to share opportunities for forest management and potential income by using wood products in woodchip bioreactors. Finally, we engage the research community to disseminate research findings and to advance nitrate removal performance of woodchip bioreactors. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Graduate students and undergraduate students have been involved in the project. The project team trained the students in various aspects including research methodologies, technical writing, critical thinking, and communication skills. The students also attended regular project meetings which exposed them to an interdisciplinary and collaborative working environment, participated in fieldwork and sampling collections, and engaged in workshops, such as the Rapid Needs Assessment and Response (RNR) workshop, organized by the research team. Furthermore, the research staff assisted the graduate student in drafting the research protocols and standard operating procedures (SOPs) for each method used in the characterization of wood species. The project team trained the graduate student in performing the cold-water solubility extraction used for analyzing the wood metabolites; in carrying out the colorimetric Folin-Ciocalteau assay; and data analysis using R and relevant statistical methods for correlation studies. The students were given the opportunity to present the project updates during project meetings, and the project team gave feedback and suggestions for the improvement of the study. The mentoring provided by the project team will help enhance the students' skills for the continuation of the project and further research dissemination to the public. How have the results been disseminated to communities of interest?Dr. Billy Beck presented a webinar for the Iowa Learning Farms Conservation Webinar Series on November 20, 2024 titled "Trees and Tiles: 4 Years of Nitrate Removal and Root Intrusion Monitoring in a Saturated Riparian FOREST Buffer" and 195 participants joined the webinar. Nine press releases/articles were published, along with three blogs and a feature in the October Iowa Learning Farms e-news. The Iowa Learning Farms Conservation Station On The Edge made two appearances: August 19, 2024, Conservation Districts of Iowa Annual Conference with 50 participants and November 7, 2024, Leadership Iowa with 52 participants. What do you plan to do during the next reporting period to accomplish the goals?Objective 1: The next step is to perform denitrification experiments - to identify microbial communities present in woodchip bioreactors using specific wood species and to characterize genes and enzymes involved in denitrification. Apart from the carbon availability and NO3-N removal rate, the microbiome in each woodchip bioreactor will be characterized to determine the link of denitrifying communities to carbon substrate and its influence on bioreactor performance. Woodchips for the denitrification set-up will be collected this Summer 2025 (from May to July 2025). The labile carbon availability and NO3-N removal of woodchip species will be done using a beaker-scale batch bioreactor in triplicates (12 wood species x 3 replicates = 36 bioreactors). The batch reactors used for carbon availability and NO3-N removal rates will also be used for microbial analysis. Microbial characterization will be performed on woodchips after one week of incubation in synthetic tile drainage water. DNA will be extracted from both woodchips and water samples using commercially available DNA extraction kits. To identify the woodchip microbiome, genomic DNA extracts will be sequenced for 16s rRNA gene amplicon sequencing, and to characterize genes involved in denitrification, a high-throughput quantitative polymerase chain reaction (qPCR) will be used to quantify genes in the extracted DNA related to nitrate reduction (narG and napA), nitrite reduction (nirS and nirK) and nitrous oxide reduction (nosZ), using specific primer sets previously designed. With this, the microbial communities from each wood species from the batch reactor set-up will be characterized, as well as their interaction with woodchip nutrient sources such as carbon and nitrogen, for assessing denitrification efficiency. Objective 2:Two wood species based on NO3-N removal efficiency will be selected to study the impacts of blending with extracts from twelve other wood species. The impacts of blending these two woodchips with woodchips from other wood species will also be assessed. Water and woodchip blend samples from each batch bioreactor will be collected to quantify tannic acid, labile carbon, microbial communities, and NO3-N concentrations using preservation and analysis methods described in Objective 1. The statistical differences in tannic acids, labile carbon concentrations, microbial communities, and NO3-N removal between respective treatments and controls will be determined. Furthermore, the impact of lignin/cellulose and tannic acid contents on labile carbon, microbial communities, and NO3-N removal will be assessed. Overall, the aim is to assess and document the effectiveness of using high NO3-N removal woodchips to promote lignin/cellulose degradation and NO3-N removal of low-performing but perhaps readily and locally available wood species. From this experiment, five woodchip blends will be identified and further characterized in simulated bioreactors in Objective 3. Objective 4:A second RNR is being planned for July 10th near Edgewood, Iowa, for forestry professionals. Additional RNR workshops related to this project and supported by other awards will be held in the coming year. Farmers and forestry leaders will be gathered from across Iowa to participate in a Leadership Circle in Ames on February 3, 2026. Development of the On-Farm Forestry Field Guide for Iowa is planned to begin in July 2025, starting with content development and moving to graphic design and layout in late 2025 to be piloted at the Leadership Circle. The regional forestry workshops will be held during years 3-5 of the project and the training workshops for high school instructors is planned for years 4-5. Additional Forestry On Tap outreach events, webinars and outreach will continue throughout the project.
Impacts
What was accomplished under these goals?
The wood properties and extractives may affect the species' denitrification performance; thus, it is vital to understand the impacts of wood extractives on bioreactor performance by first characterizing the single wood species. The physical, chemical, and biological characteristics of twelve wood species previously selected based on abundance, availability, and vessel distribution were investigated. The wood species were Black Maple (Acer nigrum; vessel distribution - diffuse porous), Shag Bark Hickory (Carya ovata; ring porous), Hackberry (Celtis occidentalis L.; ring porous), Cottonwood (Populus deltoides; diffuse porous), Black Walnut (Juglans nigra; semi-ring porous), Black Cherry (Prunus serotina; diffuse porous), Green Ash (Fraxinus pennsylvanica; ring porous), White Oak (Quercus alba; ring porous), and Red Oak (Quercus rubra; ring porous), White Mulberry (Morus alba; ring porous), Honey Locust (Gleditsia triacanthos; ring porous), and Basswood (Tilia americana; diffuse porous). Tree cores of each wood species (three biological replicates per wood species) were collected and used for physicochemical characterization (e.g. % solubility, pH, moisture content), cold-water solubility extraction of wood extractives, total phenolics analysis using the Folin-Ciocalteau assay, and metabolite analysis using Gas Chromatography-Mass Spectrophotometry (GC-MS) and Liquid Chromatography-Mass Spectrophotometry (LC-MS) of free carbohydrates and non-targeted detection, and High-Performance Liquid Chromatography (HPLC) analysis for tannins, particularly gallic acid and ellagic acid. The initial hypothesis is that tannic acid inhibits microbial processes such as denitrification, thus wood species with high tannic acid content is expected to have lower denitrification efficiency. In addition, bioreactors have varying concentrations for carbon and nitrogen availability depending on the woodchip species used and this may affect the microbial communities present depending on the ways to access C and N under C-limiting or N-limiting conditions. To confirm this claim, carbon/carbohydrate contents, biopolymers (lignin/cellulose), and tannins in the wood extracts must be measured to determine their impacts on bioreactor microbiomes and nitrate removal. Currently, wood extractions from nine out of twelve wood species (Black Maple, Hickory, Hackberry, Cottonwood, Black Walnut, Black Cherry, Green Ash, White Oak, and Red Oak) were performed, while the extractions of the remaining three wood species (White Mulberry, Honey Locust, and Basswood) are still in the process. The first part of this section aimed to confirm if the measurements and specific parameters (total phenolics, gallic acid and ellagic acid contents, free carbohydrates, polyphenols, secondary metabolites, etc.) can differentiate the various wood species and if the biological replicates are significantly different (or similar) from each other in terms of a specific parameter. Based on the initial results, the total phenolics, gallic acid, and ellagic acid concentrations significantly differ among wood species. High total phenolics contents were measured in Black Walnut (11.095 to 13.88 mg/L), Red Oak (7.67 to 10.40 mg/L), White Oak (4.18 to 9.33 mg/L), and Black Cherry (6.10 to 9.08 mg/L), while low phenolics were measured in Cottonwood (0.36 to 0.54 mg/L), Hackberry (0.65 to 0.82 mg/L), and Hickory (1.42 to 1.56 mg/L). This is consistent with the gallic acid concentrations, where Black Walnut (99.83 to 168.98 µg/mL for total gallic acid-based; and 12.72 to 16.07 µg/mL for gallic acid content), Red Oak (19.21 to 21.02 µg/mL) and White Oak (1.86 to 9.02 µg/mL) measured the highest values. In contrast, the remaining seven wood species had lower gallic acid contents. In terms of ellagic acid content, Black Walnut is the only wood species with high concentrations, ranging from 6.82 to 10.18 µg/mL, while the rest of the wood species had lower concentrations from 0.00 to 0.59 µg/mL. However, some parameters, such as pH and % solubilities were unable to significantly differentiate all wood species. However, there were some species with distinctly high or low values varying from other species, such as in the case of Red Oak and White Oak with acidic pH values, compared to other species within the neutral pH range. Moreover, clustering and hierarchical measurements were done for the free carbohydrates, organic acids, polyphenols, and secondary metabolites, to identify which species are correlated with each other. Based on the available GC-MS data, Hickory and Black Maple had the highest measurements in terms of carbohydrates, organic acids, and polyphenols. These two species clustered together indicating relatedness between them and showed more variation in comparison to the other wood species. Meanwhile Black Walnut and White Oak had the highest concentrations for phenolics content, which is in conjunction with the total phenolics assay and HPLC results. Interestingly, Cottonwood species showed consistent results having low polyphenols, secondary metabolites, and free carbohydrates. Overall, the current results from this objective demonstrate that the wood species varied in terms of some physical characteristics and wood extractive parameters. These measurements can be used to differentiate the various wood species from each other and identify correlations with other species. Further analysis will still be done, particularly the microbiome characterization, and the effect of the wood extractives on NO3-N removal and bioreactor efficiency. The first Rapid Needs Assessment and Response (RNR) workshop was held on February 18, 2025, near Webster City, Iowa, with 27 farmer and landowner participants. Three Forestry On Tap events were held this reporting period: August 6, 2024, in Clear Lake with 30 participants, November 21, 2024, in Osage with 35 participants, and February 20, 2025, in Indianola with 76 participants. Dr. Billy Beck presented a webinar for the Iowa Learning Farms Conservation Webinar Series on November 20, 2024 titled "Trees and Tiles: 4 Years of Nitrate Removal and Root Intrusion Monitoring in a Saturated Riparian FOREST Buffer" and 195 participants joined the webinar. Nine press releases/articles were published, along with three blogs and a feature in the October Iowa Learning Farms e-news. The Iowa Learning Farms Conservation Station On The Edge made two appearances: August 19, 2024, Conservation Districts of Iowa Annual Conference with 50 participants and November 7, 2024, Leadership Iowa with 52 participants.
Publications
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