DOI: 10.5061/dryad.kprr4xhh8
title: Biochar amendments in a California salt marsh restoration reduced denitrification and supported distinct microbial community functions

description: Accelerated sea level rise, combined with human-induced changes to
 hydrology and sediment transport pathways poses an existential threat to
 the survival of coastal wetlands over the next century. To support the
 preservation of wetlands, restoration and enhancement strategies—such as
 adding sediment to marsh surfaces to boost their resilience to sea level
 rise—are being actively tested and monitored. This study aimed to assess
 how biochar derived from Eucalyptus spp. feedstock influences sediment
 properties, microbial communities, functional potential, and nitrate
 reduction pathways in a tidal wetland restoration project. Biochar-amended
 sediments showed reduced denitrification rates, no detectable DNRA
 activity, and only subtle changes in sediment properties and microbial
 nitrogen cycling, with the exception of increased carbon content and
 higher relative abundance of Cyanobacteria and Truepera, a member of the
 Deinococcota.  While there was some evidence of increased
 microbial abundance, biochar did not enhance nitrogen reduction as
 hypothesized, and in some cases appeared to reduce microbial diversity.
 While biochar additions may benefit the restored wetland by increasing
 soil organic content to enhance microbial activity, and contributing to
 carbon sequestration through direct carbon burial, our results highlight
 the importance of different short-term vs. longer-term impacts to
 function, and the value of a priori testing of biochar to determine if the
 type of biochar chosen will chemically react with sediments as intended,
 before large-scale incorporation into the restoration project.
description: Research was conducted at Elkhorn Slough (36.8129°N, -121.7556°),
 a 12,000-ha estuary tidal estuary in Central California, which was the
 site of an ambitious 24-ha sediment placement and salt marsh restoration
 project completed in 2018 (Fig. 1) (termed "Hester Marsh"; Fig.
 1). During 2017-8, 180,000 m<sup>3</sup> of sediment was used
 to rebuild a low elevation marsh plain, which was diked and drained in the
 mid-20th century. Sediments included material from Pajaro River
 floodplains along with upland soils from a nearby regraded hillside. Key
 restoration goals were to recover lost ecosystem functions such as carbon
 storage, nutrient sequestration, and denitrification.
 Biochar sediment amendments (10% v/v) were incorporated in 2019
 in ten sets of paired plots across the mid-marsh platform. Biochar plots
 (1 x 1 m) received 5000 cm<sup>3</sup> of <em>Eucalyptus
 </em>spp. biochar mixed into the 5-10 cm sediment layer, capped with
 5cm of untreated sediment to prevent washout (Fig. 1). Paired (1 x 1 m)
 plots were disturbed in a similar manner. Biochar was produced on site by
 “top-down” pyrolysis of <em>Eucalyptus</em> spp. wood stock,
 in which the wood material was ignited at the top of the pyre to produce
 high temperatures that result in an anoxic center as the fire burns
 downward. Biochar was crushed and sorted, and the &lt;1.5 cm particle
 diameter was utilized. A nearby natural marsh ("Yampah Marsh")
 was chosen as a reference marsh due to its spatial proximity to the
 restoration site. In October 2021, 160
 cm<sup>3</sup> sediments samples (four composited 40
 cm<sup>3</sup> samples from 5-10 cm depth) were collected from
 biochar-amended (<em>n</em>=10) paired, unamended restoration
 plots (<em>n</em>=10), and from the nearby natural marsh
 (<em>n</em>=5; Fig. 1). We also composited fresh biochar and
 unamended restoration sediments (<em>n</em>=5). Immediately
 upon return to the lab, natural marsh, and restoration sediments were
 subsampled for DNA extraction and frozen at -80 °C. Samples were
 immediately homogenized, and processed for denitrification and DNRA
 potential activity. The remaining sediment was kept at 4°C prior to
 physicochemical characterization. To evaluate potential
 denitrification and DNRA rates, slurry incubations with
 <sup>15</sup>N tracer were conducted on 15 restoration soils
 (five unamended restoration samples, five freshly amended biochar samples,
 five aged biochar amended samples), and five samples collected from the
 nearby natural marsh. Subsamples of field-wet sediment (2.5 ± 0.5 g) were
 mixed with 10 mL artificial seawater  purged with argon to remove oxygen,
 along with glass bead to assist in homogenization. Slurries were mixed in
 four replicate 12 mL borosilicate Exetainer vials (EA Consumables, LLC,
 Marlton, NJ), to conduct paired time point measures immediately after
 injection of the tracer and 18 hours (times <em>T0</em> and
 <em>T18</em>, respectively) of incubation for both
 denitrification and DNRA potential (two for denitrification; two for
 DNRA). Vials were sealed without headspace and vortexed for 3 minutes.
 Slurries were pre-incubated in the dark at ambient temperature overnight
 to remove background
 <sup>14</sup>NO<sub>X</sub><sup>-
 </sup>and to ensure samples were anoxic. After pre-incubation, vials
 were injected with 100 µM Ar-purged
 Na<sup>15</sup>NO<sub>3</sub> solution (98%,
 Cambridge Isotope Laboratories, Inc., Andover, MA). To halt microbial
 activity, slurries were injected with 200 µL Ar-purged ZnCl<sub>2
 </sub>solution on ice: half of the tubes were sacrificed at time 0,
 and half after 18 hours of incubation in the dark. Vials were subsequently
 stored at 4 °C submerged in artificial seawater until analysis. Dissolved
 gases (<sup>30</sup>N<sub>2</sub> and
 <sup>29</sup>N<sub>2</sub>) were measured on a
 quadrupole MIMS with an inline furnace at the Tropical Research and
 Education Center, University of Florida. Potential
 denitrification rates for each sediment type and treatment were calculated
 following the isotope pairing technique (Nielsen, 1992). Concentrations of
 dissolved gases
 <sup>29</sup>N­<sub>2</sub>
 and <sup>30</sup>N<sub>2</sub> were corrected for
 sediment and water volumes. Then, differences in concentrations were used
 to determine production rates of
 <sup>29</sup>N<sub>2</sub> and
 <sup>30</sup>N<sub>2</sub>, or
 <em>p29</em> and <em>p30</em>, respectively,
 normalized by the weights of sediment subsamples in each vial. Rates of
 denitrification of
 <sup>15</sup>NO<sub>3</sub><sup>-</sup> (<em>D<sub>15</sub></em>) and <sup>14</sup>NO<sub>3</sub><sup>-</sup> (<em>D<sub>14</sub></em>), were determined using  the following equations to calculate <em>D<sub>14</sub></em><sub> </sub>and <em>D<sub>15</sub></em>: D15 = p29 + 2p30 D14 = D15[p29/2p30]    Finally, <em>D<sub>14</sub></em> and <em>D<sub>15</sub></em><sub> </sub>were summed for total denitrification rate (<em>D<sub>total</sub></em>) for each sediment type and treatment. DNRA potential was measured using the OX/MIMS method. Half of the replicates for DNRA measurements were oxidized with hypobromite iodine solution prior to analysis of <sup>29+30</sup>N<sub>2</sub> on a MIMS with an inline furnace, to convert NH<sub>4</sub><sup>+</sup> to N<sub>2</sub><sub>. </sub>. The difference between unoxidized and oxidized samples represented the concentration of <sup>15</sup>NH<sub>4</sub><sup>+</sup>. Rates were then calculated as production of <sup>15</sup>NH<sub>4</sub><sup>+</sup> using the<sup> </sup>same methods described<sup> </sup>above. Sediment organic content, percent water content, and the field-moist/oven-dry ratio were determined by drying a 5 cm<sup>3</sup> sediment volume for 24 hours at 105 °C, followed by combustion at 550 °C for 5 hours. Sediment pH was measured using a Thermo Scientific Orion A121 meter on vortexed 1:1 (w/w) deionized water to dry sediment slurries. Slurries were produced without pre-drying samples based on the field-moist to oven-dried ratio measured above. Sediment salinity was measured using a YSI Pro Series multimeter with conductivity probe on vortexed and centrifuged 10:1 DI to dry sediment slurries. Measured salinities were adjusted to a 1:1 water:sediment ratio. Soils were extracted with a 2M KCl solution, and extractable NH<sub>4</sub>-N and NO<sub>x</sub><sup>-</sup>-N were measured on a Lachat QC8500 Flow Injection Colorimeter at Stony Brook University's Center for Clean Water Technology. Samples were additionally measured for redox potential (herein, referred to as eH) by placing the probe in sediment to a depth of 5-cm using a benchtop Oakton ORP electrode after allowing readings to stabilize for at least 10 minutes; readings were corrected using standard solution. Genomic DNA was isolated from 23 samples (10 from restoration sediments, 10 from biochar amended restoration sediments, and 3 from reference marsh) using a Takara Nucleospin Soil DNA extraction kit (Takara Bio USA, Inc., San Jose, CA) following manufacturer’s instructions. Extracted DNA was quantified with a Qubit 3.0 fluorometer. Library preparation and amplicon sequencing were conducted at the University of Minnesota Genomics Center. The amplicon library was generated for V3-V5 region of the 16S rDNA gene following protocols outlined in Gohl et al. (2016).  Amplicon libraries were sequenced using 2 x 300 PE sequencing chemistry on the MiSeq<sup>®</sup> platform (Illumina). The raw sequencing data have been deposited in the NCBI Sequence Read Archive (SRA) database under accession number PRJNA1263012. Bioinformatics and statistical analysis were conducted using R v4.3.1. Raw paired-end reads were primer-trimmed using Cutadapt  by calling Python v. 3.1.1 through R. Upon reads’ quality inspection, only the forward reads were used to generate amplicon sequence variants (ASVs) using “dada2” v1.28.0. Chimeras and singletons were filtered and produced amplicon sequence variants (ASVs) taxonomically classified using Silva reference database release 138.  Data matrices were managed using R package “phyloseq” v1.28.0 and plots were generated using R package “ggplot2” v3.4.2. Amplicon abundance data were rarefied to 500 reads per sample prior to data analysis. Principal Component Analysis (PCA) was carried with edaphic parameters to visualize differences among samples from natural and restored marsh and among control, fresh biochar and aged biochar plots. Non-metric multidimensional scaling (NMDS) analyses coupled with the “envfit” procedure that computes correlations of environmental factors with NMDS axes explored patterns of microbial assemblage composition. PCA and NMDS ordinations were conducted using R package “vegan” v2.6-4. Alpha diversity metrics were calculated to compare diversity in biochar-amended vs. restoration and reference plots as indicators of richness (Chao1), dominance (Berger-Parker), phylogenetic diversity (Faith's PD), and entropy (Shannon). Diversity indices were calculated using the packages "BiodiversityR", "vegan" and "phyloseq". To calculate Faith's PD, an alignment of bacterial sequences was generated and a phylogenetic tree constructed from these using package "phangorn". To functionally annotate Amplicon Sequence Variants (ASVs), the ASV table was formatted into appropriate input files for Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2). The ASV table, generated from DADA2, was converted into BIOM (.biom) and FASTA (.fasta) file formats using R v4.3.1. The BIOM format retains the sample-by-ASV abundance matrix, while the FASTA file provides nucleotide sequences of ASVs, both of which serve as essential inputs for PICRUSt2. These files were transferred to the Stony Brook Seawulf cluster, where PICRUSt2 was executed in a Bash shell terminal. PICRUSt2 predicts the functional potential of microbial communities by placing ASVs onto a reference phylogeny and inferring gene families and metabolic functions based on ancestral trait reconstruction. The output includes Enzyme Commission (EC) numbers and KEGG Orthology (KO) annotations, providing insight into the functional capabilities of the sampled microbial communities. The functional prediction outputs from PICRUSt2 (EC and KO abundances per sample) were analyzed in R to explore differences in functional composition across samples. To visualize pathway abundance across samples, the PICRUSt2 output was filtered to retain only pathways associated with decomposition, followed by total sum scaling (TSS) normalization. One sample (biochar amended sediment, site 7; BcharP7_S165_L001_R1_001.fastq.gz, or) was removed due to low quality. The resulting dataset was organized into a long-format table containing sample IDs, pathway names, experimental group assignments (natural marsh, restoration, and biochar amended), and TSS-normalized relative abundances. Functional beta-diversity was further examined using non-metric multidimensional scaling (NMDS), implemented with the metaMDS() function from the vegan package. Ordination plots were produced in two dimensions, with ellipses illustrating group-level dispersion. Permutational Multivariate Analysis of Variance (PERMANOVA) was used to evaluate whether differences in functional composition were significant among experimental groups. This was performed using the adonis2() function in the vegan package based on Bray-Curtis dissimilarities. Statistical significance was assessed using 999 permutations. Post hoc pairwise comparisons were conducted following a significant global PERMANOVA result using the pairwise.adonis2() function from the pairwiseAdonis package. P-values were adjusted for multiple testing using the Benjamini-Hochberg false discovery rate (FDR) method. One-way Analysis of Variance (ANOVA) tests was used to compare denitrification potential rates between sample types (unamended restoration, freshly amended restoration, aged biochar amended restoration, and natural marsh sediments), after log<sub>10</sub> transforming prior to analysis to meet assumptions for parametric tests. Non-parametric Kruskal-Wallis rank sum tests were conducted on KO/KEGG functions associated with nitrogen metabolism, alpha-diversity metrics, and edaphic parameters measured (pH, salinity, eH, moisture, LOI, NH<sub>4</sub><sup>+</sup>-N, and NO<sub>x</sub><sup>-</sup>-N). For edaphic characteristics, a Bonferroni correction was used, resulting in a corrected-α of 0.0063. Significant interactions were followed by using a post hoc Tukey’s HSD Test (R package “multcomp” v1.4.23) for parametric analyses and a Dunn Test (R package “FSA” v0.9.4) for non-parametric analyses. Correlation matrices were used to visualize relationships between edaphic parameters and denitrification potential (“corrplot” v0.92). Significance of correlation coefficients was determined using the function ‘cor.mtest’ and a confidence interval of 0.95. Resulting <em>p</em>-values (Supplemental Material) were considered significant if they were less than the Bonferroni-corrected α (<em>p</em> &lt; 0.0014) due to the multiple comparisons and possible Type I error.
description: # Biochar in a California salt marsh restoration had modest effects on
 nitrogen metabolism but supported distinct microbial communities and
 functions Dataset DOI: [10.5061/dryad.kprr4xhh8](10.5061/dryad.kprr4xhh8)
 ## Description of the data and file structure The following files include
 processed data associated with 16S amplicon sequence variants
 ("asv_table.csv", "taxa_rare.csv",
 "asv_rare.csv", "refseq.csv",
 "diversity.csv"), sediment metadata
 ("FullSeddata.csv") and functional annotations
 ("Normalized_Data_cleaned.csv", "n_met.csv",
 "n_met_paired.csv", "ko_new_annotated.csv",
 "path_abun_unstrat.csv"). Analysis is described by an annotated
 R script, 16S Ecological Analysis_9June2025.R. ### Files and variables
 #### File: FullSeddata.csv **Description:** This file contains the
 sediment metadata for the project. ##### Variables * Sample: The values
 for this field include Yampah1-5 (the natural marsh), Control 1-10 (the
 restored marsh), Fresh Biochar 1-5, or Aged Biochar 1-10 (the biochar
 ammended sediment). * Type: The values for this field include the four
 treatments: Reference (the natural marsh), Control (the restored marsh),
 FreshBiochar (the sediments freshly amended with biochar), AgedBiochar. *
 Biochar: This field has two values, With (with biochar) and Without
 (without Biochar) * pH: pH measured on sediments in a 1:1 mix of sediment
 and water * Salinity: salinity in psu measured on sediment according the
 methods described in the paper. * eH: redox measure in mV according the
 methods described in the paper. * Moisture: gravimetric moisture measured
 as the difference between field moist and dried samples divided by the
 weight of the dry sample * OC: organic content or fraction loss on
 ignition after combustion at 550C for four hours. * NH4: 2M KCl extracted
 ammonium in mg N / dry KG of sediment * NOx: 2M KCl extracted nitrate and
 nitrite in mg N / dry KG of sediment * Denit: denitrification measured
 according to the methods described in the paper in nmol per gram (dry) per
 hour. Several values are "null" as not all sediments were
 measured. #### File: taxa_rare.csv **Description:**  This file contains
 the taxonomic assignments of the ASVs. ##### Variables * ASVnum: this
 field contains the number of the ASV, from 1-8474 * Kingdom: The taxonomic
 assignment of kingdom to the ASV (e.g., Bacteria, Archea) * Phylum: The
 phylum the ASV belongs to (e.g., Acidobacteriota,) * Class: The class the
 ASV belongs to (e.g., Thermoanaerobaculia) * Order: The order the ASV
 belongs to (e.g., Thermoanaerobaculales) * Family: The family the ASV
 belongs to (e.g., Thermoanaerobaculaceae) * Genus: The genus the ASV
 belongs to (e.g., Halofilum) * Species: The species the ASV belongs to
 (e.g., ochraceum) #### File: path_abun_unstrat.csv **Description:** This
 file contains the metabolic pathway assignments of all identified ASVs,
 organized as pathways as the first column, and each of the different
 columns is a different sediment sample. The numbers of the data matrix are
 the number of reads assigned to each pathway. ##### Variables * pathway:
 all matched pathways from the KEGG-KO database. *
 BcharP1_S153_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and includes biochar amendments. *
 BcharP10_S171_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 10, and includes biochar amendments. *
 BcharP10C_S172_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 10, and includes restoration sediments. *
 BcharP1C_S154_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and includes restoration sediments. *
 BcharP2_S155_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 2, and includes biochar amendments. *
 BcharP2C_S156_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 2, and includes restoration sediments. *
 BcharP3_S157_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and includes biochar amendments. *
 BcharP3C_S158_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and includes restoration sediments. *
 BcharP4_S159_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 4, and includes biochar amendments. *
 BcharP4C_S149_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 4, and includes restoration sediments. *
 BcharP5_S161_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 5, and includes biochar amendments. *
 BcharP5C_S162_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 5, and includes restoration sediments. *
 BcharP6_S163_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 6, and includes biochar amendments. *
 BcharP6C_S164_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 6, and includes restoration sediments. *
 BcharP7_S165_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 7, and includes biochar amendments. *
 BcharP7C_S166_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 7, and includes restoration sediments. *
 BcharP8_S167_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 8, and includes biochar amendments. *
 BcharP8C_S168_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 8, and includes restoration sediments. *
 BcharP9_S169_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 9. and includes biochar amendments. *
 BcharP9C_S170_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 9, and includes restoration sediments. *
 Marsh1_S173_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and is from natural marsh. * Marsh2_S174_L001_R1_001.fastq.gz: The
 column name refers to the sample name utilized the Sequence Read Archive.
 This column is collected from site 2, and is from natural marsh. *
 Marsh3_S175_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and is from natural marsh. * Total Abundance: The sum of columns
 2-24 for each row. #### File: 16S_EcologicalAnalysis_9June2025.R
 **Description:** This R code archives the data analysis used in the
 manuscript. #### File: asv_rare.csv **Description:** This file is the
 rarefied samples, with samples with low number of reads discarded. #####
 Variables * Sample: This field is the sample code, it includes Aged
 Biochar 2, 3, 4, 5, and 9; Controls 10, 2, 3, 4, 5, 7, and 9; and Yampah
 (natural marsh). * ASV1: This column contains rarified abundance data for
 Amplicon Sequence Variant 1 * ASV2: This column contains rarified
 abundance data for Amplicon Sequence Variant 2 * ASV3: This column
 contains rarified abundance data for Amplicon Sequence Variant 3 *
 ASV4: This column contains rarified abundance data for Amplicon Sequence
 Variant 4 * ASV5: This column contains rarified abundance data for
 Amplicon Sequence Variant 5 * ASV6: This column contains rarified
 abundance data for Amplicon Sequence Variant 6 * ASV7: This column
 contains rarified abundance data for Amplicon Sequence Variant 7 *
 ASV8: This column contains rarified abundance data for Amplicon Sequence
 Variant 8 * ASV9: This column contains rarified abundance data for
 Amplicon Sequence Variant 9 * ASV10: This column contains rarified
 abundance data for Amplicon Sequence Variant 10 * ............... *
 ASV8474: This column contains rarified abundance data for Amplicon
 Sequence Variant 8474 #### File: diversity.csv **Description:** This file
 contains data used for estimating, alpha diversity (using Chao1),
 dominance (using Berger-Parker), phylogenetic diversity (using
 Faith's PD), and entropy (using Shannon). ##### Variables * sample:
 This field contains sample ids, using the sample names utilized the
 Sequence Read Archive. * treatment: This field contains the treatment
 type, includes biochar, control, and natural. * S.chao1: Contains numeric
 data for computing Chao1 Index. * Shannon: Contains numeric data for
 computing Shannon index. * Berger: Contains numeric data for computing
 Berger-Parker index. * Faiths PD: Contains numeric data for computing
 Faith's PD. #### File: ko_new_annotated.csv **Description:** This
 file contains KEGG orthologs (KOs), their abundance per sample, and
 corresponding pathways they are associated with, according to the KEGG
 database. ##### Variables * function: This field contains numeric data ,
 which keeps track of the number of KOs in the file. *
 BcharP1_S153_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and includes biochar amendments. *
 BcharP10_S171_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 10, and includes biochar amendments. *
 BcharP10C_S172_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 10, and includes restoration sediments. *
 BcharP1C_S154_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and includes restoration sediments. *
 BcharP2_S155_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 2, and includes biochar amendments. *
 BcharP2C_S156_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 2, and includes restoration sediments. *
 BcharP3_S157_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and includes biochar amendments. *
 BcharP3C_S158_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and includes restoration sediments. *
 BcharP4_S159_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 4, and includes biochar amendments. *
 BcharP4C_S149_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 4, and includes restoration sediments. *
 BcharP5_S161_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 5, and includes biochar amendments. *
 BcharP5C_S162_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 5, and includes restoration sediments. *
 BcharP6_S163_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 6, and includes biochar amendments. *
 BcharP6C_S164_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 6, and includes restoration sediments. *
 BcharP7_S165_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 7, and includes biochar amendments. *
 BcharP7C_S166_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 7, and includes restoration sediments. *
 BcharP8_S167_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 8, and includes biochar amendments. *
 BcharP8C_S168_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 8, and includes restoration sediments. *
 BcharP9_S169_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 9. and includes biochar amendments. *
 BcharP9C_S170_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 9, and includes restoration sediments. *
 Marsh1_S173_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and is from natural marsh. * Marsh2_S174_L001_R1_001.fastq.gz: The
 column name refers to the sample name utilized the Sequence Read Archive.
 This column is collected from site 2, and is from natural marsh. *
 Marsh3_S175_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and is from natural marsh. - type: This field contains the KO ids.
 - Pathway_IDs: This field contains pathway ids. - KEGG_Pathway: This field
 contains the mapped paths for associating KEGG pathways with each KO. -
 annotations: This field contains the pathways associated with each KO.
 #### File: refseq.csv **Description:**  This file contains reference data
 of the ASV ids and their corresponding sequences. ##### Variables * ASV:
 This field contains the ASV ids. * x: This field contains the sequences
 associated with each ASV id. #### File: asv_table.csv
 **Description:** This file contains data with ASV abundances in each
 sample. ##### Variables * sample: This field contains sample ids, using
 the sample names utilized the Sequence Read Archive. - ASV1: This column
 contains rarified abundance data for Amplicon Sequence Variant 1. * ASV2:
 This column contains rarified abundance data for Amplicon Sequence Variant
 2. * ASV3: This column contains rarified abundance data for Amplicon
 Sequence Variant 3. * ASV4: This column contains rarified abundance data
 for Amplicon Sequence Variant 4. * ASV5: This column contains rarified
 abundance data for Amplicon Sequence Variant 5. * ASV6: This column
 contains rarified abundance data for Amplicon Sequence Variant 6. *
 ASV7: This column contains rarified abundance data for Amplicon Sequence
 Variant 7. * ASV8: This column contains rarified abundance data for
 Amplicon Sequence Variant 8. * ASV9: This column contains rarified
 abundance data for Amplicon Sequence Variant 9. * ASV10: This column
 contains rarified abundance data for Amplicon Sequence Variant 10. *
 ............... * ASV8566: This column contains rarified abundance data
 for Amplicon Sequence Variant 8566. #### File: n_met_paired.csv
 **Description:**  This file is a comparison of sediment functional reads
 in pared biochar amended and restoration plots, to permit a varied
 analysis. ##### Variables * sample: plot number, 1-10. * N-fix_b: number
 of functional copies of n-fixation genes in the biochar plots (as listed
 in the manuscript, figure 9). * N-fix_r: number of functional copies of
 n-fixation genes in the control restoration plots (as listed in the
 manuscript, figure 9). * ANRA_b: number of functional copies of genes
 associated with ANRA in the biochar plots (as listed in the manuscript,
 figure 9). * ANRA_r: number of functional copies of genes  associated with
 ANRA in the control restoration plots (as listed in the manuscript, figure
 9). * DNRA_b: number of functional copies of genes associated with DNRA in
 the biochar plots (as listed in the manuscript, figure 9). *
 DNRA_r: number of functional copies of genes  associated with DNRA in the
 control restoration plots (as listed in the manuscript, figure 9). *
 DNR_b: number of functional copies of genes associated with
 denitrification in the biochar plots (as listed in the manuscript, figure
 9). * DNF_r: number of functional copies of genes  associated with
 denitrification in the control restoration plots (as listed in the
 manuscript, figure 9). * Nitr_b: number of functional copies of genes
 associated with nitrification in the biochar plots (as listed in the
 manuscript, figure 9). * Nitr_r: number of functional copies of genes
  associated with nitrification in the control restoration plots (as listed
 in the manuscript, figure 9). * ANA_b: number of functional copies of
 genes associated with annamox  in the biochar plots (as listed in the
 manuscript, figure 9). * ANA_r: number of functional copies of genes
  associated with annamox in the control restoration plots (as listed in
 the manuscript, figure 9). #### File: n_met.csv **Description:** This file
 contains nitrogen metabolism pathways used for analysis. ##### Variables *
 row: This field contains numeric data , which keeps track of the number of
 KOs in the file * function: This field contains the function ids, could
 also be called KO ids. * BcharP1_S153_L001_R1_001.fastq.gz: The column
 name refers to the sample name utilized the Sequence Read Archive. This
 column is collected from site 1, and includes biochar amendments. *
 BcharP10_S171_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 10, and includes biochar amendments. *
 BcharP10C_S172_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 10, and includes restoration sediments. *
 BcharP1C_S154_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and includes restoration sediments. *
 BcharP2_S155_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 2, and includes biochar amendments. *
 BcharP2C_S156_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 2, and includes restoration sediments. *
 BcharP3_S157_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and includes biochar amendments. *
 BcharP3C_S158_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and includes restoration sediments. *
 BcharP4_S159_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 4, and includes biochar amendments. *
 BcharP4C_S149_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 4, and includes restoration sediments. *
 BcharP5_S161_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 5, and includes biochar amendments. *
 BcharP5C_S162_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 5, and includes restoration sediments. *
 BcharP6_S163_L001_R1_001.fastq.gz: he column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 6, and includes biochar amendments. *
 BcharP6C_S164_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 6, and includes restoration sediments. *
 BcharP7_S165_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 7, and includes biochar amendments. *
 BcharP7C_S166_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 7, and includes restoration sediments. *
 BcharP8_S167_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 8, and includes biochar amendments. *
 BcharP8C_S168_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 8, and includes restoration sediments. *
 BcharP9_S169_L001_R1_001.fastq.gz: the column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 9. and includes biochar amendments. *
 BcharP9C_S170_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 9, and includes restoration sediments. *
 Marsh1_S173_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 1, and is from natural marsh. * Marsh2_S174_L001_R1_001.fastq.gz: The
 column name refers to the sample name utilized the Sequence Read Archive.
 This column is collected from site 2, and is from natural marsh. *
 Marsh3_S175_L001_R1_001.fastq.gz: The column name refers to the sample
 name utilized the Sequence Read Archive. This column is collected from
 site 3, and is from natural marsh. - type: This field contains the KO ids.
 - Pathway_IDs: This field contains pathway ids. - KEGG_Pathway: This field
 contains the mapped paths for associating KEGG pathways with each KO. -
 annotations: This field contains the pathways associated with each KO.
 #### File: Normalized_Data_cleaned.csv **Description:** This file contains
 data used for statistical analysis and visualization, after qualitative
 assessment. ##### Variables * row: This field contains numeric data ,
 which keeps track of the number of items in the file. * pathway: This
 field contains pathways * SampleID: This field contains sample ids, using
 the sample names utilized the Sequence Read Archive. * Abundance: This
 field contains the abundance values for each pathway. * Group: This field
 contains the treatment types as grouping variables. * Abundance_TSS: This
 field contains the relative abundance values for each pathway #### File:
 Normalized_Data.csv **Description:** This file contains data used for
 creating Normalized_Data_cleaned.csv. ##### Variables * row: This field
 contains numeric data , which keeps track of the number of items in the
 file. * pathway: This field contains pathways * SampleID: This field
 contains sample ids, using the sample names utilized the Sequence Read
 Archive. * Abundance: This field contains the abundance values for each
 pathway. * Group: This field contains the treatment types as grouping
 variables. * Abundance_TSS: This field contains the relative abundance
 values for each pathway #### File: otu2.biom **Description:** This file
 contain the ASV abundance format require as input for PICRUSt #### File:
 otu_fasta.fasta **Description:** This file contains ASV ID linked to
 sequences. It is used as reference input in PICRUSt for annotation. ####
 File: PICRUSt_4_20_2025.R **Description:** This R code contain the dada
 pipeline initially used to create the ASV table. Later parts contains how
 the .biom and .fasta were created, and the line of code for running
 PICRUSt. #### File: Analysis_V3.R **Description:** This R codes used for
 statistical analysis and visualization. ## Code/software Three R scripts
 to calculate relevant data analysis metrics are provided. ## Access
 information Other publicly accessible locations of the data: * None Data
 was derived from the following sources: * Genomic data is archived in the
 sample read
 archive. [https://www.ncbi.nlm.nih.gov/sra/PRJNA1263012](https://www.ncbi.nlm.nih.gov/sra/PRJNA1263012)