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Harvard Forest Data Archive


Effects of Soil Warming on Bacterial Degradation of Carbohydrates at Harvard Forest 2011

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  • Lead: Grace Pold, Serita Frey, Jerry Melillo, Kristen DeAngelis
  • Investigators: Andrew Billings, Daniel Burkhardt, Julia Schnabel, Linda van Diepen
  • Contact: Information Manager
  • Start date: 2011
  • End date: 2011
  • Status: complete
  • Location: Prospect Hill Tract (Harvard Forest)
  • Latitude: +42.54 degrees
  • Longitude: -72.18 degrees
  • Elevation: 365 meter
  • Datum: WGS84
  • Taxa:
  • Release date: 2023
  • Language: English
  • EML file: knb-lter-hfr.371.2
  • DOI: digital object identifier
  • EDI: data package
  • DataONE: data package
  • Related links:
  • Study type: long-term measurement
  • Research topic: large experiments and permanent plot studies; physiological ecology, population dynamics and species interactions
  • LTER core area: population studies, organic matter movement
  • Keywords: bacteria, carbohydrates, carbon, cellulose, climate change, decomposition, microbes, soil warming
  • Abstract:

    As Earth’s climate warms, soil carbon pools and the microbes that process them may change, altering the way in which carbon is recycled in soil. We used bacterial cultivation to evaluate the hypothesis that experimentally raising soil temperatures by 5°C for 20 years increased the potential for temperate forest soil microbial communities to degrade carbohydrates. A greater proportion of the 295 bacteria from 6 phyla (10 classes, 14 orders, and 34 families) isolated from heated plots in the 20-year experiment were able to depolymerize cellulose and xylan than bacterial isolates from control soils. These findings indicate that the enrichment of bacteria capable of degrading carbohydrates could be important for accelerated carbon cycling in a warmer world.

    Data for the isolates from the Harvard Forest culturing project is archived at

  • Methods:

    These methods are published, and the references listed in parentheses can be found in the original publication: Pold, G., Billings, A. F., Blanchard, J. L., Burkhardt, D. B., Frey, S. D., Melillo, J. M., Schnabel, J., van Diepen, L. T. A., DeAngelis, K. M. 2016. Long-term warming alters carbohydrate degradation potential in temperate forest soils. Applied and Environmental Microbiology 82: 6518-6530.

    Isolate cultivation

    Dominant members of the bacterial communities at Prospect Hill (20 years of warming) were targeted for cultivation, with successful isolation of 537 dominant and rare organisms in 6 phyla from both warming treatment and control treatment soils (see Fig. S5 in the supplemental material). Soils from Prospect Hill were selected for cultivation because microbial communities are known to have shifted with warming at this site (4, 16, 22). Soils were collected on 22 October 2013, 28 April 2014, and 30 June 2014 when we were sampling the site for other purposes. On these instances, soil samples were collected with 1/2-in. tubular soil corers to a depth of 10 cm and split by eye into organic and mineral soils. The soil corer was washed with 70% ethanol between plots to minimize cross-contamination.

    Various methods known to be effective for isolating the closest cultured relatives of dominant Acidobacteria, Actinobacteria, and Alphapro- teobacteria identified in a previous study of these soils were used (19). Our overall approach was to maximize the total number and diversity of bacteria isolated from soil, with the intention of examining how warming changed the phylogeny of biopolymer-degrading traits. We used low- nutrient media and long incubation times (weeks to months), as these have been shown to increase up to a hundredfold the number and diversity of bacteria that can be cultured (47). Soil treatment methods prior to isolation on solid medium included (i) placing lignin-amended Bio-Sep beads into warmed and control plots for 3 months (4) and then transfer- ring them to a minimal medium (48) with Kraft lignin as sole C source under anaerobic conditions for 6 weeks, (ii) diluting soil to extinction in a soil solution mimic (49) and growing the resultant dilutions under aerobic conditions at room temperature prior to plating on solid medium, (iii) drying soils at 120°C for 1 h with or without a 1-h 30°C phenol treatment designed to select for Gram-positive organisms, (iv) surface-sterilizing and then grinding plant roots in an extraction solution to select for endo- phytic bacteria (50), and (v) vigorously stirring soil in the presence of 2.24 mM dithiothreitol and 1 mM sodium pyrophosphate for 1 h prior to plating to enable the separation of bacteria from soil while minimizing oxidative stress (51). Media used included oatmeal agar (52), humic acid vitamin agar (53), lignin soybean flour vitamin agar (54), MM1 (55), 1% nutrient agar (56), modified carboxymethyl cellulose (CMC) (50), water agar plus yeast extract, and VL-55 with gellan gum, xylan from birchwood, carboxymethyl cellulose, pectin, xanthan gum, or readily oxidized carbon (51). Plates were incubated in the dark at 23 to 25°C in the laboratory at room temperature in an anaerobic Coy chamber filled with 5% H2, 5% CO2, 90% N2, or with switching between these conditions every 2 to 3 days to mimic the reduced oxygen conditions characteristic of soil. We also prepared roll tubes with a 1 to 2% oxygen headspace (57) and incubated them at 18°C. In all instances, no-soil negative controls were prepared in order to rule out the possibility of cross-contamination between warmed and control plot soils. Plates and tubes were incubated from 1 week to 6 months before colonies were picked. No obligate anaerobes were identified. All bacteria were streaked to isolation under aerobic laboratory conditions and then cryopreserved in 15% glycerol at -80°C. Freezer stocks were identified and verified as pure by sequencing of 16S rRNA gene PCR products using the primers 27F (5=-AGAGTTTGATCMTGGCTCAG-3=) and 1492R (5=-TACGGYTACCTTGTTACGACTT-3=) (58). Sequences were trimmed to include only an average quality score of >60 and examined for contamination in 4Peaks (version 1.7.2 [Nucleobytes]) before being assigned taxonomic categories using EzTaxon (59). Closely related isolates were identified by clustering 16S rRNA sequences at 99% identity using cd-hit (60), and BLASTn (61) was subsequently used to map meta- genome 16S rRNA reads to these clusters.

    A phylogenetic tree (Fig. 4) of isolate sequences was built with RAxML version 7.7.2 (62) (100 bootstraps, GTRGAMMA model of nucleotide substitution) using sequences aligned using the bacterial model in RDP (33). We only included those 295 bacteria that both demonstrated growth in at least two of the three substrate use assays and for which we had viable freezer stocks verified as pure by sequencing of the 16S rRNA gene in our tree. Methanocaldococcus jannaschii DSM 2661 was included as the outgroup. The 16S rRNA sequences of the following strains were extracted from NCBI’s 16S prokaryotic rRNA database (63) and included in the isolate alignment to assist with tree building: Terriglobus roseus KBS 63, Burkholderia soli GP25-8, Nevskia terrae KIS13-15, Bradyrhizobium lablabi CCBAU 23086, Flavobacterium soli KCTC 12542, Opitutus terrae PB90-1, Bacillus subtilis NRS 744, Isosphaera palida ATCC 43644, and Mycobacterium smegmatis 987-M10. The phylogenetic tree was drawn and annotated using iTOL (64). Isolation information for bacteria used in this analysis can be found in Data Set S2 in the supplemental material.

    Physiological characterization of isolates

    Isolates were characterized for the ability to depolymerize three polysaccharides if their cryopreserved cultures had been validated as pure and growth was observed on the assay medium used. Our objective was to evaluate how long-term warming has affected the ability to degrade polysaccharides in phylogenetically diverse organisms isolated from soil, with the expectation that a greater fraction of bacterial genotypes from heated plots would be able to degrade biopolymers. All incubations and assays were completed aerobically at room temperature (23 to 25°C), as these are the conditions under which all isolates had been maintained following isolation. With a few exceptions for very slow-growing isolates, isolates were streaked from freezer stocks onto 10% tryptic soy agar (TSA) 6 days prior to characterization. Three distinct colonies were selected, and each was inoculated into 10 ml of 10% tryptic soy broth (TSB) and allowed to grow for 24 h with gentle shaking (120 to 150 rpm). Ten microliters of this culture was inoculated onto solidified medium with 0.1% chitin, carboxymethyl cellulose (TCI C0045), or xylan (catalog no. X4252; Sigma) for evidence of depolymerization, where depolymerization is seen as zones of transparency around growth on otherwise-opaque medium for chitin, and as zones of yellow on otherwise-purple-brown medium following staining with Gram’s iodine for xylan and cellulose (65). Chitin from crab shells (catalog no. C-7170; Sigma) was colloidized for inclusion in medium by soaking 15 g in 112.5 ml of 12 M hydrochloric acid with periodic stirring for 1 to 2 h, and then precipitating it in 3.375 liters of ice-cold distilled water overnight, filtering the pellet, and rinsing it with ice cold phosphate-buffered saline (pH 7.5) until the eluent reached circumneutral pH. We used MM medium (66) as the base for plate-based assessment of zones of clearance. Chitin degradation was examined after 11 days of growth, while xylan and cellulose degradation was assessed after 4 days. The bioinformatic tool consenTRAIT (5) was used to calculate the mean phylogenetic trait depth for groups where at least 90% of the members are able to use a substrate (tao-D). Significant differences in isolate potential for biopolymer utilization were as- sessed using a phylogenetic logistic regression method implemented in the phylolm package in R (version 2.4 [ /phylolm/index.html]), using 1,000 bootstraps (67).

  • Organization: Harvard Forest. 324 North Main Street, Petersham, MA 01366, USA. Phone (978) 724-3302. Fax (978) 724-3595.

  • Project: The Harvard Forest Long-Term Ecological Research (LTER) program examines ecological dynamics in the New England region resulting from natural disturbances, environmental change, and human impacts. (ROR).

  • Funding: National Science Foundation LTER grants: DEB-8811764, DEB-9411975, DEB-0080592, DEB-0620443, DEB-1237491, DEB-1832210.

  • Use: This dataset is released to the public under Creative Commons CC0 1.0 (No Rights Reserved). Please keep the dataset creators informed of any plans to use the dataset. Consultation with the original investigators is strongly encouraged. Publications and data products that make use of the dataset should include proper acknowledgement.

  • License: Creative Commons Zero v1.0 Universal (CC0-1.0)

  • Citation: Pold G, Frey S, Melillo J, DeAngelis K. 2023. Effects of Soil Warming on Bacterial Degradation of Carbohydrates at Harvard Forest 2011. Harvard Forest Data Archive: HF371 (v.2). Environmental Data Initiative:

Detailed Metadata

hf371-01: bacterial isolates

  1. isolate_id: unique identifier for each isolate bacterial strain
  2. 16s_rrna_sequence: full sequence for the 16S ribosomal RNA gene, obtained through amplicon sequencing and assembled as a contig of the full gene when possible
  3. sequence_length: length (in base pairs) of the 16S rRNA sequence (unit: number / missing value: NA)
  4. taxonomy: taxonomic identification based on BLAST results for nearest neighbor at time of isolation
  5. soil_horizon: origin of the isolate
    • mineral: mineral soil
    • organic: organic soil
    • roots: roots
    • subsurface: mineral soil below about 5 cm depth
    • surface: surface soil
    • UMass: originated from Harvard Forest soils transported to UMass and grown as windowsill communities for about 6 months
    • unk: unknown
  6. warming_treatment: origin of the isolate
    • control: Prospect Hill long-term warming study, from the disturbance control plots
    • outside: Prospect Hill, outside the plots
    • UMass: UMass
    • warm: Prospect Hill long-term warming study, from the +5oC heated plot soils
    • unk: unknown
  7. morphology: description of the colony morphology of the isolate when grown on 10% strength tryptic soy agar medium
  8. cmc_xylan_chitin_util: description of cellulose (CMC), hemicellulose (xylan) or chitin utilization
  9. note: notes