Keeping it cool: Soil sample cold pack storage and DNA shipment up to 1 month does not impact metabarcoding results.

With the advances of sequencing tools, the fields of environmental microbiology and soil ecology have been transformed. Today, the unculturable majority of soil microbes can be sequenced. Although these tools give us tremendous power and open many doors to answer important questions, we must understand how sample processing may impact our results and interpretations. Here, we test the impacts of four soil storage methods on downstream amplicon metabarcoding and qPCR analyses for fungi and bacteria. We further investigate the impact of thaw time on extracted DNA to determine a safe length of time during which this can occur with minimal impact on study results. Overall, we find that storage using standard cold packs with subsequent storage at -20°C is little different than immediate storage in liquid nitrogen, suggesting that the historical and current method is adequate. We further find evidence that storage at room temperature or with aid of RNAlater can lead to changes in community composition and in the case of RNAlater, lower gene copies. We therefore advise against these storage methods for metabarcoding analyses. Finally, we show that over 1 month, DNA extract thaw time does not impact diversity or qPCR metrics. We hope that this work will help researchers working with soil bacteria and fungi make informed decisions about soil storage and transport to ensure repeatability and accuracy of results and interpretations.

The Charosphere Promotes Mineralization of 13C-Phenanthrene by Psychrotrophic Microorganisms in Greenland Soils.

When soil is frozen, biochar promotes petroleum hydrocarbon (PHC) degradation, yet we still do not understand why. To investigate microbial biodegradation activity under frozen conditions, we placed 60-µm mesh bags containing 6% (v/v) biochar created from fishmeal, bonemeal, bone chip, or wood into PHC-contaminated soil, which was then frozen to -5°C. This created three soil niches: biochar particles, the charosphere (biochar-contiguous soil), and bulk soil outside of the bags. After 90 d, C-phenanthrene mineralization reached 55% in bonemeal biochar and 84% in bone chip biochar charosphere soil, compared with only 43% in bulk soil and 13% in bone chip biochar particles. Soil pH remained near neutral in bone chip and bonemeal biochar treatments, unlike wood biochar, which increased alkalinity and likely made phosphate unavailable for microorganisms. Generally, charosphere soil had higher aromatic degradative gene abundances than bulk soil, but gene abundance was not directly linked to C-phenanthrene mineralization. In bone chip biochar-amended soils, phosphate successfully predicted microbial community composition, and abundances of and increased in charosphere soil. Biochar effects on charosphere soil were dependent on feedstock material and suggest that optimizing the charosphere in bone-derived biochars may increase remediation success in northern regions.

Application Method and Biochar Type Affect Petroleum Hydrocarbon Degradation in Northern Landfarms.

To integrate biochar as a practical and successful remedial amendment at northern landfarms, components of its formulation and application must be optimized for site-specific environmental conditions. Different biochar amendments were applied to petroleum hydrocarbon (PHC)-contaminated soil at two northern field sites (Iqaluit and Whitehorse) and in a laboratory study at -5°C to determine the effects of application method (injection or incorporation) and biochar type (wood, fishmeal, bonemeal, and/or compost) on PHC degradation and associated soil properties. Incorporation decreased F2 (equivalent C-C) and F3 (equivalent C-C) PHC concentrations in soil after 31 d, whereas injection did not decrease PHC concentrations until Day 334. Bonemeal-derived biochar selectively stimulated F3-PHC degradation in frozen soil over 90 d under controlled laboratory conditions. In the field, there was little difference in PHC degradation between biochar types and the fertilizer control. Incorporation also increased NO availability, and in field trials, all biochars increased NO availability relative to the fertilizer control, whereas the effects of biochars on NH and PO were variable. Aromatic functional gene abundance was enhanced when treatments were incorporated, compared with when injected. In field trials, 6% Zakus wood plus fertilizer inhibited aliphatic and aromatic gene abundance. Liquid water content increased in incorporated treatments, specifically those amended with fishmeal biochar. Incorporation was the most successful application method for these northern soils, and although biochar amendments are not clearly effective in reducing PHC concentrations, there is evidence to suggest it can beneficially influence soil properties and PHC degradation under specific environmental conditions.

Petroleum hydrocarbon remediation in frozen soil using a meat and bonemeal biochar plus fertilizer.

Petroleum hydrocarbon (PHC) degradation slows significantly during the winter which substantially increases the time it takes to remediate soil in Arctic landfarms. The aim of this laboratory trial was to assess the potential of a meat and bonemeal (MBM) biochar to stimulate PHC degradation in contaminated soil collected from Iqaluit, Canada. Over 90 days, 3% (w/w) MBM biochar significantly increased F3- (equivalent nC16-C34) PHC degradation rate constants (k) in frozen soils when compared to the fertilizer (urea and monoammonium phosphate) control. Taking into consideration extensive variability within treatments and negative k values, this difference may not reflect significant remediation. Decreasing C17/Pr and C18/Ph ratios in the frozen soil suggest that this reduction is a result of microbial degradation rather than volatilization. Amendment type and application rate affected the immediate abiotic losses of F2 and F3-PHC in sterile soils, with the greatest losses occurring in compost-amended treatments in the first 24 h. In frozen soils, MBM biochar was found to increase liquid water content (θliquid) but not nutrient supply rates. Under frozen but not thawed conditions, genes for aromatic (C2,3O and nahAc) but not aliphatic (alkB) PHC degradation increased over time in both biochar-amended and control treatments but total viable PHC-degrading populations only increased in biochar-amended soils. Based on these results, it is possible that PHC degradation in biochar-amended soils is active and even enhanced under frozen conditions, but further investigation is required.