Microspectroscopic visualization of how biochar lifts the soil organic carbon ceiling.

The soil carbon (C) saturation concept suggests an upper limit to the storage of soil organic carbon (SOC). It is set by the mechanisms that protect soil organic matter from mineralization. Biochar has the capacity to protect new C, including rhizodeposits and microbial necromass. However, the decadal-scale mechanisms by which biochar influences the molecular diversity, spatial heterogeneity, and temporal changes in SOC persistence, remain unresolved. Here we show that the soil C storage ceiling of a Ferralsol under subtropical pasture was raised by a second application of Eucalyptus saligna biochar 8.2 years after the first application-the first application raised the soil C storage ceiling by 9.3 Mg new C ha-1 and the second application raised this by another 2.3 Mg new C ha-1. Linking direct visual evidence from one-, two-, and three-dimensional analyses with SOC quantification, we found high spatial heterogeneity of C functional groups that resulted in the retention of rhizodeposits and microbial necromass in microaggregates (53-250 µm) and the mineral fraction (<53 µm). Microbial C-use efficiency was concomitantly increased by lowering specific enzyme activities, contributing to the decreased mineralization of native SOC by 18%. We suggest that the SOC ceiling can be lifted using biochar in (sub)tropical grasslands globally.

An agricultural practise with climate and food security benefits: "Claying" with kaolinitic clay subsoil decreased soil carbon priming and mineralisation in sandy cropping soils.

As the agricultural sector seeks to feed a growing global population, climate-smart agriculture offers opportunities to concurrently mitigate climate change by reducing greenhouse gas emissions and/or increasing carbon storage in soils. This study examined the potential for clay addition to reduce CO2 emissions from plant residues and soil organic matter in a sandy soil. Soils were sourced from a 15-year-old field trial where claying (200 t ha-1) had already demonstrated improvements in water infiltration, grain yield and profits. Isotopically labelled plant residues (wheat, canola, or pea) were used to separate residue-derived and soil-derived CO2 sources from a nil-clay control, a historically clayed, and two freshly created soils with either high (10%) or low (3%) subsoil clay additions. Laboratory incubations demonstrated that historically clayed soils released less CO2 from plant residues and soil organic matter. Clay addition also decreased the priming effect of adding fresh residue to soils. The results from clay experimentally added in the laboratory varied. Differences in chemical and biological indicators (pH, microbial biomass C and N, extractable organic C and N, NO3-, NH4+, abundance of bacterial, archaeal, fungal, LMCO, GH48 and CbhI genes) did not correlate with patterns of CO2 emissions across treatments. While claying practices have previously demonstrated benefits to crop productivity, this research demonstrates long-term changes in carbon-cycling that could promote greater carbon sequestration.

Soil Microbial Community Responses After Amendment with Thermally Altered Pinus radiata Needles.

Post-fire litter layers are composed of leaves and woody debris that predominantly fall during or soon after the fire event. These layers are distinctly different to pre-fire litters due to their common origin and deposition time. However, heterogeneity can arise from the variable thermal conditions in the canopy during fire. Therefore, in this study, we used thermally altered pine needles (heated to 40 °C, 150 °C, 260 °C and 320 °C for 1 h) in a laboratory incubation study for 43 days. These samples were measured for respiration throughout and extracted for DNA at the experiment's end; soil ribosomal RNA was analysed using Illumina sequencing (16S and internal transcribed spacer amplicons). The addition of pine needles heated to 40 °C or 150 °C caused a substantial shift in community structure, decreased alpha diversity and significantly increased soil respiration relative to the control treatment. In contrast, pine needles heated to 260 °C or 320 °C had little effect on microbial community structure or soil respiration. These results indicate that highly thermally altered needles are not microbially decomposed during the first 43 days of exposure and therefore that biomass temperature may have significant effects on post-fire litter decomposition and carbon flux. This research outlines an important knowledge gap in forest fire responses that may affect post-fire carbon emission estimates.

Earthworm-induced shifts in microbial diversity in soils with rare versus established invasive earthworm populations.

European earthworms have colonised many parts of Australia, although their impact on soil microbial communities remains largely uncharacterised. An experiment was conducted to contrast the responses to Aporrectodea trapezoides introduction between soils from sites with established (Talmo, 64 A. trapezoides m-2) and rare (Glenrock, 0.6 A. trapezoides m-2) A. trapezoides populations. Our hypothesis was that earthworm introduction would lead to similar changes in bacterial communities in both soils. The effects of earthworm introduction (earthworm activity and cadaver decomposition) did not lead to a convergence of bacterial community composition between the two soils. However, in both soils, the Firmicutes decreased in abundance and a common set of bacteria responded positively to earthworms. The increase in the abundance of Flavobacterium, Chitinophagaceae, Rhodocyclaceae and Sphingobacteriales were consistent with previous studies. Evidence for possible soil resistance to earthworms was observed, with lower earthworm survival in Glenrock microcosms coinciding with A. trapezoides rarity in this site, lower soil organic matter and clay content and differences in the diversity and abundance of potential earthworm mutualist bacteria. These results suggest that while the impacts of earthworms vary between different soils, the consistent response of some bacteria may aid in predicting the impacts of earthworms on soil ecosystems.

In Situ Persistence and Migration of Biochar Carbon and Its Impact on Native Carbon Emission in Contrasting Soils under Managed Temperate Pastures.

Pyrogenic carbon (PyC) is an important component of the global soil carbon (C) pool, but its fate, persistence, and loss dynamics in contrasting soils and environments under planted field conditions are poorly understood. To fill this knowledge gap, a 13C-labelled biochar, as a surrogate material for PyC, produced from Eucalyptus saligna by slow pyrolysis (450°C; δ13C -36.7‰) was surface (0-10 cm) applied in C3 dominated temperate pasture systems across Arenosol, Cambisol and Ferralsol. The results show a low proportion of the applied biochar-C mineralised over 12 months in a relatively clay- and C-poor Arenosol (i.e., 2.0% loss via mineralisation), followed by a clay- and C-rich Cambisol (4.6%), and clay-, C- and earthworm-rich Ferralsol (7.0%). The biochar-C mean residence time (MRT), estimated by different models, varied between 44-1079 (Arenosol), 18-172 (Cambisol), and 11-29 (Ferralsol) years, with the shorter MRT estimated by a one-pool exponential and the longer MRT by an infinite-pool power or a two-pool exponential model. The two-pool model was best fitted to biochar-C mineralisation. The biochar-C recovery in the 12-30 cm soil layer varied from between 1.2% (Arenosol), 2.5-2.7% (Cambisol) and 13.8-15.7% (Ferralsol) of the applied biochar-C after 8-12 months. There was a further migration of biochar-C below the 50-cm depth in the Arenosol, as the combined biochar-C recovery in the mineralised pool and soil profile (up to 30 or 50 cm) was 82%, in contrast to 101% in the Cambisol and 104% in the Ferralsol after 12 months. These results indicate that the downward migration of biochar-C was greatest in the Arenosol (cf. Cambisol and Ferralsol). Cumulative CO2-C emission from native soil-plant sources was lower (p <0.10) in the biochar-amended vs. non-amended Ferralsol. This field-based study shows that the downward migration of biochar-C exceeded its loss via mineralisation in the Arenosol and Ferralsol, but not in the Cambisol. It is thus important to understand biochar-soil interactions to maximise long-term biochar C sequestration potential in planted soil systems.

Predicting the origin of soil evidence: High throughput eukaryote sequencing and MIR spectroscopy applied to a crime scene scenario.

Soil can serve as powerful trace evidence in forensic casework, because it is highly individualistic and can be characterised using a number of techniques. Complex soil matrixes can support a vast number of organisms that can provide a site-specific signal for use in forensic soil discrimination. Previous DNA fingerprinting techniques rely on variations in fragment length to distinguish between soil profiles and focus solely on microbial communities. However, the recent development of high throughput sequencing (HTS) has the potential to provide a more detailed picture of the soil community by accessing non-culturable microorganisms and by identifying specific bacteria, fungi, and plants within soil. To demonstrate the application of HTS to forensic soil analysis, 18S ribosomal RNA profiles of six forensic mock crime scene samples were compared to those collected from seven reference locations across South Australia. Our results demonstrate the utility of non-bacterial DNA to discriminate between different sites, and were able to link a soil to a particular location. In addition, HTS complemented traditional Mid Infrared (MIR) spectroscopy soil profiling, but was able to provide statistically stronger discriminatory power at a finer scale. Through the design of an experimental case scenario, we highlight the considerations and potential limitations of this method in forensic casework. We show that HTS analysis of soil eukaryotes was robust to environmental variation, e.g. rainfall and temperature, transfer effects, storage effects and spatial variation. In addition, this study utilises novel analytical methodologies to interpret results for investigative purposes and provides prediction statistics to support soil DNA analysis for evidential stages of a case.

C/N ratio drives soil actinobacterial cellobiohydrolase gene diversity.

Cellulose accounts for approximately half of photosynthesis-fixed carbon; however, the ecology of its degradation in soil is still relatively poorly understood. The role of actinobacteria in cellulose degradation has not been extensively investigated despite their abundance in soil and known cellulose degradation capability. Here, the diversity and abundance of the actinobacterial glycoside hydrolase family 48 (cellobiohydrolase) gene in soils from three paired pasture-woodland sites were determined by using terminal restriction fragment length polymorphism (T-RFLP) analysis and clone libraries with gene-specific primers. For comparison, the diversity and abundance of general bacteria and fungi were also assessed. Phylogenetic analysis of the nucleotide sequences of 80 clones revealed significant new diversity of actinobacterial GH48 genes, and analysis of translated protein sequences showed that these enzymes are likely to represent functional cellobiohydrolases. The soil C/N ratio was the primary environmental driver of GH48 community compositions across sites and land uses, demonstrating the importance of substrate quality in their ecology. Furthermore, mid-infrared (MIR) spectrometry-predicted humic organic carbon was distinctly more important to GH48 diversity than to total bacterial and fungal diversity. This suggests a link between the actinobacterial GH48 community and soil organic carbon dynamics and highlights the potential importance of actinobacteria in the terrestrial carbon cycle.

Network analysis reveals that bacteria and fungi form modules that correlate independently with soil parameters.

Network and multivariate statistical analyses were performed to determine interactions between bacterial and fungal community terminal restriction length polymorphisms as well as soil properties in paired woodland and pasture sites. Canonical correspondence analysis (CCA) revealed that shifts in woodland community composition correlated with soil dissolved organic carbon, while changes in pasture community composition correlated with moisture, nitrogen and phosphorus. Weighted correlation network analysis detected two distinct microbial modules per land use. Bacterial and fungal ribotypes did not group separately, rather all modules comprised of both bacterial and fungal ribotypes. Woodland modules had a similar fungal : bacterial ribotype ratio, while in the pasture, one module was fungal dominated. There was no correspondence between pasture and woodland modules in their ribotype composition. The modules had different relationships to soil variables, and these contrasts were not detected without the use of network analysis. This study demonstrated that fungi and bacteria, components of the soil microbial communities usually treated as separate functional groups as in a CCA approach, were co-correlated and formed distinct associations in these adjacent habitats. Understanding these distinct modular associations may shed more light on their niche space in the soil environment, and allow a more realistic description of soil microbial ecology and function.

Changes in the nature of dissolved organics during pulp and paper mill wastewater treatment: a multivariate statistical study combining data from three analytical techniques.

The paper-making process can produce large amounts of wastewater (WW) with high particulate and dissolved organic loads. Generally, in developed countries, stringent international regulations for environmental protection require pulp and paper mill WW to be treated to reduce the organic load prior to discharge into the receiving environment. This can be achieved by primary and secondary treatments involving both chemical and biological processes. These processes result in complex changes in the nature of the organic material, as some components are mineralised and others are transformed. In this study, changes in the nature of organics through different stages of secondary treatment of pulp and paper mill WW were followed using three advanced characterisation techniques: solid-state (13)C nuclear magnetic resonance (NMR) spectroscopy, pyrolysis-gas chromatography mass spectrometry (py-GCMS) and high-performance size-exclusion chromatography (HPSEC). Each technique provided a different perspective on the changes that occurred. To compare the different chemical perspectives in terms of the degree of similarity/difference between samples, we employed non-metric multidimensional scaling. Results indicate that NMR and HPSEC provided strongly correlated perspectives, with 86 % of the discrimination between the organic samples common to both techniques. Conversely, py-GCMS was found to provide a unique, and thus complementary, perspective.

Microbial utilisation of biochar-derived carbon.

Whilst largely considered an inert material, biochar has been documented to contain a small yet significant fraction of microbially available labile organic carbon (C). Biochar addition to soil has also been reported to alter soil microbial community structure, and to both stimulate and retard the decomposition of native soil organic matter (SOM). We conducted a short-term incubation experiment using two (13)C-labelled biochars produced from wheat or eucalypt shoots, which were incorporated in an aridic arenosol to examine the fate of the labile fraction of biochar-C through the microbial community. This was achieved using compound specific isotopic analysis (CSIA) of phospholipid fatty acids (PLFAs). A proportion of the biologically-available fraction of both biochars was rapidly (within three days) utilised by gram positive bacteria. There was a sharp peak in CO2 evolution shortly after biochar addition, resulting from rapid turnover of labile C components in biochars and through positive priming of native SOM. Our results demonstrate that this CO2 evolution was at least partially microbially mediated, and that biochar application to soil can cause significant and rapid changes in the soil microbial community; likely due to addition of labile C and increases in soil pH.

Poor efficacy of herbicides in biochar-amended soils as affected by their chemistry and mode of action.

We evaluated wheat straw biochar produced at 450°C for its ability to influence bioavailability and persistence of two commonly used herbicides (atrazine and trifluralin) with different modes of action (photosynthesis versus root tip mitosis inhibitors) in two contrasting soils. The biochar was added to soils at 0%, 0.5% and 1.0% (w/w) and the herbicides were applied to those soil-biochar mixes at nil, half, full, two times, and four times, the recommended dosage (H(4)). Annual ryegrass (Lolium rigidum) was grown in biochar amended soils for 1 month. Biochar had a positive impact on ryegrass survival rate and above-ground biomass at most of the application rates, and particularly at H(4). Within any given biochar treatment, increasing herbicide application decreased the survival rate and fresh weight of above-ground biomass. Biomass production across the biochar treatment gradient significantly differed (p<0.01) and was more pronounced in the case of atrazine than trifluralin. For example, the dose-response analysis showed that in the presence of 1% biochar in soil, the value of GR(50) (i.e. the dose required to reduce weed biomass by 50%) for atrazine increased by 3.5 times, whereas it increased only by a factor of 1.6 in the case of trifluralin. The combination of the chemical properties and the mode of action governed the extent of biochar-induced reduction in bioavailability of herbicides. The greater biomass of ryegrass in the soil containing the highest biochar (despite having the highest herbicide residues) demonstrates decreased bioavailability of the chemicals caused by the wheat straw biochar. This work clearly demonstrates decreased efficacy of herbicides in biochar amended soils. The role played by herbicide chemistry and mode of action will have major implications in choosing the appropriate application rates for biochar amended soils.