Zinc toxicity stimulates microbial production of extracellular polymers in a copiotrophic acid soil

The production of extracellular polymeric substances (EPS) is crucial for biofilm structure, microbial nutrition and proximal stability of habitat in a variety of environments. However, the production patterns of microbial EPS in soils as affected by heavy metal contamination remain uncertain. Here we investigate the extracellular response of the native microbial biomass in a grassland soil treated with refined glycerol or crude unrefined biodiesel co-product (BCP) with and without ZnCl2. We extracted microbial EPS and more readily soluble microbial products (SMP), and quantified total polysaccharide, uronic acid, and protein content in these respective extracts. Organic addition, especially BCP, significantly stimulated the production of EPS-polysaccharide and protein but had no impact on EPS-uronic acids, while in the SMP-fraction, polysaccharides and uronic acids were both significantly increased. In response to the inclusion of Zn2+, both EPS- and SMP-polysaccharides increased. This implies firstly that a tolerance mechanism of soil microorganisms against Zn2+ toxicity exists through the stimulation of SMP and EPS production, and secondly that co-products of biofuel industries may have value-added use in bioremediation efforts to support in-situ production of microbial biopolymers. Microbial films and mobile polymers are likely to impact a range of soil properties. The recent focus on EPS research in soils is anticipated to help contribute an improved understanding of biofilm dynamics in other complex systems - such as continuously operated bioreactors

Soil organic matter and the extracellular microbial matrix show contrasting responses to C and N availability

AbstractAn emerging paradigm in soil science suggests microbes can perform ‘N mining’ from recalcitrant soil organic matter (SOM) in conditions of low N availability. However, this requires the production of extracellular structures rich in N (including enzymes and structural components) and thus defies stoichiometric expectation. We set out to extract newly synthesised peptides from the extracellular matrix in soil and compare the amino acid (AA) profiles, N incorporation and AA dynamics in response to labile inputs of contrasting C/N ratio. Glycerol was added both with and without an inorganic source of N (10% 15N labelled NH4NO3) to a soil already containing a large pool of refractory SOM and incubated for 10 days. The resulting total soil peptide (TSP) and extracellular pools were compared using colorimetric methods, gas chromatography, and isotope ratio mass spectrometry. N isotope compositions showed that the extracellular polymeric substance (EPS) contained a greater proportion of products formed de novo than did TSP, with hydrophobic EPS-AAs (leucine, isoleucine, phenylalanine, hydroxyproline and tyrosine) deriving substantially more N from the inorganic source provided. Quantitative comparison between extracts showed that the EPS contained greater relative proportions of alanine, glycine, proline, phenylalanine and tyrosine. The greatest increases in EPS-peptide and EPS-polysaccharide concentrations occurred at the highest C/N ratios. All EPS-AAs responded similarly to treatment whereas the responses of TSP were more complex. The results suggest that extracellular investment of N (as EPS peptides) is a microbial survival mechanism in conditions of low N/high C which, from an evolutionary perspective, must ultimately lead to the tendency for increased N returns to the microbial biomass. A conceptual model is proposed that describes the dynamics of the extracellular matrix in response to the C/N ratio of labile inputs

Biodiesel co-product (BCP) decreases soil nitrogen (N) losses to groundwater

This study compares a traditional agricultural approach to minimise N pollution of groundwater (incorporation of crop residues) with applications of small amounts of biodiesel co-product (BCP) to arable soils. Loss of N from soil to the aqueous phase was shown to be greatly reduced in the laboratory, mainly by decreasing concentrations of dissolved nitrate-N. Increases in soil microbial biomass occurred within 4 days of BCP application—indicating rapid adaptation of the soil microbial community. Increases in biomass-N suggest that microbes were partly mechanistic in the immobilisation of N in soil. Straw, meadow-grass and BCP were subsequently incorporated into experimental soil mesocosms of depth equal to plough layer (23 cm), and placed in an exposed netted tunnel to simulate field conditions. Leachate was collected after rainfall between the autumn of 2009 and spring of 2010. Treatment with BCP resulted in less total-N transferred from soil to water over the entire period, with 32.1, 18.9, 13.2 and 4.2 mg N kg(−1) soil leached cumulatively from the control, grass, straw and BCP treatments, respectively. More than 99 % of nitrate leaching was prevented using BCP. Accordingly, soils provided with crop residues or BCP showed statistically significant increases in soil N and C compared to the control (no incorporation). Microbial biomass, indicated by soil ATP concentration, was also highest for soils given BCP (p < 0.05). These results indicate that field-scale incorporation of BCP may be an effective method to reduce nitrogen loss from agricultural soils, prevent nitrate pollution of groundwater and augment the soil microbial biomass

Soil organic carbon, extracellular polymeric substances (EPS), and soil structural stability as affected by previous and current land-use

While soil microbial ecology, soil organic carbon (SOC) and soil physical quality are widely understood to be interrelated — the underlying drivers of emergent properties, from land management to biochemistry, are hotly debated. Biological binding agents, microbial exudates, or ‘extracellular polymeric substances’ (EPS) in soil are now receiving increased attention due to several of the existing methodological challenges having been overcome. We applied a recently developed approach to quantify soil EPS, as extracellular protein and extracellular polysaccharide, on the well-characterised soils of the Highfield Experiment, Rothamsted Research, UK. Our aim was to investigate the links between agricultural land use, SOC, transient binding agents known as EPS, and their impacts on soil physical quality (given by mean weight diameter of water stable aggregates; MWD). We compared the legacy effects from long-term previous land-uses (unfertilised grassland, fertilised arable, and fallow) which were established >50 years prior to investigation, crossed with the same current land-uses established for a duration of only 2.5 years prior to sampling. Continuously fallow and grassland soils represented the poorest and greatest states of structural integrity, respectively. Total SOC and %N were found to be affected by both previous and current land-uses, while extractable EPS and MWD were driven primarily by the current land-use. Land-use change between these two extremes (fallow→grass; grass→fallow) resulted in smaller SOC differences (64% increase or 37% loss) compared to MWD (125% increase or 78% loss). SOC concentration correlated well to MWD (adjusted R2 = 0.72) but the high SOC content from previous grassland was not found to contribute directly to the current stability (p < 0.05). Our work thus supports the view that certain distinct components of SOC, rather than the total pool, have disproportionately important effects on a soil’s structural stability. EPS-protein was more closely related to aggregate stability than EPS-polysaccharide (p values of 0.002 and 0.027, respectively), and ranking soils with the 5 highest concentrations of EPS-protein to their corresponding orders of stability (MWD) resulted in a perfect match. We confirmed that both EPS-protein and EPS-polysaccharide were transient fractions: supporting the founding models for aggregate formation. We suggest that management of transient binding agents such as EPS —as opposed to simply increasing the total SOC content— may be a more feasible strategy to improve soil structural integrity and help achieve environmental objectives

Engineering soil organic matter quality: Biodiesel Co-Product (BCP) stimulates exudation of nitrogenous microbial biopolymers

Biodiesel Co-Product (BCP) is a complex organic material formed during the transesterification of lipids. We investigated the effect of BCP on the extracellular microbial matrix or ‘extracellular polymeric substance’ (EPS) in soil which is suspected to be a highly influential fraction of soil organic matter (SOM). It was hypothesised that more N would be transferred to EPS in soil given BCP compared to soil given glycerol. An arable soil was amended with BCP produced from either 1) waste vegetable oils or 2) pure oilseed rape oil, and compared with soil amended with 99% pure glycerol; all were provided with 15N labelled KNO3. We compared transfer of microbially assimilated 15N into the extracellular amino acid pool, and measured concomitant production of exopolysaccharide. Following incubation, the 15N enrichment of total hydrolysable amino acids (THAAs) indicated that intracellular anabolic products had incorporated the labelled N primarily as glutamine and glutamate. A greater proportion of the amino acids in EPS were found to contain 15N than those in the THAA pool, indicating that the increase in EPS was comprised of bioproducts synthesised de novo. Moreover, BCP had increased the EPS production efficiency of the soil microbial community (μg EPS per unit ATP) up to approximately double that of glycerol, and caused transfer of 21% more 15N from soil solution into EPS-amino acids. Given the suspected value of EPS in agricultural soils, the use of BCP to stimulate exudation is an interesting tool to consider in the theme of delivering sustainable intensification

Sequestration of C in soils under Miscanthus can be marginal and is affected by genotype-specific root distribution

AbstractMiscanthus is a low input energy crop suitable for low fertility marginal arable land and thought to provide carbon sequestration in soil. We analysed a long-term field experiment (14-year) to determine whether differences in genotype, growth habit, and root distribution affected soil carbon spatially under different Miscanthus genotypes. Soil cores were taken centrally and radially to a depth of 1m, and divided into six vertical segments. Total root length (TRL), root dry matter (RDM) and δ13C signature of soil organic carbon (SOC) were measured directly, and root length density (RLD), fractions of Miscanthus-derived soil organic C (SOCM), and residual soil carbon (SOCorig) were calculated. Genotype was found to exhibit a statistically significant influence on spatial allocation of SOC. Grouping varieties into ‘tuft-forming’ (T) and ‘non-tuft-forming’ (NT) phenotypes revealed that respective groups accumulated similar amounts of RDM over 14 years (11.4±3.3 vs. 11.9±4.8Mgha−1, respectively). However, phenotype T allocated more carbon to roots in the subsoil than NT (33% vs. 25%). Miscanthus genotypes sequestered between 4.2 and 7.1gC4-SOCkg−1 soil over the same period, which was more than the average loss of C3-derived SOC (3.25gkg−1). Carbon stocks in the ‘A horizon’ under Miscanthus increased by about 5Mgha−1 above the baseline, while the net increase in the subsoil was marginal. Amounts of Miscanthus root C in the subsoil were small (1.2–1.8MgCha−1) but could be important for sustainable sequestration as root density (RLD) explained a high percentage of SOCM (R2=0.66)

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts

Measuring the soil-microbial interface: extraction of extracellular polymeric substances (EPS) from soil biofilms

Many soil microbes exist in biofilms. These biofilms are typified by variable quantities of extracellular polymeric substances (EPS: predominantly polysaccharides, glycoconjugates, and proteins) and the embedded microbial cells. A method to measure soil-EPS (the biofilm exclusive of microbial cells) has not yet been described. The present work investigates the potential of five extraction methods to estimate changes in soil-EPS content. A rationale for selection of appropriate EPS extraction and methodology is discussed, including the crucial consideration of both intracellular and extracellular contamination. EPS was developed in situ by provision of labile C (glycerol) to the microbial biomass of a moist soil and then applying desiccation stress. Only two out of the five extraction methods showed statistically significant increases in polysaccharide production responding to substrate addition. Humified organic matter, estimated by its humic acid equivalent (HAE) was used to indicate the degree of extracellular contamination, and/or creation of humic artefacts – both of which affect detection of changes in EPS. The HAE concentration was very high when applying original and modified methods designed to extract glomalin related soil protein (GRSP). Extraction methods involving heating with dilute sulphuric acid appeared to overestimate EPS-polysaccharide. Using microbial ATP as an indicator of cell-lysis, confidence could only be ascribed to EPS extraction with cation exchange resin. Using this method, the expected increases in EPS-polysaccharide were clearly apparent. The HAE/protein ratios of EPS extracts were also lowest with cation exchange – indicating this method did not cause excessive contamination from humified soil organic matter or create related artefacts

Effects of cropping systems upon the three-dimensional architecture of soil systems are modulated by texture

Soil delivers fundamental ecosystem functions via interactions between physical and biological processes mediated by soil structure. The structure of soil is also dynamic and modified by natural factors and management intervention. The aim of this study was to investigate the effects of different cropping systems on soil structure at contrasting spatial scales. Three systems were studied in replicated plot field experiments involving varying degrees of plant-derived inputs to the soil, viz. perennial (grassland), annual (arable), and no-plant control (bare fallow), associated with two contrasting soil textures (clayey and sandy). We hypothesized the presence of plants results in a greater range (diversity) of pore sizes and that perennial cropping systems invoke greater structural heterogeneity. Accordingly, the nature of the pore systems was visualised and quantified in 3D by X-ray Computed Tomography at the mm and μm scale. Plants did not affect the porosity of clay soil at the mm scale, but at the μm scale, annual and perennial plant cover resulted in significantly increased porosity, a wider range of pore sizes and greater connectivity compared to bare fallow soil. However, the opposite occurred in the sandy soil, where plants decreased the porosity and pore connectivity at the mm scale but had no significant structural effect at the μm scale. These data reveal profound effects of different agricultural management systems upon soil structural modification, which are strongly modulated by the extent of plant presence and also contingent on the inherent texture of the soil

Soil Characteristics Overwhelm Cultivar Effects on the Structure and Assembly of Root-Associated Microbiomes of Modern Maize

Modern breeding primarily targets crop yield traits and is likely to influence root-associated microbiomes, which play significant roles in plant growth and health. The relative importance of soil and cultivar factors in shaping root-associated microbiomes of modern maize (Zea mays L.) remains uncertain. We conducted a pot experiment in a controlled environment using three soils (Mollisol, Inceptisol, and Ultisol) and four contrasting cultivars, Denghai 605, Nonghua 816, Qiaoyu 8, and Zhengdan 958, which are widely planted in China. We used 16S rRNA gene amplicon sequencing to characterize the bacterial communities in the bulk soil, rhizosphere, and endosphere. Our results showed that the four cultivars had different shoot biomass and root exudate total organic carbon and organic acid contents. The microbiomes in the bulk soil, rhizosphere, and endosphere were different. We observed apparent community divergence between soils rather than cultivars, within which edaphic factors substantially contributed to microbiome variation. Moreover, permutational multivariate analysis of variance corroborated significant contributions of soil type but not cultivar on the root-associated microbiome structure. Differential abundance analysis confirmed that each soil presented a distinct root microbiome, while network analysis indicated different co-occurrence patterns of the root microbiome among the three soils. The core root microbiome members are implicated in plant growth promotion and nutrient acquisition in the roots. In conclusion, root-associated microbiomes of modern maize are much more controlled by soil characteristics than by cultivar root exudation. Our study is anticipated to help improve breeding strategies through integrative interactions of soils, cultivars, and their associated microbiomes