Assessing biogas digestate, pot ale, wood ash and rockdust as soil amendments: effects on soil chemistry and microbial community composition

<div><p>Applying by-products as soil amendments to agricultural systems is growing in popularity. We aimed to assess the efficacy of some contemporary by-products to provide nutrients to crops as well as the potential harm of adding toxic elements to the environment. Four different by-products widely available in Northern Europe were tested for their effects on two nutrient-poor agricultural soils in terms of increasing available macro- and micro-nutrients as well as toxic elements. Assessing soil microbial community as a sensitive tool for evaluating soil quality was conducted with the focus on microbial activity, carbon metabolism and on <i>Rhizobium/Agrobacterium</i>. Wood ash increased pH and Ca_EDTA, K_EDTA and Mg_EDTA in the soils. The only increase in EDTA-extractable micronutrients in the soils was observed by applying pot ale, increasing Cu. None of the amendments increased the availability of Pb and Cd in the soils. Soils amended with the by-products thus remained similar to the unamended control but were quite different from fully mineral fertilised soils. There were no detectable adverse effects on the physiological and genetic profiles of microbial communities. The by-products were moderately beneficial and did not change the soil microbial community as much as the fully fertilised treatment with mineral fertilisers. Changes in the microbial community profiles were probably due to direct effects on microbes limited by K, Ca and N as opposed to indirect effects on plant growth. This is potentially significant in understanding how to improve impoverished and marginal soils as microbial activity affects many other ecosystem functions.</p></div

Terminal Restriction Fragment (TRF) detection for DNA extraction and post-amplification clean-up protocols for inoculated milk experiments.

<p>A: TRFs detected for both <i>S. enterica</i> (SE) and <i>L. monocytogenes</i> (LM), and are indicated as described in the legend, with three SE TRFs and five LM TRFs expected for all samples. TRFs detected are shown for the mean averages of three individual replicates in each case. Error bars shown are +/-1SD for both TRFs detected values. Milk was inoculated with five strains each of <i>S. enterica</i> (SE), <i>E. coli</i> (EC) and <i>L. monocytogenes</i> (LM) at 4.4, 3.2 and 5.4 cfu per 25 ml of milk respectively for the lowest level of initial inoculum (i.e. 10⁻⁷ dilutions) and tenfold this for 10⁻⁶ dilutions. These were used to make up inoculum ratios, shown as either Cn or Wn, where C indicates Chelex extracted and cleaned samples, W indicates Wizard extracted and uncleaned samples and n indicates ratios (SE:EC:LM respectively) as follows: 1, 1∶1:1 (10⁻⁷ dilutions); 2, 1∶1:1 (10⁻⁶ dilutions); 3, 10∶1:1; 4, 1∶10:1; 5, 1∶1:10; 6, 10∶10:1; 7, 10∶1:10; 8, 1∶10:10; 9, mean average of all corresponding samples. Paired student’s two-tailed t-tests were performed between all 9 corresponding C and W datasets for both SE and LM TRF types e.g. C1 LM TRF figures were paired and compared with W1 LM TRF figures. Asterisks at the top of the corresponding W bar indicate significant differences, with one asterisk indicating a P value of <0.02, two indicating P<0.01, and three indicating P<0.0001. B: Total specific TRF detection percentages across DNA extraction and post-amplification product clean-up treatments for pre-enrichment inoculum ratio experiments as above. Specified TRFs detected for both <i>S. enterica</i> (SE) and <i>L. monocytogenes</i> (LM), and are indicated as described. Error bars shown are +/-1SD for all data, derived from triplicate datasets. Paired student’s two-tailed t-tests were performed between corresponding Clean Chelex and Wizard extracted DNA datasets for all eight TRFs. Asterisks at the top of the corresponding bar indicate significant differences, with one asterisk indicating a P value of <0.05, two indicating P<0.02, and three indicating P<0.005.</p

Fungal community data from the second National Soils Inventory of Scotland

Fungal community data from the second National Soils Inventory of Scotland. 'SAMPLE.ID' refers to the unique sample identifier associated with the NSIS2 database (to be released publically by the James Hutton Institute); 'LAYER' refers to the paedological horizon sampled (L1: topsoil horizon; L2-4: second, third, and fourth horizon in depth from the topsoil horizon); all columns with prefix ITS represent OTUs derived from T-RFLP analysis, with the value representing the proportional abundance of that OTU in that sample based on peak height (the sum of these columns equals 1)

Terminal restriction fragments (TRFs) predicted and produced from five target genes following individual species amplification and <i>Hha</i>I digestion.

<p>All TRFs are listed using the code: (dye)(length in bp).</p>*<p>TRFs unique to that species and definable on analysis.</p>a<p>TRFs combined into a single bin due to lack of distinction on analysis.</p>b<p>TRFs combined into a single bin due to lack of distinction on analysis.</p><p>UTRF: TRF derived from the upstream i.e. forward primer end of the amplicon.</p><p>DTRF: TRF derived from the downstream i.e. reverse primer end of the amplicon.</p

Strains used <i>in vitro</i> for M-TRFLP testing.

*<p>Used in milk inoculation experiments. ATCC: American Type Culture Collection, USA.; CMCC: Colworth Microbiology Culture Collection, Unilever, UK.; NCIMB and FDL: NCIMB Ltd, Aberdeen, UK.; NCTC: National Collection of Type Cultures, U.K.; SPRC and TX: Food and Drug Administration, U.S.A.; MI: This study.</p

Multiplex Polymerase Chain Reaction amplification products shown following gel electrophoresis.

<p>Lanes 1 and 12 contain HyperLadder V (Bioline), with the remaining lanes containing products derived from 6 ng genomic DNA from as shown. Internal amplification control (IAC) template DNA was included in all reactions. Templates were amplified in isolation (apart from universal inclusion of the IAC) or in combination as described in the figure using a 15-primer multiplex PCR. Codes used are as follows: SE, <i>Salmonella enterica</i> MISE807439; LM, <i>Listeria monocytogenes</i> CMCC2993; LW, <i>Listeria welshimeri</i> CMCC3366; LG, <i>Listeria grayi</i> CMCC3362. Patterns for <i>L. seeligeri</i>, <i>L. murrayi</i>, <i>L. ivanovii</i> and <i>L. innocua</i> were identical to that of <i>L. welshimeri</i>, and all patterns shown were representative of all other strains tested of the same species. Size standards are descirbed on the left of the figure in base pairs (bp), while PCR products are described on the right of the figure with both their name and size in bp. ² indicates the <i>prs</i> amplimer from <i>Listeria grayi</i> only, with ¹ denoting the corresponding product from all other <i>Listeria</i> species. Electrophoresis was performed on a 1.7% agarose gel at 70 volts for 1.5 hrs with EtBr, with 4 ul of each product loaded.</p

Bacterial community data from the second National Soils Inventory of Scotland

Bacterial community data from the second National Soils Inventory of Scotland. 'SAMPLE.ID' refers to the unique sample identifier associated with the NSIS2 database (to be released publically by the James Hutton Institute); 'LAYER' refers to the paedological horizon sampled (L1: topsoil horizon; L2-4: second, third, and fourth horizon in depth from the topsoil horizon); all columns with prefix S16 represent OTUs derived from T-RFLP analysis, with the value representing the proportional abundance of that OTU in that sample based on peak height (the sum of these columns equals 1)

Average species richness (mean ± s.e., n = 8) at 0–3 cm depth found in the heterogeneous treatment for the biotic groups sampled.

<p>Both sampling regimes used in the heterogeneous treatment are presented: d3 represents the same depth as sampled in the homogeneous treatment, whereas mix represents pooled samples collected across shallow (3 cm), medium (7.5 cm) and deep (12 cm) organic horizons.</p

Average species richness (mean ± s.e., n = 8) at 0–6 cm depth found in the two treatments for the biotic groups sampled.

<p>Letters indicate significant within-group differences between treatments (one-way ANOVA with blocks).</p

Summary of abundance data (mean ± s.e., n = 8) of mesostigmatid and oribatid mites, collembolans and nematodes (ind. ×10³ m⁻²) in the top 0–6 cm of the organic horizon in treatments representing either homogeneous or heterogeneous soil environment.

<p><i>F</i> statistic and associated significance level (one-way ANOVA with blocks; bold if significant) are presented.</p