Analysis of physical pore space characteristics of two pyrolytic biochars and potential as microhabitat

Biochar amendment to soil is a promising practice of enhancing productivity of agricultural systems. The positive effects on crop are often attributed to a promotion of beneficial soil microorganisms while suppressing pathogens e.g. This study aims to determine the influence of biochar feedstock on (i) spontaneous and fungi inoculated microbial colonisation of biochar particles and (ii) physical pore space characteristics of native and fungi colonised biochar particles which impact microbial habitat quality. Pyrolytic biochars from mixed woods and Miscanthus were investigated towards spontaneous colonisation by classical microbiological isolation, phylogenetic identification of bacterial and fungal strains, and microbial respiration analysis. Physical pore space characteristics of biochar particles were determined by X-ray μ-CT. Subsequent 3D image analysis included porosity, surface area, connectivities, and pore size distribution. Microorganisms isolated from Wood biochar were more abundant and proliferated faster than those from the Miscanthus biochar. All isolated bacteria belonged to gram-positive bacteria and were feedstock specific. Respiration analysis revealed higher microbial activity for Wood biochar after water and substrate amendment while basal respiration was on the same low level for both biochars. Differences in porosity and physical surface area were detected only in interaction with biochar-specific colonisation. Miscanthus biochar was shown to have higher connectivity values in surface, volume and transmission than Wood biochars as well as larger pores as observed by pore size distribution. Differences in physical properties between colonised and non-colonised particles were larger in Miscanthus biochar than in Wood biochar. Vigorous colonisation was found on Wood biochar compared to Miscanthus biochar. This is contrasted by our findings from physical pore space analysis which suggests better habitat quality in Miscanthus biochar than in Wood biochar. We conclude that (i) the selected feedstocks display large differences in microbial habitat quality as well as physical pore space characteristics and (ii) physical description of biochars alone does not suffice for the reliable prediction of microbial habitat quality and recommend that physical and surface chemical data should be linked for this purpose.


Introduction
space is actually accessible to soil life due to size limitations (Hattori, 1988). As many 80 microorganisms show movement which is passive by water flow rather than active motility, 81 spread along particle surfaces is considered a major means of movement, rendering pore 82 space characteristics such as surface or directional connectivity more meaningful to microbial

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There is broad agreement that fungal hyphae can access biochar for habitat (Ascough et al.   We expect the biochars of two different feedstocks to be different in pore geometry for all 121 investigated parameters. Since fungal inoculation enters biochars' pores, it is assumed that 122 porosities would be reduced but analysed surface and directional connectivities would be 123 increased due to the establishment of pathways via fungal growth.

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Biochars and treatments 128 Chars representing different feedstocks and being commonly applied as soil amendments 129 were used in order to account for differences in the investigated properties. Commercial biochars from mixed deciduous and coniferous woods (W; Schottdorf, Romania) and Miscanthus (M; delinat, Switzerland) chips were purchased and shipped in sealed big-bags 132 directly after production to the University of Bremen where they were stored for 3 years in a          The samples were introduced as random factor in the model.    No systematic effect of the biochar and fungal inoculation on porosity was found (p > 0.05, 277 Figure 5). However, a significant interaction between the two factors was observed (p < 0.05).

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The post-hoc test revealed significant differences (p < 0.05) between both native biochars. For           Figure S1). Due to the high similarity in optical density between biochar and the mycelium no quantification of fungal biomass or habitat access was possible. Nevertheless, changes in 337 functional pore space characteristics between biochar colonised by fungi and native biochar 338 particles is indicative of extensive habitat access by the fungus.

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Our microbiological approach of testing bacterial presence on the biochars' surface was 340 influenced by mycelial structures on the agar plates which proliferated much faster than 341 emerging bacterial colonies. However, fungal habitat potential of the two biochars is 342 accounted for by the indication of fungal hyphae in the biochar particles via X-ray µ-CT and 343 the related changes in pore space characteristics.. 344 We did not find differences between Wood and Miscanthus biochar regarding porosity or 345 physical surface area as determinants of habitable space available for microbial colonisation.  Naturally, our approach of placing biochar particles on agar plates and investigating emerging colonies is constraint by the limited contact surface (less than 50% of the particles' surface) 389 between the biochar particles and the medium. However, assuming all parts of a biochar 390 particle have the same probability of exposure towards microbial colonisation, our partial 391 insights can be regarded as representative for the entire biochar particles. Nevertheless, 392 oligotrophic microorganisms are substantially neglected using a standard nutrient medium for 393 cultivation as we did (Atlas 2010).  While the biochar itself probably exerts a selective influence on microbial attachment and 403 colonisation, it must not be neglected that every colonisation reflects the materials' exposure 404 history e.g. during quenching with water after pyrolysis as a further selective factor. As both 405 biochars were stored under the same conditions, they either exert a very strong selective 406 influence on their spontaneous colonisation or have been exposed to colonisation between 407 pyrolytic production and packing. Either case is important for practitioners because biochars Miscanthus has a tendency towards larger pores and higher connectivities than Wood biochar.

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While Wood biochar is a rather homogeneous material, biochar derived from the grass 432 Miscanthus displays a higher variability, probably due to low mechanical stability and 433 subsequent breaking. But habitat features such as porosity, physical surface area, and pore 434 size distribution can be influenced by colonising organisms, as access by fungal hyphae 435 shows. This renders habitat quality as a dynamic feature, prone to constant change as 436 colonisation takes place. 437 We also revealed bacterial presence on the biochar surface to be biochar-specific. Rapidly

S3: DNA extraction and PCR/DGGE analysis
DNA from selected isolates was extracted by a bead-beating procedure in 2 ml reaction cups. After centrifugation and removal of liquid medium the cell pellet was resuspended in extraction buffer (100 mM Tris, 50 mM EDTA, 50 mM NaCl, 0.5 % SDS (w/v),100 µg ml-1 Proteinase K, final concentrations) and incubated at 50°C for 10 min. Sterile glass beads were added (700 mg, 1 mm diameter; 400 mg, 0.1 mm diameter) and the cups were shaken in a mixer mill (MM200, Retsch, Germany) at 25 Hz for 30 s. Proteins were removed by ammonium acetate and DNA was precipitated by the addition of one volume of isopropanol. The DNA was washed with 70 % ethanol, air dried, dissolved in TE buffer and stored at 20°C. For fungal DNA extraction the mycelium was first air dried and disrupted by pestling in extraction buffer followed by the glass bead extraction as described above.
The 16S rRNA genes were amplified using universal bacterial primers Gm5F (with gc clamp) and 907r (Muyzer et al. 1995). A touchdown program was conducted with an initial denaturation at 95°C for 60 s, followed by 13 cycles of 30 s denaturation at 95°C, annealing for 25 s at 57°C with a decrement of 0.5°C per cycle and an extension at 72°C for 13 s. The PCR fragments were separated by denaturing gradient gel electrophoresis (DGGE) with a 50 to 70 % denaturing gradient (100 % denaturant contained 7 mol l-1 urea and 40 % (v/v) deionized formamide) at 60°C and 60 V for 16 h using a DGGE 2001 apparatus (CBS Scientific, USA). Selected bands of different gel positions were excised, reamplified by PCR and purified for later sequencing.
The fungal strains were selected by colony morphology. The 18S rRNA genes were PCR amplified using the NS1 and EF3 primers (Hoshino & Morimoto 2008). The PCR programme was conducted with an initial denaturation at 94°C for 120 s, followed by 25 cycles of 15 s denaturation at 94°C, annealing for 30 s at 47°C and an extension at 72°C for 120 s followed by a final extension of 8 min. The content of the PCR reactions were the same as for bacteria with the exception that the final MgCl2 concentration was 3 mM.