microscopy observations of habitable space in biochar for colonization by fungal hyphae from soil

Journal of Integrative Agriculture2014, 13(3): 483-490 March 2014RESEARCH ARTICLE

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.doi: 10.1016/S2095-3119(13)60703-0

Microscopy Observations of Habitable Space in Biochar for Colonization by Fungal Hyphae From Soil

Noraini M Jaafar1, 3, Peta L Clode2 and Lynette K Abbott1

1 Soil Biology and Molecular Ecology Group, School of Earth and Environment and UWA Institute of Agriculture, The University of Western Australia, Crawley 6009, Australia

2 Centre for Microscopy, Characterisation and Analysis, The University of Western Australia, Crawley 6009, Australia3 Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, Serdang, Selangor 43400, Malaysia

Abstract

Biochar is a potential micro-environment for soil microorganisms but evidence to support this suggestion is limited. We explored imaging techniques to visualize and quantify fungal colonization of habitable spaces in a biochar made from a woody feedstock. In addition to characterization of the biochar, it was necessary to optimize preparation and observation methodologies for examining fungal colonization of the biochar. Biochar surfaces and pores were investigated using several microscopy techniques. Biochar particles were compared in soilless media and after deposition in soil. Scanning electron microscopy (SEM) observations and characterization of the biochar demonstrated structural heterogeneity within and among biochar particles. Fungal colonization in and on biochar particles was observed using light, fluorescence and electron microscopy. Fluorescent brightener RR 2200 was more effective than Calcofluor White as a hyphal stain. Biochar retrieved from soil and observed using fluorescence microscopy exhibited distinct hyphal networks on external biochar surfaces. The extent of hyphal colonization of biochar incubated in soil was much less than for biochar artificially inoculated with fungi in a soilless medium. The location of fungal hyphae was more clearly visible using SEM than with fluorescence microscopy. Observations of biochar particles colonized by hyphae from soil posed a range of difficulties including obstruction by the presence of soil particles on biochar surfaces and inside pores. Extensive hyphal colonization of the surface of the biochar in the soilless medium contrasted with limited hyphal colonization of pores within the biochar. Both visualization and quantification of hyphal colonization of surfaces and pores of biochar were restricted by two-dimensional imaging associated with uneven biochar surfaces and variable biochar pore structure. There was very little colonization of biochar from hyphae in the agricultural soil used in this study.

Key words: biochar, agriculture, fungi, soil, habitable pore space, microscopy

INTRODUCTION

Biochar is the product of biological materials that have undergone pyrolysis to create stable, carbonized

organic matter that is heterogeneous and porous in nature. Due to its potential as a soil amendment, biochar has been extensively studied using a range of microscopy techniques (Bird et al. 2008; Chia et al. 2010; Joseph et al. 2010; Moskal-del Hoyo

Received 9 October, 2013 Accepted 18 December, 2013Correspondence Noraini M Jaafar, Tel: +61-603-89474953, Fax : +61-603-89408316, E-mail: [email protected]

484 Noraini M Jaafar et al.

© 2014, CAAS. All rights reserved. Published by Elsevier Ltd.

et al. 2010). The pore and surface structures of biochars potentially provide suitable habitats for soil microorganisms (Warnock et al. 2007; Downie et al. 2009). However, the extent of colonization of these potential habitats is not well understood. There are relatively few microscopic evaluations of biochar interactions with soil conducted under controlled conditions (Joseph et al . 2010). Furthermore, changes in biochar structural characteristics over time and microbial colonization of biochar pores after incubation in soil have been unclear and inadequately investigated (Hockaday et al. 2007; Bird et al. 2008; Ascough et al. 2010b).

Some s tud ie s conc luded tha t b iocha r had potential as a habitat for soil microorganisms based on observations of fresh and pyrogenic biochars (Hockaday et al . 2007; Lehmann et al . 2011). Speculation on the possible role of biochar as a soil microbial habitat linked to enhancement of soil health has mainly been based on indirect measurements such as quantification of soil microbial biomass and other microbial indicators (Zackrisson et al. 1996; Pietikäinen et al. 2000; Hockaday et al. 2007; Lehmann et al. 2011). Fungal growth on biochar surfaces and decomposition of biochar evaluated in laboratory Petri dish study (Ascough et al. 2010b) showed biochar surfaces could be colonized by fungal hyphae. Light microscopy and scanning electron microscopy (SEM) showed that fungi colonized both the surface and interior of charcoals prepared at 300 and 400°C over three months (Ascough et al. 2010b). However, laboratory inoculation of biochar may not be representative of soil conditions. Examination of pyrogenic biochar materials retrieved from forest fire sites clearly demonstrated evidence of biochar changes and fungal colonization (Hockaday et al. 2007; Ascough et al. 2010a).

Hyphal colonization on biochar surfaces and along cracks has been reported (Ascough et al. 2010b). Cracks in biochar particles may facilitate microbial colonization (Verheijen et al. 2009; Ascough et al. 2010b). Pore connectivity and pore size are also likely to influence microbial colonization of biochar but this has received little attention. Pyrogenic biochar have been shown to be colonized by fungi on both interior and external surfaces (Hockaday et al. 2007).

However, the preferential nature of fungal colonization of biochar pores is unclear.

Colonization of habitable biochar pore spaces by soil microorganisms may depend on microbial community composition as well as on changes that occur to biochar particles in soil over time (Ascough et al. 2010a, b; Jindo et al. 2012a, b). Once biochar interacts with soil, modification of physical and chemical characteristics of biochar can occur and attachment of soil particles to biochar surfaces may alter habitat suitability and microbial activity (Thies and Rillig 2009; Lehmann et al. 2011). Further investigation of the fate of biochar particles once deposited in soil is needed to clarify the extent to which biochar pores, structural cracks, pore connectivity and surfaces contribute to biochar as a habitat for soil microorganisms (Thies and Rillig 2009; Lehmann et al. 2011).

In our study, microscopy techniques were used to estimate habitable space within biochar prior to its application in soil as well as to explore challenges associated with visualization of hyphae inhabiting pores and surfaces of biochar retrieved from soil. The aim was to assess a range of methodologies for biochar sample preparation and microscopy techniques for visualizing and quantifying the potential habitat properties of biochar. The objectives were: 1) to characterize potential habitable spaces in biochar suitable for colonization by fungal hyphae; 2) to optimize methods for visualizing hyphae associated with biochar surfaces and inside biochar particles using optical fluorescence and scanning electron microscopy, and 3) to characterize the nature of hyphal colonization of biochar both in the absence of soil and after its incubation in an agricultural soil.

RESULTS

Biochar surface and pore structures varied within a single source of a biochar that originated from Eu-calyptus wood (Fig. 1, Fig. 2-A-C). The crushing of particles into smaller particles may have contributed to heterogeneity in pore and surface structure, including cracks (Fig. 2-D). Exposed pores were visible in sur-face observations (Fig. 1, Fig. 2-A-C). Some particles had rough surfaces while others were smooth (Fig. 1).

Microscopy Observations of Habitable Space in Biochar for Colonization by Fungal Hyphae From Soil 485


tribution were similar when estimated using either the cross-sections of resin-embedded biochar or biochar mounted on stubs (Table 1).

Fig. 1 Scanning electron micrograph of Simcoa biochar showing extensive variation in particle size range (0.5-1 mm) and heterogeneity within a single biochar source. Scale bar=1 mm.

Visible pores were limited in some particles while in others, pores were highly exposed.

Biochar derived from this woody feedstock var-ied in pore size distribution for samples examined in cross-section and as whole particles (Fig. 2-E and F). Cross-sections of biochar prepared using the resin-em-bedded method (Fig. 2-E) demonstrated the internal porous architecture and pore networking whereas whole biochar fragments mounted on stubs enabled visualization of cracks (Fig. 2-D) and the structure of surface pores (Fig. 2-F). Estimates of the porosity dis-

A B C

D E F

Fig. 2 Scanning electron micrographs showing heterogeneity in surface and pore structure of biochar for particles in the range of 0.5-1 mm. A-C and F, whole biochar. D-E, cross-sections of resin-embedded biochar. Scale bars=100 µm.

Table 1 Comparison of the percentage pore-size distribution for Simcoa biochar determined by SEM imaging of whole fragments and cross-sections of biochar after resin-embedding1)

Biochar preparation

Numbers of pores measured

Pore size<50 µm 50-100 µm >100 µm

Whole 500 86% 9% 5%Cross sectioned 150 87% 7% 6%1) The minimum pore size measured was 4 µm, while the maximum pore size

measured was 210 µm.

In the soilless Petri dish study, fluorescent stains Calcofluor White and RR 2200 confirmed the presence of hyphae on biochar particles, but RR 2200 stained the hyphae more clearly (Fig. 3). There were some problems in using Calcofluor White as a fluorescent stain for hyphae (Fig. 3-A-D). Background staining was evident on biochar stained with Calcofluor White (Fig. 3-A-C) making it difficult to observe the hyphae. Residues and traces of crystals formed by Calcofluor White were also observed (Fig. 3-D). RR 2200 did not exhibit these characteristics and clear staining of fungal hyphae was observed (Fig. 3-E and F), thus RR 2200 was the preferred method for staining fungal hyphae on biochar in the soil-based incubation studies.



Biochar particles incubated in soilless media were ex-tensively colonized by hyphae but this was restricted to the biochar surface. There was little occurrence of fungal penetration into pores within the biochar (Fig. 3-E); the fungal colonization was confined pri-marily to biochar surfaces (Fig. 3-F).

Biochar retrieved from soil and observed using fluorescence microscopy exhibited distinct hyphal networking on external biochar surfaces (Fig. 4-A and B). Fluorescence microscopy allowed observa-tion of larger areas of fungal colonization across the entire biochar particle. Fungal hyphae were observed in some larger biochar pores after incubation in soil (Fig. 4-C-E). SEM imaging allowed observation and confirmation of fungal colonization earlier observed using fluorescence microscopy. The location of hy-phae both upon the surface and within pore spaces was clearly observed using SEM, and allowed visualization of biochar-hyphal interactions. Soil particles could also be clearly observed in smaller and larger biochar pores (Fig. 4-F).

SEM imaging allowed visualization of smaller ar-eas in greater detail than did fluorescence microscopy (Fig. 5-A and B). Fracturing biochar particles to ob-serve internal regions via SEM enabled observation of fungal colonization within the biochar. Detection

and quantification of soil hyphae inside biochar pores was difficult due to soil interaction with the biochar (Fig. 4-D-F). Thus, accurate quantification of hyphae was not possible. Despite the difficulty of quantifying microorganisms in biochar pores, SEM observation enabled location of fungal hyphae in larger pores but not in smaller pores. The main obstacle to observation of hyphae was the presence of soil particles within the smaller pores. Most of the pores filled with soil parti-cles were <20 µm.

DISCUSSION

The structural characteristics of biochar provide potential micro-habitats for soil organisms. SEM imaging enabled visualization of surface and pore structure of biochar following appropriate sample preparation. Considerable structural variability in biochar particles was observed. This was consistent with previous observations (Abdullah and Wu 2009; Downie et al. 2009). The woody biochar used in this study has potential as a fungal habitat based on characterization of biochar pore sizes (Warnock et al. 2007). Woody biochar can enhance colonization of roots by mycorrhizal fungi in soil (Solaiman et al.

A B C

D E F

Fig. 3 Fluorescence micrographs of biochar particles from soilless media stained with Calcofluor White (A-D); or RR 2200 (E and F). Background staining and artefactual crystallization is evident in Calcofluor White stained samples. Hyphae (arrows) colonizing the surface of biochar particles were readily observed (C, E and F), but little occurrence of hyphae penetration into biochar pores was observed in fractured samples (E). Scale bars=100 µm.



2010) but it is not clear whether biochar provides a habitat advantage for these fungi during mycorrhiza formation.

Biochar particles are usually fractured or sectioned for observation (Moskal-del Hoyo et al. 2010). For the biochar used here, particles were either fractured

A B

C D

E F

Fig. 4 Fluorescence (A and B) (stained with RR 2200) and scanning electron (C-F) micrographs of colonized biochar particles. Soil fungal networks (arrows) in biochar pores, especially larger pores, and soil particles in pores (E and F) are seen in biochar incubated in an agricultural soil for 56 d. Scale bars=100 µm.

Fig. 5 Fluorescence (A) and SEM (B) micrographs of biochar incubated in an agricultural soil for 12 wk. Fungal hyphae were stained with RR 2200. Arrows highlight difference in depth of focus and difficulty in using fluorescence imaging for understanding hyphal-biochar surface interactions. Scale bars=100 µm.

A B



or embedded in resin and polished in cross section to provide additional information on the pore connectiv-ity, which is an important criterion for microbial col-onization. Indeed, this form of visualization showed that limited connectivity of pores in this biochar could restrict colonization by fungal hyphae, and confine it to surface layers. Surface cracks in biochar particles may be important points of entry for fungal hyphae for colonization of internal spaces within forms of biochar that have limited pore connectivity.

Visualization of fungal hyphae on biochar surfaces was successful using both fluorescence microscopy and SEM. It was easy to locate fungal hyphae on the surface of biochar as demonstrated in our soilless Petri dish study and in previous investigations (Ascough et al. 2010b; Moskal-del Hoyo et al. 2010). Further-more, the level of fungal colonization in soilless media was much greater than that which occurred on biochar incubated in soil. This was confirmed in our study using a combination of fluorescence microscopy and SEM techniques. Fluorescence microscopy highlight-ed fungal colonization while SEM provided greater detail into the structural relationship between the fun-gal hyphae and biochar surface.

Soil particles were observed adhering to surfaces for biochar retrieved from soil. The impact of soil particles that enter biochar pores following incubation in soil is not known but it would likely influence the capacity of biochar to act as a habitat for fungal hy-phae. Some smaller pores were often covered by soil particles. Soil particles entering these pores include clay, silt and organic compounds (Joseph et al. 2010). Soil particles could either introduce microorganisms into the internal environment of biochar, or alterna-tively, limit their access. Predictions based on labo-ratory studies of inoculated biochar in soilless media could overestimate the role of biochar as a habitat for soil microorganisms if soil particles limited access or if there is little connectivity between pores within the biochar.

CONCLUSION

The preparation and observation of the intact biochar particles colonized by fungal hyphae in soil posed a range of difficulties including obstruction by the

presence of soil particles. The extent of hyphal colonization of biochar incubated in soil was much less than for biochar artificially inoculated with fungi in a soilless medium. Further observation of biochar surfaces and fractured biochar that exposed the internal biochar structures raised questions about the effects of interactions between soil particles and biochar for microbial colonization. The role of biochar as a habitat appeared to be minimal after incubation in this agricultural soil for 56 d.

MATERIALS AND METHODS

One gram of biochar obtained from Simcoa Ltd (Bunbury, WA) that originated from Eucalyptus sp. (Blackwell et al. 2010) was air-dried to remove the majority of moisture prior to sample preparation. Two methods of preparation were used for observation of biochar surface and pores: 1) whole and fractured pieces of biochar, and 2) resin embedded and cross sectioned biochar.

SEM of whole and fractured biochar

For SEM, 10 particles of biochar sized 0.5-1 mm were randomly selected, mounted on a carbon stub and coated with 5-10 nm Au. Observation by SEM was performed using a Phillips XL 30 ESEM or a Zeiss 55 VP-FESEM. Images of surface structure and pore structure of biochars were captured from 5 points on each particle at 3 fixed magnifications using 5-15 kV accelerating voltage.

SEM of embedded biochar

Biochar particles were dehydrated in a series of increasing ethanol concentrations (30, 50, 70 and 90% ethanol, anhydrous 100% ethanol). Biochar particles of more than 2 mm were treated differently as the time required for dehydration process was longer and each step was repeated. After dehydration, biochar particles were infiltrated in Procure Araldite resin and acetone mixtures at a ratio of 2:1, 1:1 and 1:2 for 2 h each before being embedded in 100% and cured in an oven at 60°C under vacuum overnight. Coarse polishing was done with a series of sandpapers (100, 240, 400, 1 000, 2 000, 4 000 grade of grit). These polished resin embedded biochars provided oriented cross sections through the biochar particles allowing visualization of pore connectivity. For imaging, polished sections were coated with 20 nm carbon. SEM was performed using either a Phillips XL 30 ESEM or a Zeiss 55 VP-FESEM. Surface structure and pore structure of biochars were captured from 5 points on each particle at 3 fixed magnifications at 5-15 kV accelerating voltage. The NIH freeware package



Image J was used to measure the diameter of all visible pores in the SEM images. Comparisons were made to compare the pore size distribution between scanning electron micrographs of both whole biochar mounted on stubs and cross sectioned resin embedded biochar. The pores were scored against 3 pore size ranges (<50 µm, 50-100 µm and >100 µm).

Fluorescence imaging of biochar colonization

Preparation of biochar for examining hyphal colonization was performed on both a laboratory-based Petri dish (soilless) study and from a soil-based incubation experiment.

For the Petri dish (soilless) study, 20 biochar particles 1-4 mm in size were randomly selected, spread and placed on potato dextrose agar (PDA) media and inoculated with Sclerotinia sclerotiorum culture in Petri dishes (3 replicates) in a 25°C temperature controlled room. After 3 d incubation, the biochar particles were retrieved, immediately preserved in 2.5% glutaraldehyde in phosphate buffered saline (PBS) solution (pH 7.2), and stored at 4°C.

For the soil-based incubation experiment, 10 biochar particles (1-4 mm) were randomly selected after 56 d incubation in an agricultural soil (loamy sand) from Moora, Western Australia. The soil and biochar mixture had been incubated at 25°C. These biochar particles were also preserved in 2.5% glutaraldehyde in PBS solution and stored at 4°C.

Sampled biochar particles were then rinsed with PBS solution for 3-5 times to remove excess glutaraldehyde. To optimize staining, two fluorescent brighteners were investigated based on previous reports of crystallization problems with Calcofluor White M2R (Sigma Chemical, Poole). Fluorescent brightener Calcofluor White M2R and SCRI Renaissance 2200 (FBA 220 liquid), supplied as a 20% aqueous solution (Renaissance Chemicals), were diluted to a 0.2% working dilution (Harris et al. 2002) and stored at 4°C until use in a covered bottle to prevent light transmission during storage. Biochar particles from the laboratory-based soilless Petri dish study were used to compare the effectiveness of the fluorescent brighteners. Fluorescent brightener RR 2200 and Calcofluor White M2R were each applied separately to five biochar particles for 10 min. They were rinsed 3 times with PBS to remove excess stain and observed using a Zeiss Axioplan 2 microscope. All images were captured via a Zeiss Axiocam digital camera.

Correlative examination of fungal hyphae on biochar by fluorescence and SEM

Ten colonized biochar particles from the incubated soil were stained with RR 2200 as detailed above before being dehydrated in a series of increasing ethanol concentrations (30, 50 and 70%). For biochar particles greater than 2 mm

in diameter, the time required for dehydration process was longer and each step was repeated. For initial fluorescence imaging, colonized biochar particles were submerged in 70% ethanol solution for observation under the fluorescence microscope. The biochar particles were then dehydrated completely in 100% ethanol and critical point dried for 2 h before being mounted on carbon stubs and coated with 5-10 nm Au for SEM imaging.

AcknowledgementsThe Universiti Putra Malaysia and Government of Malaysia are acknowledged for providing a postgraduate scholarship and study leave to Noraini M Jaafar. The University of Western Australia provided access to facilities, research funds and postgraduate student support. Dr. Zakaria Solaiman from the University of Western Australia provided laboratory advice and Dr. Paul Blackwell from the Department of Agriculture and Food Western Australia provided the biochar. The authors acknowledge the use of facilities within the Australian Microscopy & Microanalysis Research Fac i l i ty a t the Cen t re fo r Microscopy , Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State and Commonwealth Governments.

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microscopy observations of habitable space in biochar for colonization by fungal hyphae from soil

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