Soil Microbial Responses to Biochars Varying in Particle Size, Surface and Pore Properties

Pedosphere 25(5): 770–780, 2015 ISSN 1002-0160/CN 32-1315/P c 2015 Soil Science Society of China ⃝ Published by Elsevier B.V. and Science Press

Soil Microbial Responses to Biochars Varying in Particle Size, Surface and Pore Properties Noraini M. JAAFAR1,3,∗ , Peta L. CLODE2 and Lynette K. ABBOTT1 1 Soil

Biology and Molecular Ecology Group, School of Earth and Environment (M087) and UWA Institute of Agriculture, The University of Western Australia, Crawley 6009 (Australia) 2 Centre for Microscopy, Characterisation and Analysis (M010), The University of Western Australia, Crawley 6009 (Australia) 3 Department of Land Management, Faculty of Agriculture, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor (Malaysia) (Received May 11, 2015; revised July 23, 2015)

ABSTRACT Biochars are known for their heterogeneity, especially in pore and surface structure associated with pyrolysis processes and sources of feedstocks. The surface area of biochar is likely to be an important determinant of the extent of soil microbial attachment, whereas the porous structure of biochar is expected to provide protection for soil microorganisms. Potential interactions between biochars from different sources and with different particle sizes were investigated in relation to soil microbial properties in a short-term incubation study. Three particle size (sieved) fractions (0.5–1.0, 1.0–2.0 and 2.0–4.0 mm) from three woody biochars produced from jarrah wood, jarrah and wandoo wood and Australian wattle branches, respectively, were incubated in soil at 25 ◦ C for 56 d. Observation by scanning electron microscopy (SEM) and characterisation of pore and surface area showed that all three woody biochars provided potential habitats for soil microorganisms due to their high porosity and surface areas. The biochars were structurally heterogeneous, varying in porosity and surface structure both within and between the biochar sources. After the 56-d incubation, hyphal colonisation was observed on biochar surfaces and in larger biochar pores. Soil clumping occurred on biochar particles, cementing and covering exposed biochar pores. This may have altered surface area and pore availability for microbial colonisation. Transient changes in soil microbial biomass, without a consistent trend, were observed among biochars during the 56-d incubation. Key Words:

microbial biomass, microbial colonisation, microbial habitats, porosity, scanning electron microscopy, surface structure

Citation: Jaafar N M, Clode P L, Abbott L K. 2015. Soil microbial responses to biochars varying in particle size, surface and pore properties. Pedosphere. 25(5): 770–780.

INTRODUCTION Feedstock characteristics and pyrolysis conditions contribute to biochar heterogeneity (Downie et al., 2009). Previous research on biochar as a soil amendment showed the potential of biochar to improve soil microbial properties (Glaser et al., 2002; Oguntunde et al., 2004; Yamato et al., 2006; Jones et al., 2011) and the occurrence of microorganisms in biochar or coal obtained after fire aging from 100 to 300 years (Zackrisson et al., 1996; Hockaday et al., 2007). Biochar can interact with soil particles and influence soil microbial communities by creating microhabitats (Zackrisson et al., 1996; Wardle et al., 2008), introducing labile organic compounds for microbial growth (Graber et al., 2010) and activating nutrient retention (Cornelissen et al., 2005; Keech et al., 2005). Contradictory observations of the effects of biochar may be attributed to heterogeneity of pyrolysed materials (Singh et al., 2010). In general, woody biochars are porous with ∗ Corresponding

author. E-mail: j [email protected].

high surface areas and could provide habitats for soil microorganisms (Thies and Rillig, 2009; Graber et al., 2010). Microbial biomass may or may not be influenced by addition of biochar to soil (e.g., Rousk et al., 2013). Both decreases (e.g., Dempster et al., 2012a) and increases (e.g., Zhou et al., 2015a) in soil microbial biomass have been associated with biochar application to soil. Responses appear to differ with soil type and form of biochar. For example, in an incubation study, recently prepared biochar increased soil microbial biomass carbon and nitrogen but no such effect was observed for aged biochar (Zhou et al., 2015a). Specific effects of biochar heterogeneity, especially variations in porosity and surface structure, on soil microbial communities are largely unknown. The high surface area of biochar is a potential determinant of soil microbial attachment, whereas porosity and particle size may affect provision of microbial habitat and protection (Thies and Rillig, 2009; Lehmann et al.,

SOIL MICROBIAL RESPONSES TO BIOCHARS

2011; Jaafar et al., 2014). Surface attachment may offer protection to soil microorganisms and opportunities for their interactions with biochar. Surface-associated fungi and bacteria could degrade nutrients on biochar surfaces and variations in surface area of biochar particles may influence microbial requirements for water and nutrients (Atkinson et al., 2010; Sohi et al., 2010; Lehmann et al., 2011). Biochar can adsorb cations and organic matter (Liang et al., 2006), and soil pH changes were shown to change with time during a biochar-soil incubation study (Zhou et al., 2015b). There have been few investigations of biochar particle size in relation to microbial response in soil. Biochars occur as large (> 4 mm) to fine particles (< 20 µm) (Glaser et al., 2000). Commonly, biochar contains a mixture of particle size (Downie et al., 2009) or it is ground after production into smaller fractions (Sohi et al., 2010). Woody biochars normally occur in large fragments (Blackwell et al., 2009; Downie et al., 2009) and may be less practical for agricultural use (Blackwell et al., 2009). Biochar surfaces can gradually oxidise with exposure to air, activities of soil microorganisms or roots, thereby increasing their cation exchange capacity (Joseph et al., 2010). Changes to the surface of biochar after exposure to the soil environment may also alter water and nutrient retention properties of the biochar (Joseph et al., 2010). The size of the biochar particles applied to soil is not expected to greatly affect nutrient uptake but may alter surface properties that influence microbial attachment (Verheijen et al., 2009). The aims of this study were: i) to characterise three woody biochars varying in particle size and determine their potential as microbial habitats in soil, ii) to observe changes in biochar and fungal colonisation during a short-term (56 d) incubation through microscopy observation and iii) to monitor potential effects of biochar source and particle size on soil microbial biomass. It was expected that the potential of biochar as a microbial habitat in soil would differ among biochar sources

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and particle sizes. It was hypothesized that: i) woody biochars would provide suitable habitats for soil microorganisms based on their high porosity, pore size distribution and surface area and ii) among woody biochar particles, those with higher porosity or smaller particle size would harbour more microbial biomass than those with smaller pores or larger particle size. MATERIALS AND METHODS Soil and biochar sources The soil (0–10 cm) used was collected from Moora, Western Australia. Soil samples were sieved (< 2 mm) and kept at 4 ◦ C until used in the experiment. Subsamples of the soil were sent to the Soil and Plant Laboratory of the CSBP Ltd. (Kwinana, Western Australia) for basic soil chemical analysis and characterisation (Rayment and Lyons, 2011). Ammonium and nitrate were determined by steam distillation and automated colour finishes after extraction with 2 mol L−1 KCl, phosphorus using bicarbonate extraction (Colwell, 1963) and organic carbon using the method of Walkley and Black (1934). Soil pH was measured using a soil to solution ratio of 1:5 and texture was assessed by particle distribution. The soil was acidic (pH 4.3 in CaCl2 ) and contained 30 mg kg−1 carbon, 12 mg kg−1 phosphorus, 65 mg kg−1 nitrate and 1 mg kg−1 ammonium. Three woody biochars (Saligna, Wundowie and Simcoa, Table I) were used in this study. The Simcoa biochar was made from jarrah wood (Eucalyptus marginata) in 2008 by the Simcoa Ltd. (Bunbury, Australia). The Wundowie biochar was collected from a 35year-old stockpile of metallurgical charcoal made with a Lambiotte carbonisation reactor from jarrah (Eucalyptus marginata) and wandoo (Eucalyptus wandoo) wood at Wundowie Foundry (Wundowie, Australia) (Blackwell et al., 2010). Both Wundowie and Simcoa biochars were produced at 550–650 ◦ C for 24 h. The Saligna biochar was produced from Australian wattle

TABLE I Some basic characteristics of the three woody biochar types tested Characteristic

Feedstock Pyrolysis process C content (g kg−1 ) Electrical conductivity (mS cm−1 ) pH in H2 O (1:5) pH in CaCl2 (1:5) Calcium carbonate equivalent (%)

Biochar type Simcoa

Wundowie

Saligna

Jarrah wood 550–650 ◦ C for 24 h 738 0.54 8.65 7.62 27.7

Jarrah and wandoo wood 550–650 ◦ C for 24 h 611 0.07 4.89 3.74 30.8

Australian wattle branches 380 ◦ C for 2 h 582 5.73 8.15 7.42 63.4

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(Acacia saligna) branches at 380 ◦ C for 2 h using a laboratory scale pyrolysis unit at the Department of Agriculture and Food, Western Australia. The biochars were sieved and collected in three particle size ranges (0.5–1.0, 1.0–2.0 and 2.0–4.0 mm). Biochar particles between 0.5–4.0 mm were selected and applied to soil for easier retrieval after incubation for microscopy and analytical purposes. The biochars were crushed and characterised by X-ray diffraction analysis. Carbonate analysis was conducted using the method of Rayment and Lyons (2011) and reported as calcium carbonate equivalent. Incubation experiment A laboratory incubation experiment involving 3 particles size ranges (0.5–1.0, 1.0–2.0 and 2.0–4.0 mm) for each woody biochar type was conducted at The University of Western Australia, Crawley, Australia. The biochars were sieved through a series of sieves (4.0, 2.0, 1.0 and 0.5 mm) into three categories (0.5–1.0, 1.0–2.0 and 2.0–4.0 mm). Biochars were added to soil at an amount equivalent to 50 t ha−1 . The soil and biochar incorporated and mixed by hand were incubated aerobically in individual jars in a temperature (25 ◦ C)-controlled room for 56 d. The soil-biochar mixtures were destructively collected for analysis on days 14, 28 and 56 after the start of incubation. An equivalent set of soil was incubated in glass jars with a gas septum, adjusted to 45% water-holding capacity by adding water and sealed to trap CO2 for measurement of microbial respiration (Anderson, 1982). Biochar microscopy observations For microscopy imaging, biochar pore and surface characteristics were observed using scanning electron microscopy (SEM) before (on the original biochar stocks) and 56 d after incubation in soil (Jaafar et al., 2014). Pore size distribution of biochar was determined based on examination of 10 particles (5–10 µm diameter) using SEM. Measurements and analyses were made from micrographs using the NIH freeware package Image J software (Research Services Branch (RSB) of the National Institute of Mental Health (NIMH), Maryland, USA). After the 56-d incubation, the biochar particles were retrieved and fixed in glutaraldehyde, critical point dried, mounted on carbon tabs and examined with SEM (Jaafar et al., 2014). The observations were performed on both intact and manually broken biochar particles (from the original biochar stocks) to verify the degree of porosity. In addition, fixed Simcoa biochar particles were stained with fluorescent brightener SR 2200, before imaging using fluo-

rescence microscopy (Jaafar et al., 2014). Determinations of biochar surface area and pore volumes were made with the Micromeritics Gemini III 2375 instrument (Micromeritics Instrument Corporation, Norcross, USA) using the Brunauer-EmmettTeller (BET) surface area calculations (Brunauer et al., 1938) and the Barrett-Joyner-Halenda (BJH) pore volume and distribution calculations (Barrett et al., 1951), respectively. The biochar particles were maintained in their sieved fractions (0.5–1.0, 1.0–2.0 and 2.0–4.0 mm), prepared in quantities of less than 0.1 g and outgassed at 300 ◦ C for 8 h (Yu et al., 2006). Measurements were calculated based on 5 analysis points. Soil microbial biomass analysis Soil samples collected on days 14, 28 and 56 after the start of the incubation were immediately analysed for microbial biomass carbon (MBC) and phosphorus (MBP). Soil MBC was determined using the fumigation extraction method (Vance et al., 1987). Fumigated (CHCl3 ) and non-fumigated soil samples were placed in vials containing 20 g soil (dry weight equivalent), with 80 mL of 0.5 mol L−1 potassium sulfate and shaken for 1 h. The dilution ratio of 1:7 was used to determine organic carbon with a Shimadzu TOC-5000a analyzer (Shimadzu Scientific Instruments, Kyoto, Japan). The difference in organic carbon between the fumigated and non-fumigated samples was calculated as MBC. A factor of 0.45 was applied to data for adjustment as recommended for agricultural soils (Wu et al., 1990; Joergensen, 1996; Joergensen and Mueller, 1996). Soil MBP was determined using the anion exchange membrane method (Kouno et al., 1995). Anion exchange membrane (AEM) strips were shaken with suspensions of 2 g dried soil in 30 mL distilled water with and without addition of 1 mL hexanol for the samples fumigated with CHCl3 . After shaking for 16 h, the AEM strips were rinsed with 30 mL distilled water to remove soil. Phosphorus adsorbed by the AEM strips was then eluted using 0.5 mol L−1 HCl with shaking for 2 h and determined using the colorimetric molybdenum blue method (Murphy and Riley, 1962). The amount of CHCl3 -released phosphorus was calculated from the difference between the amounts of inorganic phosphorus adsorbed by the AEM in the non-fumigated and fumigated soils. Soil cumulative CO2 emission was measured according to Anderson (1982). Statistical analysis Analysis of variance (ANOVA) was performed using the Statistical Analysis System (SAS) software

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version 8.02 for Windows (SAS Institute Inc., Cary, USA). Comparison of treatment means (within each biochar) was made using the Duncan’s multiple range test (DMRT) at a 95% confidence level.

TABLE III Pore size distribution of the three woody biochar types determined from scanning electron microscopy micrographs using the Image J software Biochar typea)

RESULTS

Pore size distribution < 50 µm

50–100 µm

Characteristics of the woody biochars tested The Simcoa biochar had the highest C content (740 g kg−1 ), followed by the Wundowie and Saligna biochars (Table I). The Simcoa and Saligna biochars were alkaline, while the Wundowie biochar had a lower pH. Prior to application to soil, sieved fractions of each biochar revealed high variations in particle size distribution (Table II). All three biochars had a similar proportion of particles sized 1.0–2.0 mm. Wundowie biochar had the smallest proportion of particles in the 2.0–4.0 mm range. The Saligna and Wundowie biochars had no particles greater than 4.0 mm. TABLE II Particle size distribution of the three woody biochar types determined using the sieving method Biochar typea)

Particle size distribution > 4.0 mm

2.0–4.0 mm

1.0–2.0 mm

7b) – –

28 8 23

28 29 30

0.5–1.0 mm

< 0.5 mm

16 23 28

21 40 19

% Simcoa Wundowie Saligna a) See

> 100 µm

%

Table I for characteristics of each biochar type. are means (n = 10).

b) Values

Pore size distribution varied among the three woody biochars (Table III). The SEM micrographs showed that there was a high variability among the woody biochars in porosity, pore size and surface area (Tables III and IV). All three woody biochars were macroporous. The Saligna biochar, made from the woody plant Acacia saligna, had pores that were most uniform in size. The percentage of pores sized less than 50 µm estimated from electron micrographs was the highest in Saligna biochar, followed by Simcoa and Wundowie biochars (Table III). Characterisation of morphological heterogeneity in pore and surface structures using SEM demonstrated differences both within and among biochars. The Simcoa biochar (Fig. 1a) had fewer large pores than the Wundowie biochar (Fig. 1b). The Saligna biochar had the least number of large pores (Fig. 1c). Unknown compounds (tar or condensed volatile) were observed inside pores of the Simcoa biochar (Fig. 1d). Fungal hyphae were observed inside pores of the Simcoa biochar

Simcoa Saligna Wundowie a) See

86b) 95 5

9 3 85

5 2 10

Table I for characteristics of each biochar type. are means (n = 10).

b) Values

prior to its application to soil (Fig. 1e). The woody biochars were heterogeneous in surface characteristics and pore volume, and this heterogeneity was observed for all particles (Table IV). Problems related to degassing and determination of biochar surface area at multiple points were encountered. The BET surface area of the biochars ranged from 5.32 (Wundowie) to 452 (Simcoa) m2 g−1 . The highest external surface area was found in the 0.5–1.0 mm particles of the Saligna and Wundowie biochars. However, for the Simcoa biochar, the highest external surface area was generated by the particles in the size range of 1.0–2.0 mm. The BET surface area decreased with an increase in particle size, as shown for the Saligna biochar, where a 6-fold decrease in BET surface area was calculated for the particles of 2.0–4.0 m compared to those of 0.5– 1.0 mm. Most of the surface area was associated with biochar pores (micropore surface area). The Saligna and Wundowie biochars, despite being derived from different feedstocks, both had a lower micropore surface area, external surface area and BET surface area with the highest surface area found in the particles within the range of 0.5–1.0 mm than those within 2.0–4.0 mm. In contrast, the highest surface area was found in particles within the range of 1.0–2.0 mm for the Simcoa biochar. Biochar interactions with soil: microscopy Comparison of the Simcoa biochar before (Fig. 1a, d, e) and after (Fig. 1f) application to soil showed that the pore availability for microbial habitat could be affected by the presence of soil particles within biochar pores. After 56 d of incubation in soil, both small and large pores of biochar were clogged by soil particles. Blockage of smaller pores by soil particles was greater than that of larger pores (Fig. 1f). Soil particles were attached to the external surfaces of the Simcoa biochars after 56 d of incubation in soil (Figs. 1 and 2).

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Fig. 1 Scanning electron microscopy (SEM) micrographs of the pores in the three woody biochar types tested, Simcoa biochar (a), Wundowie biochar (b) and Saligna biochar (c), the Simcoa biochar pores filled with unknown material (d), the fungal network (arrow) in larger pores (100 µm) of the initial Simcoa biochar stock (e) and soil particles adhering onto/into the pores of the Simcoa biochar incubated in soil for 56 d (f). See Table I for characteristics of each biochar type. TABLE IV Surface area and pore volume of biochar particles of the three woody biochar types tested Biochar typea) Saligna

Simcoa

Wundowie

Particle size range mm 0.5–1.0 1.0–2.0 2.0–4.0 Mean 0.5–1.0 1.0–2.0 2.0–4.0 Mean 0.5–1.0 1.0–2.0 2.0–4.0 Mean

Micropore surface area −15.51b)

External surface area

16.92 3.36

50.30 6.79 2.91

314.98 355.58 243.06

83.46 96.83 92.07

16.33 11.72 7.72

26.86 −6.40 18.27

BET surface area m2 g−1 34.79 23.71 6.26 21.59 398.44 452.41 335.13 395.33 43.19 5.32 25.99 24.83

Langmuir surface area 49.90 31.15 8.27 29.77 521.45 592.21 439.93 517.86 57.64 6.73 34.75 33.04

Cumulative surface area

Cumulative pore volume

Micropore volume

29.14 3.92 1.60

0.014 0.002 0.001

cm3 g−1 −0.008 0.0078 0.0016

49.44 57.93 55.17

0.024 0.028 0.026

0.1448 0.1635 0.1116

15.93 NAc) 10.65

0.008 NA 0.005

0.0074 0.0054 0.0035

a) See

Table I for characteristics of each biochar type. are means (n = 5). c) Not available.

b) Values

Fungal hyphae on the Simcoa biochar after it was retrieved from soil could not be distinguished from those present on biochar prior to its application to soil (Fig. 1). Hyphal networks were observed via fluorescence and SEM techniques (Fig. 2). Problems were encountered with focusing on and observing hyphae on some biochar particles due to their uneven surfaces when viewed using the fluorescence microscope (Fig. 2e, f). Some fungal hyphae observed on surfaces extended into larger pores within biochar particles (Fig. 3). Larger pores of some particles of the Simcoa

biochar incubated had fewer attached soil particles and fungal hyphae were visible (Fig. 3a–c). These hyphae were attached to the wall of larger pores (about 100 µm diameter). In contrast, smaller pores (20 µm diameter) were clogged by soil particles (Fig. 3c–f), limiting observation of fungal hyphae. Soil aggregates of more than 20 µm diameter were associated with biochar surfaces (Fig. 3c, e, f). Biochar interactions with soil: soil microbial biomass The three types of biochars applied to soil all signi-

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Fig. 2 Scanning electron microscopy micrographs (a–c) and fluorescent micrographs (d–f) of incubated (56 d) and colonised Simcoa biochar particles with soil particles and fungal networks (arrow) on biochar external surfaces. Micrographs taken from a similar spot of one biochar particle (e and f) highlight the problem associated with focusing on and observing microorganisms on particular biochar particles and with uneven surfaces. See Table I for characteristics of the Simcoa biochar.

Fig. 3 Scanning electron microscopy micrographs of incubated (56 d) and colonised Simcoa biochar particles with soil particles cementing the surfaces and pores: fungal hyphae (arrow) observed in the biochar pores (a–c) and soil particles on the biochar surfaces (d) and pores (e, f). See Table I for characteristics of the Simcoa biochar.

ficantly (P ≤ 0.05) changed soil microbial biomass and activity (Tables V and VI). Throughout the 56d incubation, significant (P ≤ 0.05) interactions were found among days of incubation, biochar type and particle size for MBC and MBP. However, varying biochar particle sizes in each biochar type did not change (P ≥ 0.05) soil microbial respiration or soil pH. There was no consistent trend with increasing particle size of each biochar type for MBC or MBP after

incubation for 14 d (Tables VII and VIII). After 28 d, MBC increased only for the Saligna biochar in fractions greater than 1.0 mm diameter, whereas MBP increased with increasing particle size for all three biochars (Tables VII and VIII). Little change was observed in either MBC or MBP after the 56-d incubation (data not shown). There was no effect of biochar type or biochar particle size on soil respiration or soil pH at any measurement time during the incubation (data not shown).

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TABLE V Effects of biochar particle size and biochar type on soil microbial biomass and activity and soil pH throughout the 56-d incubation in soil Item Biochar typea) Simcoa Saligna Wundowie Biochar particle size range 0.5–1.0 mm 1.0–2.0 mm 2.0–4.0 mm

Microbial biomass C

Microbial biomass P

Cumulative CO2 emission

mg C kg−1 dry soil

mg P kg−1 dry soil

µg CO2 -C g−1 dry soil d−1

161.28ab) 139.80b 139.71b

1.44b 1.57b 1.80a

70.15a 66.41b 66.16b

4.72a 4.78a 4.59a

128.64B 150.66A 161.47A

1.62A 1.56A 1.63A

67.97AB 65.47B 69.28A

4.71A 4.72A 4.66A

pH in water

a) See

Table I for characteristics of each biochar type. followed by the same lowercase (n = 3) or uppercase (n = 9) letter(s) within a column are not significantly different at P < 0.05 by Duncan’s multiple range test.

b) Means

TABLE VI Analysis of variance (ANOVA) on the effects of biochar particle size, biochar type and incubation days and their interactions on soil microbial biomass and activity and soil pH throughout the 56-d incubation in soil Variable

Incubation days (ID) Biochar type (BT) Biochar particle size (PS) ID × BT ID × PS BT × PS ID × BT × PS

Microbial biomass C

Microbial biomass P

Cumulative CO2 emission

pH in water

P value

Significance level

P value

Significance level

P value

Significance level

P value

Significance level

< 0.0001 0.0012 < 0.0001 0.0973 0.0123 0.0050 < 0.0001

*** ** *** ns * ** ***

< 0.0001 0.0066 0.7953 < 0.0001 < 0.0001 0.1358 0.0443

*** ** ns *** *** ns *

< 0.0001 0.0049 0.0168 0.7604 0.3118 0.0121 0.4508

*** ** * ns ns * ns

0.0607 < 0.0001 0.2092 0.6063 0.7888 0.9531 0.7462

nsa) *** ns ns ns ns ns

*, **, ***Significant at P < 0.05, P < 0.01 and P < 0.001, respectively. a) Not significant. TABLE VII Effects of biochar particle size and biochar type on soil microbial biomass after 14 and 28 d of incubation in soil Biochar typea)

Particle size range

After 14 d of incubation Microbial biomass C

Saligna

Wundowie

Simcoa

mm 0.5–1.0 1.0–2.0 2.0–4.0 0.5–1.0 1.0–2.0 2.0–4.0 0.5–1.0 1.0–2.0 2.0–4.0

kg−1

mg C dry soil 131.03b) bc) 224.79a 152.48b 118.50b 225.35a 186.70a 187.81b 129.41c 235.95a

After 28 d of incubation Microbial biomass P mg P 1.63a 2.07a 1.98a 1.57a 1.21b 1.77a 1.88a 1.78a 1.96a

kg−1

dry soil

Microbial biomass C kg−1

mg C 92.72b 155.61a 182.26a 156.18a 152.73a 165.84a 164.14a 176.88a 197.85a

dry soil

Microbial biomass P mg P kg−1 dry soil 0.76b 0.92b 1.83a 0.65b 1.39a 1.65a 0.87b 1.61a 1.33a

a) See

Table I for characteristics of each biochar type. are means (n = 3). c) Means followed by the same letter within a column for each biochar type are not significantly different at P < 0.05 by Duncan’s multiple range test.

b) Values

DISCUSSION Characterisation of three woody biochars was made based on the hypothesis that woody biochars had the

potential to provide habitats for soil microorganisms because of having a high porosity and high surface areas. This hypothesis was supported by microscopic observation and surface area analyses of the biochars. The

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TABLE VIII Analysis of variance (ANOVA) on the effects of biochar particle size and biochar type and their interaction on soil microbial biomass after 14 and 28 d of incubation in soil Variable

Biochar type (BT) Particle size (PS) BT × PS

After 14 d of incubation

After 28 d of incubation

Microbial biomass C

Microbial biomass P

Microbial biomass C

Microbial biomass P

P value

Significance level

P value

Significance level

P value

Significance level

P value

Significance level

0.4658 0.0010 < .0001

nsa) ** ***

0.0947 0.4103 0.4278

ns ns ns

0.0404 0.0117 0.1541

* * ns

0.7684 <0.0001 0.0331

ns *** *

*, **, ***Significant at P < 0.05, P < 0.01 and P < 0.001, respectively. a) Not significant.

Simcoa and Wundowie biochars investigated in this study had high surface areas as found previously (Blackwell et al., 2010). Blackwell et al. (2010) reported 0.49 m2 g−1 of pores 2–5 µm measured by porosimetry for the Simcoa biochar. The 86% of the pores sized less than 50 µm diameter in the Simcoa biochar estimated in this study included the pore size range of 2–5 µm diameter that generated the highest BET surface area. Previous observations have shown that woody biochars are more porous than biochars produced from organic sources such as manures, leaves and other wastes (Bird et al., 2008; Downie et al., 2009). Heterogeneity in pore size distribution and chemical composition among the woody biochars observed in this study was expected to correspond to characteristics of the different feedstocks. The Wundowie biochar was a mixture of two Eucalyptus spp. and differed in pore distribution from the Simcoa biochar prepared from one Eucalyptus sp. and from the Saligna biochar which was originated from Acacia saligna. The Saligna and Simcoa biochars both had a high porosity with a lower percentage of larger pores. In contrast, the Wundowie biochar had a low porosity and a high percentage of smaller pores. The physical heterogeneity of these woody biochars was in line with the finding that heterogeneity of biochar pore structure across sources was mostly attributed to pyrolysis conditions ¨ cimen and Ersoy-Meri¸cboyu, and parent material (Oz¸ 2010). A pyrolysis temperature of 400 ◦ C had been used to produce the Saligna biochar and a higher temperature (> 550 ◦ C) was used for the Wundowie and Simcoa biochars (Blackwell et al., 2010). Similar effects of varying pyrolysis conditions and feedstocks on biochar responses in soil have been observed previously (Chan et al., 2007, 2008; Downie et al., 2009; Uchimiya et al., 2010). The Simcoa biochar consistently revealed higher

pore surface areas than external surface areas and showed three-fold greater internal micropore surface areas than external surface areas for all particle size ranges examined. This biochar had a higher porosity or larger pores and was expected to have a greater effect on microbial biomass; this was not always the case throughout this short-term 56-d incubation. Larger pores of the Simcoa biochar were observed to be colonised by fungi, but surface area and pore availability for fungal colonisation may be visually restricted if only determined using 2D microscopy techniques (Jaafar et al., 2014). After the 56-d incubation, few and inconsistent effects of these three woody biochars on microbial respiration and microbial biomass were observed. Woody biochars can increase microbial activity in soils by providing basic microbial requirements including habitat, water, labile source and nutrient (Lehmann et al., 2003; Wardle et al., 2008; Jones et al. 2011). Although the effects of soil particle accumulation on biochar after incubation for 56 d are not known, the duration of 56 d has been justified to be sufficient to observe the biochar effects on soil biology and nutrient availability based on previous studies (Cheng et al., 2008; Nelson et al., 2011). Cheng et al. (2008) found that a small amount of wood-derived biochar is mineralized during incubation for 56 d. The Simcoa biochar which had a higher porosity and a higher BET surface area than the Saligna and Wundowie biochars did not have higher MBC after 28 d of incubation. With more pores of < 50 µm in diameter, it was expected that the Simcoa biochar would have more potential to protect microorganisms, leading to greater soil microbial biomass. Pores of 2– 80 µm in diameter are known to occur in most woodderived biochars and this pore size range could support fungal activity (Thies and Rillig, 2009; Hammer et al., 2014). Such pores would be accessible to soil bacte-

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ria and fungal hyphae (Swift et al., 1979). Recently, Hammer et al. (2014) found that mycorrhizal fungi colonizing biochar surfaces and microsites can improve phosphorus acquisition, supporting the suggestion that biochars from woody feedstocks with high porosity and surface area could benefit the growth of fungal hyphae. External surface area decreased with increasing particle size according to characterisation using the BET measurement, highlighting the potential importance of smaller size biochar particles. Smaller particle size fractions of biochar applied to soil have the potential to harbour more microbial biomass through microbial surface attachment and habitable space. However, there were few effects of particle size within each biochar source measured on MBC and MBP after the 56-d incubation, although transient effects of particle size within each biochar sources were observed at earlier stages of the incubation. Although the biochar particle size may not strongly affect microbial biomass in soil, alterations in surface properties of biochar in soil may influence microbial attachment (Verheijen et al., 2009). The role of soil particles in clogging biochar pores and in cementing biochar surfaces could overshadow the effects of biochar pore and surface structure, but this is yet to be understood in relation to the microbial biomass in soil. Furthermore, the influence of soil attachment to biochar on microbial activity needs to be demonstrated. Soil attachment could play a role in introducing microorganisms, nutrients and water sources into biochar micro-environments, facilitating colonisation of internal surfaces. Subsequent interactions of biochar with soil could also change the porosity of biochar, as adsorption of organic matter to biochar can decrease porosity by blocking pores (Kwon and Pignatello, 2005). Soil nutrient retention on biochar (Cornelissen and Gustafsson, 2004, 2005; Cornelissen et al., 2005; Keech et al., 2005) or use of biochar as a metabolic substrate could also influence microbial activity. Future examination on biochar as a habitat for soil microorganisms could include pre- and post-determination of changes in biochar surface area. However, technical limitations could pose a challenge. In addition to characterisation of biochar, adequate sampling and replication is required for accuracy, especially for surface area measurement. Technical issues related to measurement of surface area using the BET machine, as noted in this study, must be recognised to improve understanding of the relationships between biochar surface area and porosity measurements. This includes concerns associated with the outgassing processes for

determination of biochar porosity and surface area at multiple points as observed in previous studies (Braida et al., 2002; Badalyan and Pendleton, 2003; Yu et al., 2006). Other methodological uncertainties include thermal transpiration and outgassing mass of charred material associated with the measurement. Various degassing methods and heating temperatures in BET measurement have been reported (Chun et al., 2004; Yu et al., 2006). Chun et al. (2004) used approximately 0.2 g char samples with overnight outgassing (more than 15 h) at 105 ◦ C, but no information on particle size of the biochar was mentioned. In contrast, outgassing of ground charcoal was carried out at 300 ◦ C for 8 h by Yu et al. (2006). However, the biochars used in this study were not ground; instead, they were maintained in their respective particle sizes, so that it would be possible to estimate external area of different particle sizes as well as micropore surface area which generated BET surface area. Different degassing techniques and analysis methods applied may vary for surface area determination for biochar samples, either ground or unground, which could be the underlying reason for the negative values obtained for micropore surface area analysis in this study. This aspect remains to be investigated. CONCLUSIONS All three woody biochar sources used in the 56-d incubation experiment of this study could contribute potential habitats for soil microorganisms because of their high porosities and surface areas. The biochars were heterogeneous, varying in porosity and surface structure both within and between the biochar sources. Once the biochar particles were deposited in soil, soil clumping and attachment to the biochar particles were observed, which could affect the biochar surface area by blocking smaller pores and overshadowing the effects of variations in particle size of biochar. Particle sizes of the three biochar sources had few effects on soil MBC and MBP after the 56-d incubation, but some transient effects were observed. ACKNOWLEDGEMENTS The Universiti Putra Malaysia and the 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, The University of Western Australia, provided laboratory advice. Dr. Paul Blackwell, the Department of Agri-

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culture and Food Western Australia, provided the biochars. The authors acknowledge the scientific and technical assistance from Ms. Georgina Holbech and Mr. Michael Smirk for sample analyses and the use of facilities within the Australian Microscopy & Microanalysis Research Facility at the Centre for Microscopy, Characterisation & Analysis, The University of Western Australia, a facility funded by the University, State and Commonwealth Governments, Australia. REFERENCES Anderson J P E. 1982. Soil respiration. In Page A L, Miller R H, Keeney D R (eds.) Methods of Soil Analysis. Part 2. Chemical and Microbiological Properties. 2nd Ed. Agronomy Monograph 9. ASA-SSSA, Madison. pp. 831–871. Atkinson C J, Fitzgerald J D, Hipps N A. 2010. Potential mechanisms for achieving agricultural benefits from biochar application to temperate soils: a review. Plant Soil. 337: 1–18. Badalyan A, Pendleton P. 2003. Analysis of uncertainties in manometric gas-adsorption measurements. I: Propagation of uncertainties in BET analyses. Langmuir. 19: 7919–7928. Barrett E P, Joyner L S, Halenda P P. 1951. The determination of pore volume and area distributions in porous substances. 1. Computations from nitrogen isotherms. J Am Chem Soc. 73: 373–380. Bird M I, Ascough P L, Young I M, Wood C V, Scott A C. 2008. X-ray microtomographic imaging of charcoal. J Archaeol Sci. 35: 2698–2706. Blackwell P, Krull E, Butler G, Herbert A, Solaiman Z. 2010. Effect of banded biochar on dryland wheat production and fertiliser use in south-western Australia: an agronomic and economic perspective. Soil Res. 48: 531–545. Blackwell P, Riethmuller G, Collins M. 2009. Biochar application to soil. In Lehmann J, Joseph S (eds.) Biochar for Environmental Management: Science and Technology. Earthscan, London. pp. 207–226. Braida W J, Pignatello J J, Lu Y, Ravikovitch P I, Neimark A V, Xing B. 2002. Sorption hysteresis of benzene in charcoal particles. Environ Sci Technol. 37: 409–417. Brunauer S, Emmett P H, Teller E. 1938. Adsorption of gases in multimolecular layers. J Am Chem Soc. 60: 309–319. Chan K Y, Van Zwieten L, Meszaros I, Downie A, Joseph S. 2007. Agronomic values of greenwaste biochar as a soil amendment. Soil Res. 45: 629–634. Chan K Y, Van Zwieten L, Meszaros I, Downie A, Joseph S. 2008. Using poultry litter biochars as soil amendments. Soil Res. 46: 437–444. Cheng C, Lehmann J, Thies J E, Burton S D. 2008. Stability of black carbon in soils across a climatic gradient. J Geophys Res. 113: G02027. Chun Y, Sheng G Y, Chiou C T, Xing B S. 2004. Compositions and sorptive properties of crop residue-derived chars. Environ Sci Technol. 38: 4649-4655. ¨ 2004. Sorption of phenanthrene to Cornelissen G, Gustafsson O. environmental black carbon in sediment with and without organic matter and native sorbates. Environ Sci Technol. 38: 148–155. ¨ 2005. Importance of unburned Cornelissen G, Gustafsson O. coal carbon, black carbon, and amorphous organic carbon to phenanthrene sorption in sediments. Environ Sci Technol. 39: 764–769.

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