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Biochar and the Soil Nitrogen Cycle:
Unravelling the Interactions
Daniel Norman Dempster
BSc (Agric, Hons); BComm
This thesis is presented for the degree of
Doctor of Philosophy
to The University of Western Australia
School of Earth and Environment
Faculty of Natural and Agricultural Sciences
2013
ii
iii
Table of Contents
Table of Contents ....................................................................................................... iii
Acknowledgements ..................................................................................................... ix
Declaration .................................................................................................................. xi
Publications arising from this Thesis ..................................................................... xiii
List of Abbreviations .............................................................................................. xvii
Abstract ..................................................................................................................... xxi
1. General Introduction and Literature Review ................................................. 1
1.1 General Introduction .................................................................................. 1
1.2 Western Australian Agriculture ................................................................. 1
1.3 Biochar ....................................................................................................... 4
1.3.1 Introduction to biochar ..................................................................... 4
1.3.2 Biochar in the agricultural system: focus on broadacre agriculture in
Western Australia ............................................................................. 8
1.3.3 Soil biological fertility changes with biochar addition ..................... 9
1.4 The Nitrogen Cycle .................................................................................. 13
1.5 Sorption of nitrogen to biochar ................................................................ 18
1.5.1 Mechanisms of sorption and properties influencing sorption to
biochar ............................................................................................ 18
1.5.2 The role of nitrogen sorption to biochar in ecosystem processes ... 21
1.6 Desorption ................................................................................................ 21
1.7 The influence of biochar on nitrogen leaching......................................... 22
1.7.1 Leaching of nitrogen decreases due to sorption.............................. 22
1.7.2 Leaching of nitrogen decreases due to increasing water holding
capacity ........................................................................................... 23
iv
1.7.3 Leaching of nitrogen changes due to nitrification and mineralisation
......................................................................................................... 23
1.7.4 Leaching of nitrogen changes due to microbial immobilisation ..... 25
1.8 Changes to nitrogen immobilisation induced by biochar application ...... 25
1.9 Biochar influences on nitrogen mineralisation ......................................... 27
1.10 The influence of biochar on nitrification .................................................. 30
1.11 The influence of biochar on plant nitrogen uptake ................................... 32
1.12 Summary of key research areas highlighted within this review ............... 34
2. Decreased soil microbial biomass and nitrogen mineralisation with
Eucalyptus biochar addition to a coarse textured soil .................................. 39
2.1 Abstract ..................................................................................................... 39
2.2 Introduction............................................................................................... 40
2.3 Materials and methods .............................................................................. 43
2.3.1 Biochar characterisation .................................................................. 43
2.3.2 Soil preparation and description ...................................................... 44
2.3.3 Pot experiment ................................................................................ 45
2.3.4 Plant analysis ................................................................................... 46
2.3.5 Microbial biomass ........................................................................... 47
2.3.6 Soil carbon dioxide evolution ......................................................... 48
2.3.7 Community level physiological profiles (CLPP) ............................ 48
2.3.8 DNA extraction and terminal restriction fragment length
polymorphism (T-RFLP) profiles ................................................... 48
2.3.9 Net nitrogen mineralisation ............................................................. 50
2.3.10 Organic carbon sorption experiment ........................................... 50
2.3.11 Nitrogen sorption experiment ..................................................... 50
2.3.12 Statistical analysis ....................................................................... 51
2.4 Results ...................................................................................................... 52
2.4.1 Biochar characterisation .................................................................. 52
2.4.2 Plant growth analysis ...................................................................... 53
2.4.3 Microbial biomass ........................................................................... 55
v
2.4.4 Microbial community function (CLPP) and structural diversity
(amoA T-RFLP) .............................................................................. 58
2.4.5 Carbon dioxide evolution and nitrogen mineralisation .................. 60
2.4.6 Sorption isotherms .......................................................................... 64
2.5 Discussion ................................................................................................ 67
2.5.1 Microbial biomass .......................................................................... 67
2.5.2 Carbon and nitrogen mineralisation ................................................ 69
3. Clay and biochar amendments decreased inorganic nitrogen leaching but
not dissolved organic nitrogen leaching in soil ............................................. 75
3.1 Abstract .................................................................................................... 75
3.2 Introduction .............................................................................................. 76
3.3 Materials and Methods ............................................................................. 78
3.3.1 Soil, clay and biochar ..................................................................... 78
3.3.2 Experimental design and setup ....................................................... 81
3.3.3 Nitrogen sorption characteristics .................................................... 82
3.3.4 Water retention ............................................................................... 82
3.3.5 Statistical analysis ........................................................................... 82
3.4 Results ...................................................................................................... 83
3.4.1 Nitrogen leaching ............................................................................ 83
3.4.2 Nitrogen sorption characteristics .................................................... 85
3.4.3 Water retention capacity ................................................................. 87
3.5 Discussion ................................................................................................ 89
4. Organic nitrogen mineralisation in two contrasting agro-ecosystems is
unaffected by biochar addition ....................................................................... 93
4.1 Abstract .................................................................................................... 93
4.2 Introduction .............................................................................................. 93
4.3 Materials and Methods ............................................................................. 95
4.3.1 Experimental design and characterisation of soils and biochars .... 95
4.3.2 Statistical Analysis .......................................................................... 97
4.4 Results .................................................................................................... 101
vi
4.5 Discussion ............................................................................................... 107
5. Minimal interaction between wheat chaff biochar and N fertiliser in a
broadacre field experiment ........................................................................... 111
5.1 Abstract ................................................................................................... 111
5.2 Introduction............................................................................................. 112
5.3 Materials and Methods ........................................................................... 114
5.3.1 Experimental site and design ........................................................ 114
5.3.2 Sampling times and analysis ......................................................... 118
5.3.3 Statistical analysis ......................................................................... 118
5.4 Results .................................................................................................... 119
5.4.1 Soil microbial biomass .................................................................. 119
5.4.2 Soil ammonium and nitrate ........................................................... 119
5.4.3 Plant biomass and nitrogen content ............................................... 120
5.4.4 Grain yield and nitrogen content ................................................... 122
5.5 Discussion ............................................................................................... 124
5.5.1 Interaction between nitrogen fertiliser and biochar....................... 124
5.5.2 Yield influence of biochar ............................................................. 125
5.5.3 Biochar application method .......................................................... 126
5.5.4 Conclusion ..................................................................................... 126
6. General Discussion ......................................................................................... 129
6.1 Sorption of low molecular weight nitrogen to biochar ........................... 131
6.1.1 Applying findings to other experiments ........................................ 133
6.1.2 Implications of the minimal impact of sorption of low molecular
weight nitrogen to biochar ............................................................ 134
6.2 Sorption of high molecular weight organic nitrogen compounds to
biochar .................................................................................................... 135
6.2.1 Implications of the sorption of high molecular weight compounds to
biochar ........................................................................................... 138
6.3 The addition of labile carbon contained in biochar ................................ 139
6.4 Increasing the supply of phosphorus and potassium in biochar ............. 141
vii
6.5 Water supply and water holding capacity .............................................. 142
6.6 No effect of biochar................................................................................ 143
6.7 Conclusion .............................................................................................. 145
7. References ....................................................................................................... 147
viii
Acknowledgements
ix
Acknowledgements
I acknowledge the Grains Research and Development Corporation (GRDC) for
funding my scholarship (GRS153) and for the travel awards received. I also
acknowledge the School of Earth and Environment within the University of Western
Australia for providing further operating expenditure.
I thank my supervisors Associate Professor Deirdre Gleeson, Professor Daniel
Murphy, and Winthrop Professor Lyn Abbott, especially Dan Murphy for facilitating
my collaboration with Professor Davey Jones. I also thank Professor Davey Jones for
supervising my research component at Bangor University, Wales.
My family deserve special thanks for providing me with many opportunities
throughout my life. Emily Laing has provided immense support and friendship. I also
thank many other friends for their discussion and thoughts.
x
Declaration
xi
Declaration
I, Daniel Norman Dempster, declare that this thesis was composed by me and that
research detailed within was designed, conducted and interpreted by myself, except in
instances where the work and contribution of others has been acknowledged.
Daniel N. Dempster
xii
Publications arising from this Thesis
xiii
Publications arising from this Thesis
Peer Reviewed Publications
Dempster, D.N., Gleeson, D.B., Solaiman, Z.M., Jones, D.L., Murphy, D.V. (2012)
Decreased soil microbial biomass and nitrogen mineralisation with Eucalyptus
biochar addition to a coarse textured soil. Plant and Soil 354, 311 – 324
(Chapter 2).
Dempster, D.N., Jones, D.L., Murphy, D.V. (2012) Clay and biochar amendments
decreased inorganic but not dissolved organic nitrogen leaching in soil. Soil
Research 50, 216 – 221 (Chapter 3).
Dempster, D.N., Jones, D.L., Murphy, D.V. (2012) Organic nitrogen mineralisation
in two contrasting agro-ecosystems is unchanged by biochar addition. Soil
Biology & Biochemistry 48, 47 – 50 (Chapter 4).
Conference Presentations
Dempster, D.N., Gleeson, D.B., Solaiman, Z.M., Jones, D.L., Murphy, D.V. (2010)
Biochar changed microbial dynamics and nitrogen mineralisation. 19th World
Congress of Soil Science, Brisbane, August 2010 (poster).
Publications arising from this Thesis
xiv
Dempster, D.N., Gleeson, D.B., Solaiman, Z.M., Jones, D.L., Murphy, D.V. (2011)
Biochar changed microbial dynamics and nitrogen mineralisation. UK Biochar
Conference, Edinburgh, Scotland, May 2011 (poster).
Dempster, D.N., Jones, D.L., Murphy, D.V. (2011) Sporadic effects of biochar on
organic nitrogen decomposition. Australian Society of Soil Science Inc, WA
Branch Conference, Busselton, September 2011 (oral presentation).
Dempster, D.N., Jones, D.L., Murphy, D.V. (2011) Sporadic effects of biochar on
organic nitrogen decomposition. 3rd International Rhizosphere Conference,
Perth, September 2011 (oral presentation).
Other Publications and Presentations
Dempster, D.N. (2009) Biochar attracts attention. Ground Cover: Soil Health
Supplement, May-June 2009, pp 18 – 19.
Dempster, D.N., Wherrett, A. (2010). Biochar trial update. Liebe group “Spring on
the Sandplain” field day, Buntine, October, 2010. Oral given by A. Wherrett
and D. Dempster.
Dempster, D.N. (2011). Biochar saving fertiliser? Results from the first year of the
Liebe group trial. Liebe group 2011 crop updates. Paper and presentation by D.
Dempster.
Publications arising from this Thesis
xv
Dempster, D.N. (2011). Biochar: Still much to uncover about its agronomic impact.
New Frontiers in Agriculture: The Official Journal of the Western Australian
No-till Farmers Association. June edition, pp 53-55.
Dempster, D.N., Gleeson, D.B., Abbott, L.K., Murphy, D.V. (2011). Biochar and the
nitrogen cycle: My investigation. Biochar and Soil Biology Workshop,
Mingenew (25th), August 2011. Oral presentation by D. Dempster.
Dempster, D.N., Gleeson, D.B., Abbott, L.K., Murphy, D.V. (2011). Biochar and the
nitrogen cycle: My investigation. Biochar and Soil Biology Workshop,
Dandaragan (26th), August 2011. Oral presentation by D. Dempster.
Dempster, D.N. (2012) Biochar and the nitrogen cycle: Unravelling the interaction.
Soil and Water Seminar. March 2012. Oral presentation by D. Dempster.
Dempster, D.N. (2012) Biochar and the nitrogen cycle: Unravelling the interaction.
Postgraduate Showcase; UWA Institute of Agriculture. June 2012. Oral
presentation by D. Dempster.
xvi
List of Abbreviations
xvii
List of Abbreviations
ANOVA
AOB
BC
C
CO2
CO32-
CLPP
DMRT
DOC
DON
EC
FAA
HMW
IRGA
K
LMW
MBC
MBN
MRT
N
NA
analysis of variance
ammonia oxidising bacteria
black carbon
carbon
carbon dioxide
carbonate
community level physiological profile
Duncan’s multiple range test
dissolved organic carbon
dissolved organic nitrogen
electrical conductivity
free amino acids
high molecular weight
infra-red gas analyser
potassium
low molecular weight
microbial biomass carbon
microbial biomass nitrogen
mean residence time
nitrogen
not assessed
List of Abbreviations
xviii
NH3
NH4+
NO3-
NS
P
PAHs
PCR
PERMANOVA
SIR
SOM
TN
T-RFLP
T-RFs
VM
VOCs
WA
WHC
ammonia
ammonium
nitrate
not significant
phosphorus
polycyclic aromatic hydrocarbons
polymerase chain reaction
permutational analysis of variance
substrate induced respiration
soil organic matter
total nitrogen
terminal-restriction fragment length polymorphism
terminal-restriction fragments
volatile matter
volatile organic compounds
Western Australia
water holding capacity
xix
xx
Abstract
xxi
Abstract
Methods of sequestering carbon (C) are being investigated to mitigate the current
trend of increasing atmospheric carbon dioxide concentration. Furthermore, since
the onset of intensive farming practices soil organic matter (SOM) content has
declined in many soil types throughout the world. The application of pyrolysed
organic matter, or biochar, to soil can maintain higher soil C. The application of
biochar to soil has also been shown to potentially improve soil fertility and enhance
yields of crops such as maize, wheat and peanuts. However, biochar amendment
changes many soil properties but the mechanism by which biochar alters soil
processes and plant growth is not always clear. This thesis focuses primarily on
those changes that are directly linked to the soil nitrogen (N) cycle.
Biochar has been reported to have a large nutrient and water retention capacity;
which may cause decreases in N leaching with its application to soil. Biochar can
contain labile C and is also highly porous, potentially providing a substrate for
microbial utilisation and a habitat for microbial colonisation. Biochar addition to
soil can increase microbial abundance and activity. Previous research has
hypothesised that this may be responsible for enhanced N mineralisation rates that
contribute to the demonstrated improvements of N fertiliser use efficiency (up to
90%) in glasshouse-based agronomic research.
The sandy soils that are widespread throughout the broadacre agricultural region of
Western Australia (WA) naturally have low water (2-10% v/v) and nutrient
Abstract
xxii
retention capacities and low SOM content. Thus the sandy soils of WA are an
environment in which biochar amendment is likely to influence soil N cycling. This
provided the premise for the investigation into how biochar interacts with the soil N
cycle (mineralisation, nitrification, immobilisation, N sorption, leaching, and plant
N uptake) in coarse-textured soils of Western Australian broadacre agriculture.
The influence of Jarrah (Eucalyptus marginata Donn ex Sm.) biochar on N
mineralisation and nitrification in a Tenosol (95% sand; 3.5% clay) was determined
in a glasshouse experiment combined with subsequent laboratory incubations. Net
N mineralisation and nitrification were highest in the control soil and significantly
decreased with increasing addition of biochar (Chapter 2). These decreases could
not be attributed to immobilisation of N, because microbial biomass N did not
change with biochar addition. Decreased mineralisation and nitrification was also
not caused by the sorption of amino acids, or ammonium (NH4+) to the biochar. It is
possible that toxic compounds (for example benzene) present on biochar might have
inhibited N mineralisation; however such compounds were not detected.
Biochar has been hypothesised as an alternative soil amendment (historically clay
has been used) for coarse textured soils to decrease N leaching. To test this
hypothesis Jarrah biochar (25 t ha-1) was added to a Sodosol (94% sand, 5% clay at
0-10 cm depth) in a glasshouse lysimeter experiment to determine its influence on
N leaching. Biochar amendment decreased leaching of NH4+ and nitrate (NO3
-) by
14% and 28%, respectively (Chapter 3). Sub-soil clay addition at 25 t ha-1 also
Abstract
xxiii
caused similar decreases in NH4+ and NO3
- leaching (18% and 16% respectively)
despite the large differences between their sorption capacities, as determined by
sorption isotherms. The greater decrease in NO3- leaching with biochar addition was
likely to be due to a decrease in nitrification (observed in Chapter 2). Therefore
sorption is unlikely to be the primary mechanism causing decreases in inorganic N
leaching with biochar addition. In the absence of other evidence it may be possible
that improved water holding capacity as a result of biochar addition might have
decreased inorganic N leaching.
To investigate whether organic N sorption to an alternate biochar type similarly
influenced mineralisation of organic N, wheat (Triticum aestivum L.) residue
biochar was applied to a Kandosol (93% sand) at 4 t ha-1. In a laboratory incubation,
the mineralisation rate of 14C-labelled peptides and amino acids did not change with
biochar addition (Chapter 4). Thus, the sorption of low molecular weight (LMW)
organic N to biochar is unlikely to influence N mineralisation upon addition to
sandy soil and the addition of wheat residue biochar at 4 t ha-1 is unlikely to
influence intrinsic dissolved organic N turnover.
To assess the net effect of the interaction between biochar and the soil N cycle
wheat residue biochar was applied to a Kandosol (93% sand) at 4 t ha-1 and soil
(inorganic N and microbial biomass N) and plant measurements (biomass, yield and
N) were taken over the growing season. Biochar decreased wheat yield from 2.06 t
ha-1 to 1.8 t ha-1 where biochar was spread and significantly decreased to 1.7 t ha-1
Abstract
xxiv
where biochar was banded (Chapter 5). Of the sampling periods, only one (terminal
spikelet) showed any significant differences in soil NH4+ or NO3
- with biochar
application. In the first year of application, wheat residue biochar was an ineffective
amendment for improving N cycling and synergistically interacting with N
fertiliser.
Although biochar decreased N leaching in a high rainfall controlled environment,
there were no other benefits observed from the addition of two types of biochar to
the tested coarse-textured soils used for broadacre agriculture in WA. This may be
due to the low rates of application possible in this agro-ecosystem. Results may
vary with contrasting biochar types, different soil types or higher application rates
of biochar. The sorption of low molecular weight N to biochar is unlikely to
influence N mineralisation, nitrification or be the main factor decreasing N leaching
with biochar addition to coarse-textured soil. Overall biochar is unlikely to
influence N cycling for the improvement of crop productivity on coarse-textured
soils in broadacre agriculture of WA.
General Introduction and Literature Review Chapter 1
1
Chapter 1
General Introduction and Literature Review
1.1 General Introduction
This thesis examines how biochar amendment to coarse-textured soils used for
broadacre agriculture in Western Australia (WA) impacts the terrestrial nitrogen (N)
cycle. This chapter introduces both broadacre agriculture in WA and biochar, and
examines research assessing the interaction between soil N cycling and biochar
application to soil. The examined aspects of the N cycle are: sorption of N; desorption
of N; leaching; mineralisation; nitrification; and plant N uptake. Where possible, this
review extrapolates current research to a broadacre agricultural context for WA.
1.2 Western Australian Agriculture
Broadacre agriculture, within WA and other parts of Australia, is characterised by
large scale, low productivity and infertile soils. From 2000-2001 to 2010-2011 the
total area of winter crop in Australia averaged 21.4 m ha, of which WA contributed
7.5 m ha (ABARES, 2011). In this agro-ecosystem, wheat is the dominant cropping
enterprise, occupying 12.6 and 4.6 m ha in Australia and WA, respectively
(ABARES, 2011). Broadacre agriculture within WA occurs in a mediterranean-type
climate (Rovira, 1992), with average winter-dominant rainfall ranging from less than
300 mm to around 500 mm (1996-2005 records; Bureau of Meteorology, 2012).
Average maximum and minimum temperatures range from approximately 34 and
General Introduction and Literature Review Chapter 1
2
17˚C in January and 17 and 3 ˚C in July (Bureau of Meteorology, 2012). Wheat
yields average 1.5 t ha-1 in this area (2000-2001 to 2010-2011; ABARES, 2011).
Soil types within the broadacre agricultural region of WA vary from deep sands and
texture contrast (duplex) soils, to loamy earths and cracking clays (Schoknecht, 2002)
but are dominated by coarse-textured top soils, which occupy over 14.2 m ha in the
region (Tennant et al., 1992). The depth of the coarse textured A horizon varies from
5 cm to over 100 cm, and generally has a low clay content (often less than 5%;
Tennant et al., 1992). Consequently, water holding capacities for these soil types are
low, ranging from 20-50 mm m-1 for a medium sand to 90-120 mm m-1 for a loamy
sand (Tennant et al., 1992). Soils within this region tend to be acidic; a recent survey
found that over 80% of top soil (0-10 cm) samples had a pH (CaCl2) of less than 5.5
(Gazey & Andrew, 2009). Low organic carbon is also common, typical values may
range from 1.5-2% (Hoyle et al., 2011), but varies with farming practices (Murphy et
al., 2011).
Due to the inherent low fertility of soils within this region, fertilisers, such as N and
phosphorus (P), are an important component of agricultural production. For crop N
nutrition, 0-80 kg N ha-1 is applied depending on the expected rainfall (Anderson &
Hoyle, 1999), but typically 40-80 kg N ha-1 for an expected 2 t ha-1 wheat (Triticum
aestivum L.) yield (Hoyle & Murphy, 2011). For economic reasons, many farmers
seek methods to improve productivity (Kingwell & Pannell, 2005) and decrease input
costs (Keating & Carberry, 2010) by increasing N fertiliser use efficiency (Chen et
al., 2008).
General Introduction and Literature Review Chapter 1
3
Within Australia, estimates of N fertiliser use efficiency in wheat production vary
from 0 to 43 kg grain for each kilogram of N applied with a recovery rate of applied
N ranging from 6-78% (Mason et al., 1972; McDonald, 1989; Ladha et al., 2005).
Generally, this is greater than the global estimate of 33% N fertiliser use efficiency
for cereal production (Raun & Johnson, 1999).
To improve N fertiliser use efficiency in crop production, the synchrony (both in
terms of time and location) between crop N demand and N supply (mineralised or
fertiliser) must be improved (Chen et al., 2009a). During the dry summer fallow
period, episodic rainfall events enable N to be mineralised (McNeill et al., 1998),
which, provided it is not leached, could be available for plant N uptake during the
following growing season. At the commencement of the growing season, plant (crop
and pasture) N demand is small and plant roots have a low surface area for N capture.
Coarse-textured soils are particularly prone to N leaching and acidification (Dolling
& Porter, 1994; Dolling et al., 1994; Anderson et al., 1998). Nitrate (NO3-) leaching
has been reported to range from 17-59 kg N ha-1 yr-1, with greater NO3- leaching
under crops (40-59 kg N ha-1 year-1) than pastures (17-28 kg N ha-1 yr-1; Anderson et
al., 1998). Fine-textured amendments, such as ‘red mud’ bauxite residue or subsoil
clay from the B horizon of Sodosols and Chromosols, can ameliorate nutrient
leaching (Vlahos et al., 1989) and water repellence in coarse-textured soils
(McKissock et al., 2000; McKissock et al., 2002). Biochar addition can also decrease
N leaching (Lehmann et al., 2003; Ding et al., 2010), thus may aid increased N
fertiliser use efficiency on coarse-textured soils of WA.
General Introduction and Literature Review Chapter 1
4
1.3 Biochar
1.3.1 Introduction to biochar
Current concerns about climatic change, have provided impetus for investigations
into a range of carbon (C) sequestration options such as increasing organic C in soils
(Jackson & Schlesinger, 2004). The organic matter content of many soil types
globally has declined within recent history (Dalal & Chan, 2001; Bellamy et al.,
2005). In Australia, losses of organic C can be in the range of 0.04-1.21 g C g-1 soil
yr-1, varying with soil type (greater losses in coarse textured soils; Dalal & Mayer,
1986). Consequences of declining soil organic matter (SOM) include increases in
bulk density (Dalal & Chan, 2001) and decreased energy for heterotrophic soil
microbes to catalyse nutrient transformations (Rosswall, 1982; Killham, 2006).
One method to increase SOM is by adding biochar to soil (Lehmann et al., 2006;
Sohi et al., 2010). Biochar has been defined as thermally decomposed (or pyrolysed)
organic matter, created by heating to temperatures from 250°C to 700°C in low
oxygen conditions (Lehmann & Joseph, 2009). The application of biochar to soil
provides a method to sequester C (Lehmann et al., 2006; Ogawa, et al., 2006; Sohi et
al., 2010) because biochar contains a large portion of aromatic C (Schmidt & Noack,
2000) which can be recalcitrant for thousands of years (Preston & Schmidt, 2006).
The length of time over which biochar C is stored in soil and the influence of biochar
on soil fertility vary with the properties and characteristics of the biochar (Singh et
al., 2010). Some properties of biochar that enable it to alter soil fertility are porosity
General Introduction and Literature Review Chapter 1
5
(Fig. 1.1), surface area (Downie et al., 2009; Table 1.1), pH (Laird, 2008; Sohi et al.,
2010; Table 1.1), bulk density (Laird, 2008; Sohi et al., 2010; Table 1.1), surface
functional groups (Laird, 2008; Clough & Condron, 2010; Sohi et al., 2010) and the
sizes of the fractions of recalcitrant biochar C and mineral ash (Baldock & Smernik,
2002; Bruun et al., 2008; Singh et al., 2010; Table 1.1). Biochar production variables
(e.g. pyrolysis temperature and pyrolysis time) influence biochar properties like the
size of the recalcitrant fraction of biochar C (Baldock & Smernik, 2002; Bruun et al.,
2008; Singh et al., 2010), the functional groups present on the surface of the biochar
(Boehm, 1994; Antal & Grønli, 2003) and the surface area and porosity of the biochar
(Antal & Grønli, 2003; Bornemann et al., 2007). Consequently, biochar is inherently
heterogeneous.
Changes in all production variables such as feedstock (type, water content, particle
size), peak pyrolysis temperature, type of pyrolysis equipment (batch or continuous)
and length of pyrolysis will result in biochar with varying characteristics (Antal &
Grønli, 2003). Increasing peak pyrolysis temperature will increase C concentration of
the biochar (Antal & Grønli, 2003; Table 1.1), altering its influence on C
sequestration and soil fertility (Singh et al., 2010). Contrasting feedstocks will
produce biochars with different surface areas, and hence varying sorption capacity
(Bornemann et al., 2007). The effect of biochar also varies with soil type and crop
type (Van Zwieten et al., 2010b). Therefore, to isolate the mechanisms controlling
changes to soil processes induced by biochar addition, it is important to characterise
experimental biochars. Seven properties for characterisation of biochars have been
General Introduction and Literature Review Chapter 1
6
proposed by Sohi et al. (2010): pH; the content of volatile compounds; ash content;
water holding capacity (WHC); bulk density; pore volume; and specific surface area.
Consistent characterisation of biochar could aid with determining which biochar
characteristics influence soil fertility across a range of ecosystems. The goal of
consistent and thorough characterisation of biochar could be to predict the agronomic
effectiveness of each biochar type as an amendment for varying soil or crop types.
Fig. 1.1: A scanning electron microscope illustrating the porosity of biochar. Image
source: D. Dempster and P. Clode.
Tab
le 1
.1:
Sele
cted
pro
per
ties
of a
ra
nge
of
bioc
har
s. *
No
te:
pH
fo
r th
e g
reenw
aste
bio
char
wa
s m
eas
ure
d in
Ca
Cl
2.
Fe
ed
sto
ck
Pyr
oly
sis
Te
mp
erat
ure
(°C
)
pH
(H2O
)
Bul
k
De
nsity
(g c
m-3)
Tot
al C
(%)
Tot
al N
(%)
Su
rfa
ce
Are
a
(m2 g
-1)
Vo
latil
e
Co
nte
nt
(g k
g-1)
Ash
Co
nte
nt
(g k
g-1)
Re
fere
nce
Oil
Ma
llee
- 8
.4
- -
- 8
1 1
6 5
37
Sol
aim
an e
t al
., 2
010
Mix
ed
Ha
rdw
oo
d 4
50
9.7
0
.35
76
0.6
9 3
9 -
- Jo
nes
et
al.,
201
0
Jarr
ah
600
8
.5
0.4
5 7
8 0
.38
4 -
- Jo
nes
et
al.,
201
0
Ba
mb
oo
600
8
.1
0.7
5 6
8 0
.87
330
- -
Din
g et
al.,
20
10
Ra
diat
a P
ine
300
5
.7
- 6
2 0
.04
21
- -
Ta
ghiz
ad
eh-T
oos
i et
al.,
201
2
Ra
diat
a P
ine
500
6
.6
- 8
3 0
.22
56
- -
Ta
ghiz
ad
eh-T
oos
i et
al.,
201
2
Ma
cada
mia
nu
t sh
ell
6
40
8.2
-
89
0.4
5 -
63
42
De
enik
et
al.,
20
10
Ma
cada
mia
nu
t sh
ell
4
30
5.7
-
85
0.4
5 -
22
3 D
een
ik e
t al
., 2
01
0
Gre
en
wa
ste
600
7
.5*
-
78
0.1
4 40
9 -
- V
an
Zw
iete
n et al
., 2
010
a
Gre
en
wa
ste
350
4
.9*
- 6
2 0
.21
- -
- V
an
Zw
iete
n et al
., 2
010
c
Gre
en
wa
ste
550
7
.3*
- 7
5 0
.24
- -
- V
an
Zw
iete
n et al
., 2
010
c
Sw
itch
gra
ss 2
50
9.7
-
52
1.6
0 -
40
0 -
Sm
ith e
t al
., 2
010
Pou
ltry
Litt
er
400
9
.2
- 4
3 0
.51
- -
34
6 S
ingh
et al
., 2
010
Syd
ne
y B
lue
Gu
m 4
00
6.9
-
70
0.0
2 -
- 3
5 S
ingh
et al
., 2
010
Syd
ne
y B
lue
Gu
m 5
50
8.8
-
84
0.0
3 -
- 3
3 S
ingh
et al
., 2
010
General Introduction and Literature Review Chapter 1
8
1.3.2 Biochar in the agricultural system: focus on broadacre agriculture in
Western Australia
Many renewable feedstocks can be utilised as biochar sources such as urban
greenwaste (Chan et al., 2007), sewerage sludge (Bridle & Pritchard, 2003),
agricultural wastes such as manures (Chan et al., 2008), wheat (Triticum aestivum L.)
residue (Chun et al., 2004) and silvicultural wastes such as oil mallee (Eucalyptus
sp.) residue (Solaiman et al., 2010). The most widely available feedstocks for biochar
production in WA are cereal crop residues and Eucalyptus biomass. Biochar
production from Eucalyptus residue may provide another income source for oil
mallees planted to ameliorate salinity and wind erosion. Further, more Eucalyptus
plantations in the wheatbelt of WA could provide some of the reforestation required
to return the rainfall of the south-west of WA to its long term average (Pitman et al.,
2004).
Practical rates of application of biochar within broadacre agriculture of WA are likely
to be less than 5 t ha-1 (Blackwell et al., 2010). This is low compared to other agro-
ecosystems which may use application rates of 25-50 t ha-1 (e.g. Jones et al., 2010).
The lower rates of biochar application in broadacre WA is due to a combination of
factors such as lower net primary productivity causing lower biochar production
volumes and larger acreages of land causing larger distances for transport and higher
transport costs. Due to the lower rates of application in broadacre agriculture within
WA, it is unknown whether the mechanisms suggested in other previous research
(examined in following sections) are applicable to the target agro-ecosystem.
General Introduction and Literature Review Chapter 1
9
1.3.3 Soil biological fertility changes with biochar addition
A meta-analysis examining data assessing the impact of a variety of biochar types
added to a range of soil types on many cultivated plants found that, on average,
biochar improved plant productivity by 10% and crop yield by 5% (Jeffrey et al.,
2011). The increases in crop yield and plant productivity are explained by many
changes in soil physical, chemical and biological properties induced by biochar
addition to soil (Glaser et al., 2002; Atkinson et al., 2010; Clough & Condron, 2010;
Sohi et al., 2010; Lehmann et al., 2011).
In a comprehensive review of the soil biological changes induced by biochar addition
to soil Lehmann et al. (2011) concluded that in most studies (>20) biochar
amendment increased soil microbial biomass. There are at least six mechanisms by
which biochar application can increase soil microbial abundance: (i) introduction of
nutrients and C; (ii) increasing pH in acidic soils; (iii) adhesion of microbes to pores;
(iv) provision of protection from other biota; (v) protection against desiccation; and
(vi) sorption of toxins (Lehmann et al., 2011). This review will discuss the relevance
of each of these mechanisms for broadacre agriculture in WA.
Soils within WA generally have low labile C content (e.g. Holmes et al., 2011) which
restricts the size of the heterotrophic microbial population. Labile C contained in
biochar may partially alleviate microbial substrate deficiencies increasing microbial
abundance (Lehmann et al., 2011), but the longevity of this influence is unclear. The
influence on microbial population is will vary with the quality and quantity of
General Introduction and Literature Review Chapter 1
10
nutrients contained within the biochar, the intrinsic soil nutrition and the species of
microbe (Lehmann et al. 2011). Biochar amendment may also increase microbial
abundance by increasing the nutrient retention capacity of the coarse-textured soils of
WA.
Increasing the pH of agricultural soil can have a marked effect on microbial
populations in terms of an increase in biomass and activity (Aciego Pietri & Brookes,
2008). As acidic soils dominate the broadacre agricultural region of WA (e.g.
Tennant et al., 1992), increasing soil pH through biochar amendment may increase
microbial biomass and activity similarly. The magnitude of the increase in soil pH
with biochar amendment is variable and previous studies have not determined the
proportion of the increased microbial population due to the changes in pH or the
coinciding increased labile C.
The adhesion of microbes to biochar may prevent leaching of microbes through the
soil profile, enhancing microbial abundance (Lehmann et al., 2011). The adhesion of
microbes to biochar may occur via electrostatic forces, hydrophobic interactions or
covalent bonding, causing adsorption of microbes to the biochar surface; alternatively
microbes could become trapped within the biochar matrix (Lehmann et al., 2011).
The biochar matrix could provide a habitat for microbes to avoid grazing from
mesofauna (Wardle et al., 1998). This would require pore sizes larger than the
microbes, such as 0.6 μm for N mineralising bacteria (Strong et al., 1998), but too
narrow for microbial predators to enter. The nature of pore size distribution of
General Introduction and Literature Review Chapter 1
11
biochar has been poorly documented, and is likely to vary with soil type. In soils with
low clay content, such as those in the broadacre agricultural region of WA, these
mechanisms are likely to be expecially relevant.
The protection of microbes from desiccation may not be applicable in broadacre agro-
ecosystems in WA because it is located within a mediterranean-type climate zone,
where drying is a regular occurrence. The only studies in Lehmann et al. (2011)
assessing microbial abundance in a mediterranean-type climate focused on symbiotic
microbes (mycorrhizae; e.g. Blackwell et al., 2010; Solaiman et al., 2010) rather than
free-living microbes. Due to climatic conditions the soil dries to very low water
content (<0.5% w/w) every summer, therefore free-living microbes have adapted to
desiccation. Water contained within biochar may provide microbes with an extended
period of time prior to drying, due to its large surface area and WHC, but desiccation
of microbes through drying will occur in this environment.
Biochar can sorb (adsorb or absorb) compounds that are microbially inhibitive, such
as catechol (Chen et al., 2009b; Kasozi et al., 2011), decreasing their prevalence in
the soil solution, which may increase microbial abundance (Lehmann et al., 2011).
Conventional agricultural practices use pesticides that can be microbially inhibitive,
such as tebuconazole (Muñoz Leoz et al., 2011) and diuron (Prado & Airoldi, 2001).
Whilst biochar can sorb some pesticides, such as simazine (Jones et al., 2010),
atrazine and trifluralin (Nag et al., 2010), decreasing their bio-availability to plants
(Bornemann et al., 2007; Nag et al., 2011); further research is required to determine
General Introduction and Literature Review Chapter 1
12
how biochar amendment influences the net effect of the pesticide on microbial
abundance.
The increased soil microbial biomass induced by biochar addition has a variable
effect on specific microbiological phyla and families (Lehmann et al., 2011;
Anderson et al., 2011). Biochar addition can increase the abundance of
Actinobacteria (Khodadad et al., 2011), Gemmatimonadetes (Khodadad et al., 2011),
Zygomycota (Lehmann et al., 2011) and Glomeromycota (Lehmann et al., 2011).
Similarly, pine biochar increased the abundance of Bradyrhizobiaceae,
Hyphomicrobiaceae, Streptosporangineae and Thermomonosporaceae but decreased
the abundance of Nitrosomonadaceae (nitrosovibrio), Streptomycetaceae and
Micromonosporaceae (Anderson et al., 2011). However in general, less genetically
diverse microbial communities have been found in biochar amended soils (Lehmann
et al., 2011; Khodadad et al., 2011). Further research should determine whether
changes in process rates (e.g. nitrification) induced by biochar amendment correlate
with changes in the microbial population that exhibit the genetic capabilibility of
regulating the changed process rate (e.g. the ammonia oxidiser population). Research
should also correlate changes in both the microbial community and the process rate
with the physical and chemical properties the biochar. This may facilitate the
prediction of microbial population changes and process rate changes based on the
properties of the biochar.
General Introduction and Literature Review Chapter 1
13
1.4 The Nitrogen Cycle
The N cycle is integral for all living organisms. For plants, N is contained within
compounds such as amino acids and proteins. In conventional agricultural systems,
plants have access to two sources of N, biologically derived N and inorganic fertiliser
N. Although many conventional farming systems only apply N in the form of
inorganic fertiliser (e.g. ammonium (NH4+)), the contribution of biologically
mediated plant available N can be up to 90% in Australian soils (Angus, 2001), which
constrasts with temperate systems where approximately 50% of plant N uptake is
from fertilisers (Jenkinson, 2001).
Biochar addition can alter a number of N cycling processes (Clough & Condron,
2010) but due to differences in biochar type, soil type and climatic conditions the
impact of biochar in many agro-ecosystems is unclear. This thesis focuses on the
impacts of biochar addition to soil on the N cycle for broadacre agriculture in WA.
Thus the following sections of this review synthesise research examining how
biochar addition to soil affects N sorption, desorption, N leaching, N immobilisation,
N mineralisation, nitrification, and plant N uptake and where possible extrapolate
results to a WA broadacre agricultural context (Fig. 1.2; Table 1.2).
General Introduction and Literature Review Chapter 1
14
Fig. 1.2 (page 15): A diagram describing how biochar addition to soil can influence
the N cycling processes assessed in this thesis. Note: 1 – Lehmann et al., 2003; 2–
Deenik et al., 2010; 3– Chan et al., 2008; 4– Chan et al., 2007; 5– Prendergast-
Miller et al., 2011; 6– Ding et al., 2010; 7 – Laird et al., 2010b; 8– Berglund et al.,
2004; 9 – DeLuca et al., 2006; 10 – Ball et al., 2010; 11 – Clough & Condron, 2010;
12– Lehmann et al., 2011; 13– Clough et al., 2010; 14– Spokas et al., 2010; 15–
Spokas et al., 2012.
General Introduction and Literature Review Chapter 1
15
Biochar addition to soil may influence Plant N uptake
by: Increasing immobilisation of N1;2; Increasing supply of P and K3;4;
Localising NO3- in rhizosphere biochar5.
NO3-
Biochar addition to soil may influence N leaching
by: Increasing sorption of N1;6;7;
Increasing immobilisation of N1;7; Altering mineralisation rates7;
Increasing soil water holding capacity1. Leached NH4+ Leached NO3
-
Biochar addition to soil may influence Nitrification by:
Sorbing inhibitory compounds8;9; Introducing inhibitory compounds11;13;
Altering immobilisation of N9;10; Emitting ethylene14.
Biochar addition to soil may influence Sorption
by: Adding sorptive functional groups15;
Increasing soil surface area.
Biochar addition to soil may influence Immobilisation
by: Adding labile C compounds1;2;12.
Biochar addition to soil may influence Mineralisation
by: Introducing inhibitory compounds11;13.
Sorbed N
Nitrification
Sorption
Immobilisation
Sorption
Immobilisation
Immobilised N
NH4+
Mineralisation
Organic Nitrogen
Plant N uptake Plant N uptake
Leaching Leaching
Tab
le 1
.2:
Su
mm
ary
of s
ome c
ha
nges
in N
cyc
ling
pro
cess
es in
duce
d b
y th
e a
ddi
tion
of b
ioch
ar
to s
oil.
Ital
icis
ed
sta
tem
ents
are
hyp
oth
ese
s (e
ither
hyp
oth
eses
for
cha
nge
s in
th
e pr
oce
ss r
ate o
r h
ypo
thes
ised
mech
anis
ms)
sug
gest
ed
by t
he c
orr
esp
on
ding
refe
ren
ce.
N c
ycle
pro
cess
es
Net
pro
cess
effe
ct
Me
cha
nis
ms
aff
ect
ing
pro
cess
ra
tes
Re
fere
nce
(s)
- M
iner
alis
atio
n an
d
nitr
ifica
tion
Incr
ea
se: N
itrifi
catio
n ra
tes
we
re a
bo
ut 5
times
hig
her
in f
ore
st (
low
nitr
ifica
tion
act
ivity
) so
ils.
Ch
arco
al s
orb
ed
phe
nol
ic c
ompo
und
s ca
usin
g
eith
er
incr
ease
d ni
trifi
catio
n a
nd/
or d
ecre
ase
d
imm
obili
satio
n.
De
Luca
et a
l., 2
00
6
D
ecr
ease
: At t
he
en
d o
f a 1
4 d
ay
incu
batio
n,
NH
4+ co
nte
nt w
as
25%
low
er
with
a lo
w
vola
tile
mat
ter
(VM
) b
ioch
ar
and
75%
low
er
with
a h
igh
VM
bio
cha
r
Hig
he
r vo
latil
e co
nte
nt b
ioch
ars
ad
de
d p
hen
olic
com
pou
nd
s to
soi
l, re
sulti
ng in
imm
obi
lisa
tion,
de
crea
sing
min
era
lisa
tion.
De
enik
et
al.,
20
10
D
ecr
ease
B
ioch
ar a
me
ndm
ent t
o so
il ca
uses
eth
yle
ne
em
issi
on
whi
ch c
an
dec
reas
e ni
trifi
catio
n.
Sp
oka
s et
al.,
201
0
- Im
mo
bilis
atio
n In
cre
ase
La
bile
C c
onte
nt in
bio
char
is m
icro
bial
ly
ava
ilabl
e; c
au
sin
g im
mo
bilis
atio
n o
f N.
Leh
ma
nn
et a
l., 2
003;
Lair
d e
t al
., 2
010
b;
De
enik
et
al.,
20
10
- P
lan
t up
take
In
cre
ase
d N
fe
rtili
ser
use
effi
cien
cy
Pro
visi
on o
f o
the
r n
utri
ent
s, s
uch
as P
an
d K
,
lea
din
g to
N b
ein
g a
mor
e lim
itin
g nu
trie
nt.
Ch
an
et a
l., 2
00
7;
Ch
an e
t al
., 2
008
In
cre
ase
d N
fe
rtili
ser
use
effi
cien
cy
Loca
lisat
ion
of N
O 3- w
ithin
rhi
zosp
here
bio
cha
r P
rend
erga
st-M
iller
et
al.,
20
11
- Le
ach
ing
De
crea
se -
Cu
mu
lativ
e N
H4+
lea
chin
g
de
cre
ased
by
25%
with
ch
arco
al a
dditi
on
3
we
eks
aft
er fe
rtili
satio
n
Sor
ptio
n of
NH 4
+
Leh
ma
nn
et a
l., 2
003
In
cre
ased
wa
ter
hol
din
g ca
pa
city
Le
hm
an
n et
al.,
200
3
D
ecr
ease
S
orp
tion
of N
O 3-
In
cre
ase
- O
ver
a 4
5 w
eek
incu
bat
ion,
bio
cha
r a
t th
e gr
eat
est a
dded
ap
plic
atio
n
rate
(20
g k
g-1 d
ry s
oil)
incr
ea
sed
NO 3-
lea
chin
g b
y 2
5%
Incr
eas
ed o
rgan
ic N
min
era
lisa
tion
Lair
d e
t al
., 2
010
b
D
ecr
ease
- O
ver
a 45
we
ek
incu
batio
n,
bio
cha
r a
ppl
ica
tion
with
man
ure
de
cre
ase
d
NO
3- leac
hin
g co
mp
are
d to
no
bioc
har
cont
rols
Bio
char
sor
be
d N
H 4+ a
nd
solu
ble
org
ani
c
com
pou
nd
s, in
hibi
ting
org
anic
N m
ine
ralis
atio
n
Lair
d e
t al
., 2
010
b
- S
orp
tion
NH 4
+
Incr
ea
se
Larg
e su
rfac
e ar
ea,
va
riabl
e s
urf
ace
ch
arg
e
- S
orp
tion
NO 3
- In
cre
ase
La
rge
surf
ace
are
a, v
aria
ble
su
rfa
ce c
har
ge
- D
eso
rptio
n S
mal
l de
cre
ase
- 8
0%
of t
he N
H3 so
rbe
d to
bio
cha
r in
a g
lass
hou
se e
xpe
rim
ent
wa
s
bio
ava
ilabl
e
Larg
e s
urfa
ce c
harg
e T
agh
iza
de
h-T
oos
i et
al.,
20
12
General Introduction and Literature Review Chapter 1
18
1.5 Sorption of nitrogen to biochar
Biochar, charcoal and activated C are used in industrial, medicinal and filtration
processes to sorb impurities. Their large surface area and variety of surface
functional groups enable them to sorb a range of chemicals, such as pesticides
(Yang et al., 2004; Bornemann et al., 2007), aromatic hydrocarbons (Zhu &
Pignatello, 2005; Accardi-Dey & Gschwend, 2003), metals (Ma & Rate, 2007)
nitrogenous ions such as NH4+ (Ding et al., 2010), ammonia (NH3; Kastner et al.,
2009; Taghizadeh-Toosi et al., 2012) and NO3- (Mizuta et al., 2004). The role that
sorption interactions play in altering N bioavailability is unclear because N sorption
to biochar after its addition to soil has not been assessed.
1.5.1 Mechanisms of sorption and properties influencing sorption to biochar
Biochar can both accept and donate electrons in pi bonds and electrostatic
attractions (Keiluwait & Kleber, 2009). This is because it can be comprised
primarily of aromatic C rings located within graphene sheets and can exhibit
varying charge (Schmidt & Noack, 2000). Functional groups, such as carboxyl
groups, are located on the edge of graphene sheets (Schmidt & Noack, 2000).
Acidic functional groups, and areas of the graphene sheet with impurites are
electron rich, and react with pi bond acceptors (Zhu & Pignatello, 2005). Negatively
charged functional groups enable NH3 sorption (Seredych & Bandosz, 2007;
Kastner et al., 2009; Clough & Condron, 2010; Spokas et al., 2012) and increase
CEC upon addition to soil (Boehm, 1994; Liang et al., 2006; Clough & Condron,
2010). By contrast, basic functional groups present on biochar react with pi bond
General Introduction and Literature Review Chapter 1
19
donors anabling anion exchange capacity (Boehm, 1994). As the pH of biochar
increases, its negative charge increases proportionally (Cheng et al., 2008) and
although biochar pH will be indicative of the general charge, there may be some
different functional groups of varying charge within the biochar.
The functional groups on the biochar are influenced by a number of factors
including pyrolysis temperature, feedstock and extent of oxidation. Greater
pyrolysis temperatures increase the C content of biochars, decreasing acidic
functional groups and increasing basic functional groups (Antal & Grønli, 2003;
Chun et al., 2004). Greater pyrolysis temperatures also increase the hydrophobicity
of biochar which restricts access of water soluble molecules like NH4+ (Seredych &
Bandosz, 2007) but enables hydrophobic organic matter sorption (Kasozi et al.,
2010). The feedstock used to create the biochar shall influence which functional
groups are present on the biochar surface. Over time, biochar oxidises (Cheng et al.,
2006; Cheng et al., 2008). Through changes in surface functional groups, oxidation
decreases surface positive charge and increases negative surface charge,
correspondingly increasing cation retention, but decreasing anion retention (Cheng
et al., 2008). The main reactive functional groups on aged biochar are carboxyl and
phenol groups (Liang et al., 2006). The extent of oxidation is highly correlated with
temperature (Cheng et al., 2006; Cheng et al., 2008) but the time required for
oxidation to occur is unclear.
General Introduction and Literature Review Chapter 1
20
The total surface area of biochar influences its sorption potential by providing a
larger area for reactions to occur. Although the surface area of biochar varies with
biochar type (ranging from 4-605 m2 g-1; Table 1.1; Table 1.3) it is generally greater
than quartz based sand (~1.3 m2 g-1) or kaolin (4-88 m2 g-1; Hart et al., 2002). The
feedstock used to produce the biochar influences the surface area of biochar (Table
1.3). Analysis method also influences surface area values; Bornemann et al. (2007)
used N2 adsorption on a BET surface area analyser. The surface area of biochar
generally increases with peak pyrolysis temperature (Table 1.3) to a temperature of
around 500-600°C (Antal & Grønli, 2003).
Table 1.3: Specifice surface areas of biochar produced from two feedstocks at three
pyrolysis temperatures; data from Bornemann et al. (2007).
Biochar Feedstock Pyrolysis Temperature
(°C)
Specific Surface Area
(m2 g-1)
Red Gum (Eucalyptus camaldulensis
Dehnh.)
250 8
450 34
850 605
Phalaris (Phalaris tuberosa L.) 250 4
450 20
850 371
General Introduction and Literature Review Chapter 1
21
1.5.2 The role of nitrogen sorption to biochar in ecosystem processes
The sorption of N to biochar has been suggested to play a role in ecosystem
processes (Lehmann et al., 2003), yet has not actually been tested. As discussed, it
is clear that biochar can sorb a range of compounds and that the introduction of
biochar to soil has the potential to increase nitrogen retention through electrostatic
adsorption (Keiluweit & Kleber, 2009). However the quantity of sorption that does
occur is unclear and thus, the role that N sorption to biochar actually plays within
ecosystems, whether broadacre agriculture in WA or not, is currently unknown.
1.6 Desorption
Once sorbed to biochar, for N to be bioavailable it must be desorbed. One study
demonstrated that a portion of sorbed N is bioavailable because up to 45% of N
sorbed to biochar was recovered as soil N (either organic or inorganic) or and up to
35% of sorbed N was recovered as plant N (Taghizadeh-Toosi et al., 2012). The
remaining sorbed N was not recovered. By contrast, Hina et al. (2010) found that
only 14-27% of sorbed NH4+ could be desorbed by extraction (2M potassium
chloride). It is likely that the prevalence of desorption depends on the surface
functional groups and charge properties of the biochar.
In the study of Taghizadeh-Toosi et al. (2012) all NH3 sorbed to the biochar
occurred in vitro. Thus sorption within the agro-ecosystem was not tested. As
desorption can only occur after sorption the potential for N sorption to biochar to
occur after addition to soil should also be assessed.
General Introduction and Literature Review Chapter 1
22
1.7 The influence of biochar on nitrogen leaching
Leaching of inorganic N and dissolved organic N (DON) can contribute to
eutrophication, soil acidification, and agricultural productivity losses. The deep
sands and duplex (or texture contrast) soils that occupy about 60% of the broadacre
agricultural area of WA (Tennant et al., 1992) are prone to leaching due to low
water and nutrient retention capacities (Dolling & Porter, 1994; Dolling et al., 1994;
Ridley et al., 2001). Nitrogen leaching can be decreased by the application of
biochar (Lehmann et al., 2003; Ding et al., 2010; Laird et al., 2010b). The
minimum application rate required to induce a response is unknown, but 0.5% w/w
biochar/soil (Ding et al., 2010) is the lowest applied rate of biochar demonstrated to
decrease N leaching in the published literature. Further, it is also unclear if the rate
of N leaching decreases proportionally with increasing application rate. There are
five main causes of decreased N leaching with biochar application: increased
sorption of nutrients (Lehmann et al., 2003); increased water holding capacity
(Lehmann et al., 2003); changes in organic N mineralisation (Laird et al., 2010b);
changes in nitrification; and changes in immobilisation (Lehmann et al., 2003).
1.7.1 Leaching of nitrogen decreases due to sorption
As discussed previously (Section 1.5), biochar has the ability to sorb N. This has
been hypothesised to be a cause of decreased N leaching with biochar application,
but this hypothesis has not been tested. Consequently, it is unclear how much NO3-,
NH4+ and organic N leaching is decreased due to sorption to biochar. The extent to
which sorption decreases N leaching will change with biochar type and application
General Introduction and Literature Review Chapter 1
23
rate due to varying surface areas and quantity and types of surface functional
groups.
1.7.2 Leaching of nitrogen decreases due to increasing water holding capacity
The application of biochar to coarse-textured soils increases WHC by increasing
total soil surface area, and the density of pores within the soil (Tryon, 1948)
enabling sorption of water. Increasing soil surface area and WHC by applying fine
textured amendents, like bauxite residue mud (the fine fraction of the waste
component from bauxite extraction) can decrease nutrient leaching (Vlahos et al.,
1989). Biochar is likely to decrease nutrient leaching similarly.
1.7.3 Leaching of nitrogen changes due to nitrification and mineralisation
Negatively charged clay electrostatically attracts NH4+. If a greater proportion of N
is found as NO3-, because of increased nitrification, the risk of N leaching increases.
Similarly DON generally exhibits negative charge, thus also is less attracted to clay
than NH4+. Adding biochar to soil can change nitrification and mineralisation rates
(discussed in Sections 1.9 and 1.10) and hence the N form available to leach.
In an Amazonian Ferralsol, Lehmann et al. (2003) found that although NH4+
leaching decreased with biochar addition, NO3- leaching increased. Lehmann et al.
(2003) suggest the change in leaching was due to the increased sorption capacity,
increased WHC or greater microbial immobilisation resulting from biochar
amendment, but the increased NO3 leaching is not explained. However, increased
General Introduction and Literature Review Chapter 1
24
nitrification could explain the results of Lehmann et al., (2003) because it would
result in both increased NO3- leaching and decreased NH4
+ leaching.
In a 45 week leaching experiment using soil (Typic Hapludolls) from the mid-west
of the United States of America, the increased NO3- leaching with the greatest rate
of biochar addition was attributed to increased organic N mineralisation (Laird et
al., 2010b). By contrast, with manure and biochar additions, NO3- leaching
decreased compared to manure additions alone also apparently due, in part, to less
organic N mineralisation (Laird et al., 2010b). With manure addition the decreased
organic N mineralisation was attributed to the sorption of NH4+ and DON to biochar
limiting substrate availability (Laird et al., 2010b). Laird et al. (2010b) disregard
anion retention as an influencing factor, suggesting that active sites on aged biochar
are primarily carboxylic and phenolic groups, as demonstrated in Liang et al.
(2006). The validity of this assumption is not known. Most of the leaching in Laird
et al. (2010b) occurred within the first 12 weeks without manure addition, and
between weeks 11 and 30 with manure addition. The extent of oxidation is highly
correlated with temperature (Cheng et al., 2006; Cheng et al., 2008). This leaching
experiment was conducted at 25°C (Laird et al., 2010b), but Cheng et al. (2006) did
not measure any net change in total acidic functional groups after a four month
incubation at 30°C. Future research should clarify the links between organic N
mineralisation, N leaching and the aging of biochar.
General Introduction and Literature Review Chapter 1
25
1.7.4 Leaching of nitrogen changes due to microbial immobilisation
Microbial immobilisation of N can decrease the total quantity of N available for N
leaching. The manner in which biochar addition to soil changes microbial
immobilisation of N will be discussed in a following section (Section 1.8); this
section will discuss the implications of immobilisation for N leaching. Due to the
high C content and low N content, immobilisation of N has been attributed to
decreased leaching of N (Lehmann et al., 2003; Laird et al., 2010b) yet
immobilisation was not measured in either study for verification.
1.8 Changes to nitrogen immobilisation induced by biochar application
Soil microbes require a certain portion of N for metabolism, cell maintenance and
other functions to maintain their desired C:N ratio (5:1 for bacteria, 15:1 for fungi;
Killham, 2006). For high C substrates, such as wheat residue (C:N ratio of
approximately 100:1) microbial decomposition requires available N, hence
microbial immobilistion of N occurs. Because biochar also contains a large C:N
ratio (>32.5; Table 1.1) many previous studies have attributed decreased N
leaching, or net NH4+ with biochar amendment to microbial immobisation of N (e.g.
Lehmann et al., 2003; Steiner et al., 2008b; Deenik et al., 2010; Laird et al.,
2010b). Despite this the quantity of N immobilised in the microbial biomass is
usually not measured. Literature assessing the change in the nitrogen component of
the microbial biomass could not be found.
General Introduction and Literature Review Chapter 1
26
Work that attributes results to N immobilisation because of widening C:N ratios can
be criticised in two ways. Firstly, it must be the amount of labile carbon that is
assessed, rather than the total carbon. The labile carbon on the biochar is more
likely to directly influence the microbial community, hence for immobilisation it is
more important that the biochar is assessed for a labile C:N ratio, rather than a total
C:N ratio. Secondly, for net immobilisation to occur the microbial biomass must
utilise inorganic N at a faster rate than it is being supplied via mineralisation of
SOM or from external inputs. As stated previously, this has not been reported in
published literature.
There has been assessment of changes in the soil microbial biomass with biochar
additions to soil via either the substrate induced respiration (SIR) method
(Anderson & Domsch, 1978) or the fumigation extraction method (Vance et al.,
1987). Literature mostly states that adding biochar to soil increases the microbial
biomass (Steiner et al., 2008a; Kolb et al., 2009; Lehmann et al., 2011). Caution
must be shown when interpreting the results. If the biochar contains carbonates
(CO32-; e.g. Singh et al., 2010) and is added to an acidic soil, abiotic carbon dioxide
(CO2) evolution may confound results. Despite this many studies have not assessed
biochar for CO32- (e.g. Steiner et al., 2008a). Calculating the microbial biomass
using the fumigation extraction method may require a correction factor to account
for sorption of organic C, released during chloroform induced cell lysis, to biochar
(Liang et al., 2010), but this may not be required for all biochar types (Durenkamp
et al., 2010).
General Introduction and Literature Review Chapter 1
27
This chapter shall not assess the soil biological changes due to biochar addition
further than discussed in Section 1.3.3, except to re-iterate that in a comprehensive
review Lehmann et al. (2011) concluded that in most cases biochar addition has
resulted in greater microbial abundance and that Section 1.3.3 concluded that this is
also likely to occur in the coarse-textured soil of the Western Australian wheatbelt.
If biochar amendment causes immobilisation of N, plant N availability is likely to
decrease. However the magnitude of the change is unclear, but will likely vary with
biochar application rate and type.
1.9 Biochar influences on nitrogen mineralisation
For this chapter, N mineralisation specifically refers to ammonification, the
conversion of organic N to NH4+. Microbes decompose organic matter to obtain the
energy and nutrients required for cell functioning and maintenance (Murphy et al.,
2003). Where the decomposing substrate contains N surplus to the decomposing
microbe requirements, N is released in the form of NH4+. The total production of
NH4+ over time is the gross rate of N mineralisation which contrasts with the net
rate of N mineralisation, which is the change in total NH4+ over time.
There are large deficiencies in our understanding of how biochar influences N
mineralisation because research assessing how biochar influences gross N
mineralisation is lacking. Current understanding of how biochar alters N
mineralisation is based on changes in net N mineralisation. The addition of two
types of macadamia nut shell biochar decreased soil NH4+ (by 25% with a low
General Introduction and Literature Review Chapter 1
28
volatile matter (VM) biochar and 75% with a high VM biochar) when added at 5%
w/w biochar/soil (Deenik et al., 2010). Deenik et al. (2010) attributed the decreased
NH4+ to greater microbial immobilisation of N because soil respiration either
increased or did not change with the addition of biochar; the intimate link between
the C and N cycles suggest that N mineralisation will have either increased or not
changed similarly (Manzoni et al., 2008). Biochar can contain volatile organic
compounds (VOCs) such as acetaldehyde, α-pinene, β-pinene and trans-pinocarveol
(Clough et al., 2010). Α-pinene can increase C mineralisation and decrease net N
mineralisation (Uusitalo et al., 2008). If α-pinene was present on the macadamia nut
shell biochar, decreased N mineralisation and increased CO2 evolution may also
have occurred. This would alter the interpretation of the results of Deenik et al.
(2010) to suggest that decreased N mineralisation may have occurred. As such,
biochar may contain compounds inhibitive to mineralisation and decrease intrinsic
soil N mineralisation rates upon amendment. Thus the influence of VOCs contained
within biochar, and their influence on N mineralisation requires research.
Although biochar may introduce compounds inhibitory to N mineralisation to soil,
biochar may also sorb compounds, such as phenolics that may be inhibitory to
mineralisation (Gundale & DeLuca, 2007). Although such phenolics may not be
present in agricultural soil types (DeLuca et al., 2006), other compounds, such as
pesticides (e.g. tebuconazole and diuron) that inhibit microbial activity (Prado &
Airoldi, 2001; Muñoz Leoz et al., 2011) may be present. Pesticide bio-availability
General Introduction and Literature Review Chapter 1
29
may be lessened with biochar addition (Nag et al., 2011), but whether N
mineralisation is influenced similarly is unclear.
Biochar could also change the N mineralisation rate by sorbing compounds integral
to mineralisation such as enzymes. Sorption of enzymes, like β-glucosidase, to
biochar can decrease their activity (Bailey et al., 2011; Lammirato et al., 2011).
Bailey et al. (2011) showed β-glucosidase, lipase and leucine aminopeptidase
activities decreased upon biochar exposure in vitro, but N-acetylglucosaminidase
activity increased. When biochar was added to three soils types from three different
ecosystems, arable (Ultic Haploxeroll), grassland (Xeric Torripsamment) or
shrubland soil (Xeric Haplocambid), the effect on enzyme activity was highly
variable (Bailey et al., 2011). With biochar addition to the arable soil, lipase activity
doubled; but decreased with biochar addition to the grassland and shrubland soils
(Bailey et al., 2011). Leucine aminopeptidase activity also increased with biochar
addition to the arable soil, but did not change with biochar addition to the grassland
soil, and halved with biochar addition to the shrubland soil (Bailey et al., 2011). As
SOM content of the three soil types used in Bailey et al. (2011) was not presented,
it cannot be assessed how enzyme activity varies with SOM content. Other research
has shown that SOM mineralisation decreases with biochar addition to soil correlate
with increasing SOM content (Keith et al., 2011). Whether the changes in the
activity of enzymes with biochar addition to soil correlate with changes in SOM
content is unknown. Similarly, it is unknown whether sorption of dissolved organic
General Introduction and Literature Review Chapter 1
30
C (DOC) and DON to biochar when added to soil affects mineralisation, and
whether the mineralisation changes correlate with SOM content.
Substrates for N mineralisation may also sorb to biochar restricting their mobility
within the soil solution. The sorption affinity of varying organic matter compounds,
such as humic acids and catechol, is known to vary in vitro (Kasozi et al., 2010),
thus it is also likely to vary upon amendment to soil. Although not determined, the
mineralisation of sorbed organic N could be expected to slow, but not completely
inhibited because the sorption of β-glucosidase to biochar limits its catylitic activity
but does not halt it (Lammirato et al., 2011). This topic, however, requires further
research.
1.10 The influence of biochar on nitrification
In boreal forests, biochar can increase nitrification rates because it may sorb
phenolic compounds (Berglund et al., 2004; DeLuca et al., 2006; Ball et al., 2010).
These volatile monoterpenes can inhibit nitrification through allelopathic inhibition
(Paavolainen et al., 1998). Ammonia monooxygenase activity is inhibited by
phenolic compounds or monoterpenes with a 6 C ring and an unsaturated C-C bond
close to the terminal position (White, 1988; White, 1991; White, 1994). Such
volatile monoterpenes are common in the soils of Ponderosa pine forests. In
contrast to forest soils, nitrification in grassland soils did not increase with biochar
addition possibly due to the higher inherent nitrification rates or lack of phenolic
compounds (DeLuca et al., 2006). In agricultural systems other compounds such as
General Introduction and Literature Review Chapter 1
31
pesticides such as tebuconazole can inhibit nitrification (Muñoz-Leoz et al., 2011).
Muñoz-Leoz et al. (2011) partially attribute the decreased nitrification to a killing
of fungi, resulting in a large pool of nutrients for ammonifying bacteria combined
with concomitant mineralisation of organic N to NH4+, although ammonia-oxidising
archaea are not mentioned, Muñoz-Leoz also suggest ammonia-oxidising bacteria
are severely inhibited by tebuconazole. Biochar can sorb other pesticides (trifluralin
and atrazine) negating their influence on weeds (Nag et al., 2011); whether sorption
of pesticides to biochar negates their influence on nitrification is unclear.
Biochar addition to agricultural soil may decrease nitrification if it contains VOCs
or polycyclic aromatic hydrocarbons (PAHs) (Clough & Condron, 2010).
Nitrification is sensitive to PAHs and VOCs (Svederup et al., 2002; Uusitalo et al.,
2008), with the degree of sensitivity to PAHs correlating with the lipophilicity
(Svederup et al. 2002). Sverderup et al. (2002) suggest that PAHs exhibit toxicity to
nitrifiers in a non-specfic mode of action through narcoticism, where the function
and fluidity of biological membranes are disturbed through dissolution (partial or
otherwise). The manner in which VOCs such as monoterpenes affect nitrification
has already been discussed. In a study examining the effect of biochar addition to a
soil used for dairy production on N2O emissions, nitrification was not affected by
biochar addition, even though the biochar contained pinene and acetaldehyde
(Clough et al., 2010). Thus, the influence of PAHs and VOCs on nitrification
depends on the chemistry and concentration of the compound(s). The concentration
at which PAHs influence nitrification can range from concentrations greater than 22
General Introduction and Literature Review Chapter 1
32
mg kg-1 (Sverdup et al., 2002). Whilst White (1988) suggest that only a few parts
per million of monoterpenes similar to nitrapyrin may inhibit nitrification. The
VOCs (α-pinene and β-pinene) contained in the Radiata pine biochar of Clough et
al. (2010) were not quantified, so it is unclear at which concentration thresholds
certain VOCs and PAHs can be present in biochar before nitrification is inhibited.
An important phytohormone, ethylene can induce fruit ripening, fine root hair
growth, and other pleiotropic effects (Arbeles et al., 1992) as well as inhibiting
microbial processes such as ammonia oxidation (Porter, 1992). Ethylene can be
produced upon biochar addition to soil. It is unknown whether biochar decreases
ethylene oxidation or increases ethylene production but it has been hypothesised to
contribute to decreases in nitrification and CO2 evolution (Spokas et al., 2010). Its
production upon biochar amendment is highly variable, and could not be found to
correlate with any chemical or physical properties of biochar (Spokas et al., 2010).
Thus, the impact of this mechanism on soil processes requires further research.
1.11 The influence of biochar on plant nitrogen uptake
As an agricultural amendment, the manner in which biochar addition to soil
influences plant N uptake is highly important. Within the Amazonian region,
biochar amendment at 191 t ha-1 (20% w/w) decreased plant N uptake by up to 25%
(Lehmann et al., 2003). Plant N content and uptake decreased by up to 50% and
80%, respectively, with macadamia nut shell biochar additions of 5 and 10% w/w in
a glasshouse study using an Andisol (Haplustand) from the Hawaiian Islands
General Introduction and Literature Review Chapter 1
33
(Deenik et al., 2010). Both studies attributed the decreased plant N uptake to the
biological immobilisation of N (Lehmann et al., 2003; Deenik et al., 2010) but as
stated in Section 1.8 the N content of the microbial biomass was not measured.
Biochar application in north-eastern New South Wales, Australia has increased N
fertiliser use efficiency (Chan et al., 2007; Chan et al., 2008; Van Zwieten et al.,
2010a). In particular, Van Zwieten et al. (2010) claimed that greenwaste biochar
could decrease N fertiliser requirements by up to 90% when applied at 20 and 50 t
ha-1. However the wheat in the experiment was not grown to maturity (Van Zwieten
et al., 2010a) and an explanation for the increased N fertiliser use efficiency was
not provided, but increased nitrification and microbial activity were implicated.
Increased N fertiliser use efficiency with another type of greenwaste biochar
addition occurred at rates of 50 or 100 t ha-1, but not the lowest rate of 10 t ha-1
(Chan et al., 2007). Using a poultry manure biochar application increased N
fertiliser use efficiency when applied at 10 t ha-1 (Chan et al., 2008). Biochar is
relatively deficient in N, compared to potassium (K) and P. Thus, at very high rates
of application (>20 t ha-1), N may become the limiting nutrient, resulting in the
greater efficiency of use (Chan et al., 2007; Chan et al., 2008).
An alternate mechanism by which biochar addition to soil could increase N
fertiliser use efficiency is through the localisation of NO3- in rhizosphere biochar
particles (Prendergast-Miller et al., 2011). Mass flow, caused by root nutrient
uptake, may relocate NO3- in biochar particles within the rhizosphere, potentially
General Introduction and Literature Review Chapter 1
34
allowing greater NO3- uptake (Prendergast-Miller et al., 2011). Although the
retained N is likely to be bio-available (Taghizadeh-Toosi et al., 2012), it is unclear
if this mechanism translates into improved N fertiliser use efficiency because
Prendergast-Miller et al. (2011) did not find any difference in plant N uptake.
Within broadacre agricultural areas of WA practical biochar application rates are
less than 5 t ha-1 (Blackwell et al., 2010). This is due to both the low bulk density of
the biochar and the large areas over which the biochar is to be applied. At
application rates of 5 t ha-1 or less it is unknown whether biochar induced
immobilised N, greater K and P or localisation of NO3- within the rhizosphere will
influence plant N uptake. This could be verified in laboratory and glasshouse
experiments, but field experiments are also required to verify the results where
climatic conditions are not controlled.
1.12 Summary of key research areas highlighted within this review
The addition of biochar to coarse textured soil can alter N cycling processes
because of its porous structure, large surface area and reactive surface. In the
broadacre agricultural area of WA, where more than 60% of topsoil is coarse-
textured (Tennant et al., 1992), the interaction between biochar and the N cycle has
not been investigated. As an agricultural amendment, this thesis focuses on the
interaction between biochar addition to soil and the N cycling processes that
directly influence plant N uptake. To addresses some research deficiencies, this
thesis answers questions on how biochar addition to a coarse textured soil
General Introduction and Literature Review Chapter 1
35
influences N sorption to biochar, N mineralisation, nitrification, N immobilisation
and plant N uptake as summarised in Table 1.4.
General Introduction and Literature Review Chapter 1
36
Table 1.4: A summary of the questions addressed within each thesis chapter.
Chapter & Title Research Questions Addressed in this Thesis
Chapter 2: Decreased
soil microbial biomass
and nitrogen
mineralisation with
Eucalyptus biochar
addition to soil
� When added to a coarse textured soil, does biochar
sorb NH4+, NO3
- or amino acids?
� Does biochar influence net N mineralisation when
added to a coarse textured soil?
� Does biochar addition to a coarse textured soil
induce microbial immobilisation of N?
Chapter 3: Clay and
biochar amendments
decreased inorganic but
not dissolved organic
nitrogen leaching in soil
� Does biochar addition to a coarse textured soil
decrease N leaching?
� Is sorption the main mechanism decreasing
leaching when added to a coarse-textured soil?
Chapter 4: Organic
nitrogen mineralisation
in two contrasting agro-
ecosystems in
unaffected by biochar
addition
� Does organic N mineralisation change when
biochar is added to a coarse textured soil?
� Does organic N mineralisation change over time
after biochar addition to a coarse textured soil?
Chapter 5: Minimal
interaction present
between wheat chaff
biochar and N fertiliser
in a broadacre field
experiment
� When added to a coarse textured soil in a
broadacre Western Australian agro-ecosystem,
does biochar interact with N fertiliser to increase N
fertiliser use efficiency?
� Does biochar influence plant N uptake when added
to a coarse textured soil in a broadacre Western
Australian agro-ecosystem?
37
38
Biochar decreased N mineralisation Chapter 2
39
Chapter 2
Decreased soil microbial biomass and nitrogen
mineralisation with Eucalyptus biochar addition to a coarse
textured soil
2.1 Abstract
Biochar has been shown to aid soil fertility and crop production in some
circumstances. The effects of the addition of Jarrah (Eucalyptus marginata) biochar
to a coarse textured soil on soil carbon and N dynamics were investigated. Wheat was
grown for ten weeks, in soil treated with biochar (0, 5, or 25 t ha-1) in full factorial
combination with N treatments (organic N, inorganic N, or control). Samples were
analysed for plant biomass, soil microbial biomass carbon (MBC) and nitrogen
(MBN), N mineralisation, CO2 evolution, community level physiological profiles
(CLPP) and ammonia oxidising bacterial community structure. MBC significantly
decreased with biochar addition while MBN was unaltered. Net N mineralisation was
highest in control soil and significantly decreased with increasing addition of biochar.
These findings could not be attributed to sorption of inorganic N to biochar. CO2
evolution decreased with 5 t ha-1 biochar but not 25 t ha-1. Biochar addition at 25 t
ha-1 changed the CLPP, while the ammonia oxidising bacterial community structure
changed only when biochar was added with a N source. It was concluded that the
activity of the microbial community decreased in the presence of biochar, through
Biochar decreased N mineralisation Chapter 2
40
decreased soil organic matter decomposition and N mineralisation which may have
been caused by the decreased MBC.
2.2 Introduction
Application of biochar, a by-product of organic matter pyrolysis and a form of black
carbon (BC), to agricultural soils has received considerable attention (Lehmann &
Joseph, 2009; Clough & Condron, 2010; Lehmann et al., 2011). Biochar can be stable
for thousands of years (Preston & Schmidt, 2006). It has been proposed that biochar
can improve soil fertility and enhance C sequestration (Lehmann et al., 2006; Ogawa
et al., 2006). Such effects may be explained by potential improvements in soil
structure, increases in soil pH, soil moisture and nutrient retention and decreases in
nitrous oxide (N2O) and methane (CH4) emissions (Lehmann et al., 2006; Atkinson et
al., 2010; Sohi et al., 2010). In particular, amendment of soils with biochar (in the
range 0.5 to 135 t ha-1) has been shown to increase plant yield, improve chemical and
physical properties (Glaser et al., 2002), and modify the soil physical habitat for
microbial colonisation, thus altering soil microbial activity and community structure
(Pietikäinen et al., 2000; Steinbeiss et al., 2009). However, as there is a wide range of
biochar types, application rates, soil types and plant responses (Glaser et al., 2002)
the effects of biochar in agro-ecosystems are poorly understood. This is primarily
because there have been few reported studies that aim to elucidate the mechanisms
underpinning these reported effects on plant growth and soil quality.
Biochar decreased N mineralisation Chapter 2
41
Biochar has a large surface area, especially compared to sands, due to its porous
physical structure and highly aromatic microstructure (Schmidt & Noack, 2000).
Liang et al. (2006) report an enhancement in specific surface area of a sandy soil as a
result of biochar incorporation and that as a consequence these soils had a greater
potential cation exchange capacity and greater retention of dissolved organic matter
than adjacent soil (Liang et al., 2010).This potentially leads to enhanced availability
of C substrates and nutrient source for microorganisms (Zackrisson et al., 1996).
Increases in microbial activity and biomass have been reported with biochar addition
(Lehmann et al., 2011), along with changes in microbial community composition and
abundance (Steinbiess et al., 2009). Some of these increases may be due to biochar
providing a habitat for microbial colonisation (Pietikäinen et al., 2000) and protection
from predation by soil microarthropods (Zackrisson et al., 1996) as bacteria can
potentially inhabit the pores larger than 0.6 μm (Strong et al., 1998). Alternatively, in
the short term biochar may stimulate the microbial community by providing organic
C substrates (Smith et al., 2010). More recently, compounds inhibiting microbial
activity have also been found either on biochar (Deenik et al., 2010) or released after
its introduction to soil (Spokas et al., 2010). Regardless, it is unlikely that these
changes are spread equally across different phylotypes or functional groups and very
little is known about how specific microorganisms are affected (Graber et al., 2010).
Many soil processes may be affected by the incorporation of biochar into soil, in
particular biochar addition has been reported to alter nitrification rates (Berglund et
al., 2004; DeLuca et al., 2006) and potentially adsorb nitrogenous compounds
Biochar decreased N mineralisation Chapter 2
42
(Lehmann et al., 2003; Hina et al., 2010). In forest soils, biochar has generally been
associated with increased N mineralisation (Berglund et al., 2004; DeLuca et al.,
2006), however, influences within agricultural and grassland soils appear less certain
(DeLuca et al., 2006; Deenik et al., 2010). Biochar application to such soils has
resulted in either no change (DeLuca et al., 2006) or decreases (Deenik et al., 2010)
in N mineralisation, hence lower availability of N for plants (Lehmann et al., 2003).
There is currently limited understanding of the influence of biochar on soil N
processes, and in particular the organisms involved in N transformations. Autotrophic
ammonia oxidising bacteria (AOB) are a microbial functional group that mediate
nitrification, and are influenced by a variety of environmental factors (Gleeson et al.,
2010). The addition of biochar to soil can significantly increase the pH of the bulk
soil (Chan et al., 2008) or alter the local microsite pH, potentially providing a more
favourable habitat for nitrifying organisms, in particular AOB as they are sensitive to
pH (De Boer & Kowalchuk, 2001). There have been few studies examining the effect
of biochar additions on the AOB, with only the study of Ball et al. (2010) showing an
increase in AOB abundance after fire, which they suggest may be related to the
charcoal content and resultant alterations in pH.
The impact of amending a semi-arid agricultural soil with Jarrah (Eucalyptus
marginata Donn ex Sm.) biochar was investigated, with particular focus on C and N
dynamics. A pot experiment was designed in which biochar was added at rates
equivalent to 0, 5, and 25 t ha-1 to a coarse-textured soil that was selected for its low
surface area and lack of microbial refuges, in which wheat was grown. N applied in
Biochar decreased N mineralisation Chapter 2
43
either an inorganic or organic form was used to alter the C and N availability in the
soil so as to broaden the range of conditions under which changes to the soil
microbial community and soil organic matter decomposition were assessed.Three
hypotheses were investigated: that the addition of biochar to a coarse textured soil
would (i) increase the microbial population size due to increased habitable pore
space; (ii) increase microbial activity (assessed by C and N cycling) due to the
increased microbial biomass and; (iii) cause an increase in microbial community
functional diversity (assessed by CLPP) and AOB diversity (measured by terminal
restriction fragment length polymorphism (T-RFLP) of the amoA gene).
2.3 Materials and methods
2.3.1 Biochar characterisation
Jarrah wood (trunks and large branches; Eucalyptus marginata Donn ex Sm.) was
pyrolysed at 600°C for 24 h in a Lambiotte carbonisation reactor (Simcoa Ltd,
Bunbury, WA). The biochar used in the experiment was the fraction that passed
through a 3 mm seive. Total C and N were determined by combustion analysis (vario
Macro CNS; Elementar, Germany). The ammonium (NH4+) and nitrate (NO3
-)
content were determined by extracting with 0.5 M K2SO4 and analysing the extract
colorimetrically for NH4+
(Krom, 1980; Searle, 1984) and NO3- (Kamphake et al.,
1967; Kempers & Luft, 1988) on an automated flow injection Skalar Auto-analyser
(Skalar San plus).
Biochar decreased N mineralisation Chapter 2
44
The carbonate equivalence of the biochar was assessed using the method of Rayment
and Lyons (2011). The electrical conductivity (EC) and pH of the biochar were
determined in 1:5 (w/v; g cm-3) soil-to-water or 0.01 M CaCl2 mixtures, respectively.
The gravimetric ash content of the biochar was determined by loss on combustion
(750˚C). The elemental composition of the ash was determined by X-ray fluorescence
(Philips PW1404 XRF). Random powder X-ray diffraction pattern of biochar was
obtained for the range 0-70° 2θ (Philips PW 1830 X-ray diffractometer). Biochar and
soil samples were analysed for pore size distribution and surface area using a surface
area analyser (Micrometrics Gemini 2375 instrument with a Vac Prep 061) (Brunauer
et al., 1938). The biochar was sieved to obtain a particle size distribution. The biochar
contained 27% greater than 1 mm, 23% inbetween 1 mm and 425 μm, 19%
inbetween 425 μm and 180 μm and 31% less than 180 μm. The water holding
capacity of the biochar is 64% w/w and the bulk density is 0.45 g cm-3.
2.3.2 Soil preparation and description
Field dry soil (0-10 cm) was collected from a site 20 km north-west of Moora, WA
(30°32´ S 115°49´ E), sieved (<2 mm) and stored for 4 weeks at 4°C. The soil
contained 7.5 g kg-1 total C, 0.6 g kg-1 total N and 2 mg N kg-1 of NH4+-N and NO3
--
N (Table 2.1). Surface area, pore size distribution, pH and EC of the soil and
soil/biochar mixtures (5 and 25 t ha-1 equivalent) were assessed in the same manner as
for the biochar. The soil used contained 95% sand, 1.5% silt and 3.5% clay, and was
classified as a grey orthic Tenosol (Australian Soil Classification; Isbell 1996).
Biochar decreased N mineralisation Chapter 2
45
Table 2.1: Basic chemical properties of biochar and soil.
Chemical Properties Biochar Soil
Total N (g kg-1) 3.0 1.0
NH4+ (mg kg-1) < 0.1 2.0
NO3- (mg kg-1) < 0.2 2.0
Total C (g kg-1) 750.0 8.0
pH (CaCl2) 7.4 4.8
EC (μS cm-1) 554.3 44.1
Carbonate (mg g-1) 27.0 -
2.3.3 Pot experiment
A pot experiment was conducted to assess the influence of biochar on the soil
microbial community in the presence of growing plants. The experimental design
consisted of two factors: biochar (3 levels, fixed) and N treatment (3 levels, fixed)
with four replicates in a randomised block. The biochar treatments comprised of
biochar incorporated homogenously into soil at rates equivalent to 0, 5 and 25 t ha-1
(0%, 0.45% and 2.27% w/w respectively). The N treatments were: (1) organic N
applied as 500 kg ha-1 of a wheat straw and pig manure compost (Called ‘Balance’;
Custom Composts, North Dandalup, WA), which contained 15 kg N ha-1; (2)
inorganic N, 100 kg ha-1 of NH4NO3 (containing 35 kg N ha-1) at 3 and 6 weeks; or
(3) control, defined here as those pots receiving only basal N, as described below.
Pots were prepared by adding 2.2 kg of soil or soil/biochar mixture into non-draining
pots with a resulting bulk density of 1.4 g cm-3. This soil naturally compacts to a bulk
Biochar decreased N mineralisation Chapter 2
46
density between 1.4 and 1.6 g cm3. As such biochar application did not cause any
difference in the final bulk density. Basal fertiliser was added to all pots and then
adjusted to 50% WHC, equivalent to 30% water filled pore space (WFPS). Basal
nutrients were supplied at the following rates (mg kg-1 soil or soil/biochar mixture): N
33.3 (as NH4NO3), P 12.5, K 53.2, S 20.8, Ca 41, Mg 2.4, Mn 3.3, Zn 2.05, Cu 0.5, B
0.1, Co 0.1 and Mo 0.1. Due to the low soil N content (2 mg kg-1 of both NO3- and
NH4+), all pots (i.e. all biochar and N treatments) required an initial basal addition of
33.3 mg kg-1 N (45 kg ha-1 N) as NH4NO3 to ensure adequate plant growth. Three
wheat plants were grown per pot. A near constant soil water content of 50% WHC
was maintained; the WHC was measured and adjusted accordingly for each of the
biochar application rates. Over the length of the experiment the glasshouse average
daily maximum and minimum temperatures were 25 and 13°C respectively. Soil and
plants were collected for analysis 10 weeks after planting. Soil sub-samples (5 g)
were collected for DNA extraction and T-RFLP and stored at -20°C, the remaining
soil was used for soil analyses (MBC, MBN, CLPP, CO2 evolution, N
mineralisation).
2.3.4 Plant analysis
Shoots were harvested by cutting at soil level. Roots were then collected, with soil
removed by washing over a 4 mm sieve. Plant roots and shoots were then dried at
70°C for 72 h and weighed.
Biochar decreased N mineralisation Chapter 2
47
2.3.5 Microbial biomass
Microbial biomass was determined using the fumigation-extraction method (Vance et
al., 1987). To calculate MBC the resulting flush of oxidisable-C (analysed with a
Shimadzu Model 5050, Japan) was adjusted by a factor of 0.45 (Wu et al., 1990).
MBN was determined using the ninhydrin method (Joergensen & Brookes, 1990) and
adjusted by a factor of 6.47 (Sparling et al., 1993). Sample MBC values were divided
by MBN to obtain C-to-N ratios of the microbial biomass.
To test the extraction efficiency of microbial C in the presence of biochar, 5 g soil (at
50% WHC) was incubated with 500 µL of uniformly 14C-labelled glucose (100 µM;
8.3 kBq mL-1) for 3 h at 20°C in the presence of 1 M NaOH (1 mL) to trap evolved
14CO2. After incorporation of the 14C-label into the soil microbial biomass, replicate
samples of 14C-labelled soil were extracted with 0.5 M K2SO4 for 15 min (200 rev
min-1), centrifuged (18,000 g) and the 14C content of the supernatant determined by
liquid scintillation counting (unfumigated controls). The remaining soil samples were
CHCl3-fumigated (24 h) and then extracted with 0.5 M K2SO4 as described above,
except that biochar (0, 0.45 or 2.27% w/w) was added to the soil immediately before
extraction. The amount of 14C immobilised in the microbial biomass was assumed to
equal the total amount initially added minus that recovered as 14CO2 and as 14C in the
unfumigated K2SO4 extracts. The effect of biochar and fumigation on the extraction
of amino acids was also assessed.
Biochar decreased N mineralisation Chapter 2
48
2.3.6 Soil carbon dioxide evolution
At harvest, soil samples from the pot experiment were incubated in glass jars
containing a gas septum, adjusted to 40% WHC, incubated at 15ºC, and sealed to trap
respired CO2 (Anderson, 1982). Headspace gas was analysed for CO2 with an infra-
red gas analyser (IRGA; The Analytical Development Co., Series 225). Gas samples
were collected over a period of 65 d.
2.3.7 Community level physiological profiles (CLPP)
Catabolic response profiles were measured using the method of Degens & Harris
(1997). Substrates used were those specified by Stevenson et al. (2004) at the
concentrations used by Lalor et al. (2007). Headspace gas (1 mL) was analysed for
CO2 in an IRGA. The richness, evenness and diversity of CLPP profiles were
determined using indices described by Blackwood et al. (2007): richness (S),
Shannon diversity index (H’), Simpson index (D) and Shannon evenness (J’).
2.3.8 DNA extraction and terminal restriction fragment length polymorphism (T-
RFLP) profiles
Total nucleic acids were extracted from 0.5 g soil or soil/biochar mixes using
previously described methods (Griffiths et al., 2000) with a minor modification
(samples were homogenised using a Mini-BeadBeater-8 (Biospec Products Inc.,
Bartlesville, OK, USA) at 3200 rev min-1, 2 min).
Biochar decreased N mineralisation Chapter 2
49
Polymerase chain reaction (PCR) amplification of the amoA gene was based on the
method of Horz et al. (2004), using the primer set amo-1F (5’-
GGGGTTTCTACTGGTGGT-3’) and amo-2R (5’-CCCCTCKGS
AAAGCCTTCTTC-3’) (Rotthauwe et al., 1997) as previously described (Gleeson et
al., 2010). All PCRs were performed in duplicate and pooled for subsequent
purification, restriction and fragment analysis. Purified PCR products were quantified
and approximately 100 ng of PCR product was used in a restriction digest with the
restriction endonuclease enzyme HaeIII (New England Biolabs Inc.) as per the
manufacturer’s instructions. Terminal restriction fragment lengths (T-RFs) were
determined by electrophoresis using a capillary electrophoresis system (Applied
Biosystems) and analysis of fragment profiles was carried out using Genemapper®.
Profiles were generated for each sample based on relative height of peaks indicative
of relative abundance within the sample. T-RFs were assigned using the program
RiboSort (Scallan et al., 2008) and the statistical software R (R Core Development
Team, 2007). T-RFs in different profiles that differed by less that 0.5 bp were
considered identical (Dunbar et al., 2001). For each sample, bacterial T-RFLP
profiles were standardised by total fluorescence and fragments contributing <0.1% to
the total fluorescence of that sample were removed. The richness, evenness and
diversity of amoA T-RFLP profiles were determined using indices (S, H’, D and J’)
described by Blackwood et al. (2007).
Biochar decreased N mineralisation Chapter 2
50
2.3.9 Net nitrogen mineralisation
At harvest, soil and soil/biochar mixtures from the pot experiment were adjusted to
40% WHC and incubated at 15ºC in replicate containers that allowed free air
exchange. From this incubation, samples were collected over 70 days. Soil NH4+ and
NO3- was analysed as previously mentioned. Net N mineralisation and nitrification
rates were determined as the changes in the sizes of the inorganic N (NH4+ plus NO3
-)
and NO3- pools, respectively, over time.
2.3.10 Organic carbon sorption experiment
A mixture of 16 individual uniformly 14C-labelled amino acids was added to 0.5 M
K2SO4 to give a total concentration of 40 mg C l-1 (ca. 0.4 mM; 0.6 kBq ml-1). This
solution was then mixed with either; soil; soil/biochar mixture (25 t ha-1 or 2.27%
w/w) or; biochar alone at a soil-to-solution ratio of 1:4 (w/v). The soil suspension was
then shaken for 15 min at 200 rev min-1. After shaking, the suspensions were
centrifuged (16,000 g, 5 min) and the supernatant recovered for 14C determination
using a 1404 liquid scintillation counter (Wallace EG&G Ltd., Milton Keynes, UK).
These tests were both conducted using fresh soil and fresh biochar.
2.3.11 Nitrogen sorption experiment
Sub-samples (10 g) of fresh biochar, soil, or soil/biochar mixtures (5 or 25 t ha-1) and
100 mL of organic or inorganic N (leucine-N, (NH4)2SO4, KNO3 or NH4NO3)
solution were added to 120 mL vials. Vials were shaken for 24 h after which solution
N concentrations were measured for organic N, NO3- or NH4
+ as mentioned
Biochar decreased N mineralisation Chapter 2
51
previously. To replicate the extraction methodology, this procedure was repeated,
except after shaking for 24 h, K2SO4 salt was added to a concentration of 0.5 M and
shaken for a further 1 h. The N concentrations used were 0, 0.5, 1, 1.67, 2.5, 5, 10,
25, 50 and 100 mg N L-1 for leucine, (NH4)2SO4 and KNO3. The N concentrations for
NH4NO3 were doubled to ensure the same concentrations of NH4+ and NO3
- as the
previous incubations. The dilute concentrations (0, 0.5, 1 and 1.67 mg N L-1) were
equivalent to those in the net N mineralisation incubation (0, 5, 10 and 16.7 mg N
kg-1 soil).
2.3.12 Statistical analysis
A series of general analysis of variance (ANOVA) using GENSTAT 10th Edition
(Lawes Agricultural Trust, 2007) were completed. Multiple comparisons were done
using the Duncan’s Multiple Range Test (DMRT) and significance considered at the
P ≤ 0.05 significance level.
Multivariate statistical analyses were performed on standardised, (Log (X+1))
transformed CLPP data and standardised, untransformed T-RFLP profiles using
Primer 6 (Plymouth Marine Laboratory, 2007) and Bray-Curtis similarity matrices.
Tests of the null hypothesis among a priori defined groups were examined using
permutational multivariate ANOVA (PERMANOVA) for both data sets. All
PERMANOVA tests used 9999 permutations of raw data from residuals under a
reduced model (Anderson, 2001). Pairwise comparisons were performed to determine
whether amoA communities differed significantly (P<0.05) between biochar and N-
Biochar decreased N mineralisation Chapter 2
52
treatments. Analyses of differences in richness, evenness and diversity measures
between treatments were performed using ANOVA.
Data of solution concentrations were analysed to give 95% confidence intervals for
the means; these were used to judge significance.
2.4 Results
2.4.1 Biochar characterisation
Biochar comprised 75% C and 0.3% N with a C:N ratio of 275:1 (Table 2.1); this
equated to an addition of 75 and 15 kg N ha-1 in the 25 and 5 t ha-1 biochar
treatments, respectively. However, The NH4+ and NO3
- content of the extractions
were below the detection limit (<0.2 mg L-1 NO3- and <0.1 mg L-1 NH4
+; Table 2.1).
The biochar had a pH of 7.4 and an EC of 554.3 μS cm-1 (Table 2.1). Biochar
application increased the soil pH from 4.77 to 4.88 and 5.06 with the addition of 5
and 25 t ha-1 of biochar, respectively. The carbonate content of the biochar was 27 mg
g-1 (Table 2.1). The ash content was 60 mg g-1. The major elements in the ash,
determined by X-ray fluorescence analysis, were Si (206 mg g-1), Al (127 mg g-1), Fe
(91 mg g-1), Ca (54 mg g-1) and Mg (11 mg g-1). X-ray diffraction showed that the
biochar contained amorphous C, quartz and calcite (data not shown).
The amount of pore space surface area potentially habitable by microorganisms,
determined on the proportion of pores >0.60 μm, in the biochar alone was 4.42 m2 g-1,
Biochar decreased N mineralisation Chapter 2
53
which was six times greater than the soil (0.74 m2 g-1) pore area (Table 2.2).
However, at biochar application rates of 5 and 25 t ha-1 to soil, calculations
demonstrate this equates to an increase of 0.02 and 0.08 m2 g-1 of habitable pore space
(i.e. net increase of 2.7 and 10.8%).
Table 2.2: Incremental and total pore area (m2 g-1) for soil and biochar, along with
calculated estimates for application rates of 5 and 25 t ha-1 of applied biochar, for
the range of pore diameters deemed habitable by bacteria.
Pore diameter (μm) Quantity of biochar added (5 and 25 t ha-1 values calculated)
Soil 5 t ha-1 25 t ha-1 Biochar
4.50-1.90 0.28 0.28 0.28 0.49
1.90-1.20 0.19 0.19 0.20 0.67
1.20-0.90 0.13 0.13 0.15 0.96
0.90-0.70 0.09 0.09 0.11 1.05
0.70-0.60 0.05 0.06 0.08 1.25
Total 0.74 0.76 0.82 4.42
2.4.2 Plant growth analysis
ANOVA showed significant differences due to both biochar (P<0.001) and N
(P<0.001) treatments with no interaction. Post hoc comparisons using DMRT showed
biochar addition at 25 t ha-1 was associated with a significantly lower shoot dry
weight (6.1 g) compared to the 0 and 5 t ha-1 application rates (6.9 and 6.3 g,
respectively; Fig. 2.1a). Inorganic N addition resulted in significantly greater shoot
Biochar decreased N mineralisation Chapter 2
54
dry weight (8.3 g) compared to the organic N (5.8 g) and control treatments (5.7 g;
Fig. 2.1a). No differences between root dry weights were evident (data not shown);
however the root to shoot ratio was significantly lower with inorganic N application
(P<0.001; Fig. 2.1b).
Sh
oo
t Dry
Wei
gh
t (g
)
Ro
ot:S
ho
ot R
atio
0
1
2
3
4
0 5 25 0 5 25 0 5 25
Control Compost Inorganic N
(b)
0
2
4
6
8
10 (a)
Fig. 2.1: Shoot dry weight (a) and root: shoot ratio (b) results for the control,
organic N (compost) and inorganic N treatments at three added rates of biochar (0, 5
and 25 t ha-1). Error bars represent standard errors (n=4).
Biochar decreased N mineralisation Chapter 2
55
2.4.3 Microbial biomass
Overall, 97.4 ± 0.2% of the added 14C glucose-C was taken up by the microbial
biomass after 3 h, of which 81 ± 2% was immobilised and 19 ± 2% was respired as
14CO2 (data not shown). Chloroform fumigation-extraction revealed that 21.0 ± 0.1%
of the added 14C was present in the microbial pool whilst 79.0 ± 0.1% remained non-
extractable (data not shown). Overall, the presence of biochar at both addition rates
had no significant effect on the recovery of 14C from the soil microbial biomass. In
addition, biochar did not affect the recovery of amino acids either before or after
fumigation (Table 2.3). No significant difference in the recovery of leucine-N
standards over the range 0 to 50 mg amino-N kg soil-1 in the presence of biochar
when extracted in 0.5 M K2SO4 was found (data not shown). As such the fumigation-
extraction method was not affected by the presence of the biochar.
Table 2.3: Recovery of 14C-labelled amino acids added to soil in the presence and
absence of biochar either before or after CHCl3 fumigation. Values represent means
± standard errors (n=4). There were no significant differences between any of the
treatments (P > 0.05).
Non-fumigated Fumigated
(% recovery) (% recovery)
Soil 93.5 ±2.6 95.9 ±2.4
Soil + biochar (25 t ha-1) 99.2 ±5.9 103.6 ±4.1
Biochar 102.8 ±4.7 104.6 ±4.8
Biochar decreased N mineralisation Chapter 2
56
There was a significant effect of biochar rate on MBC (P=0.05), and of N treatment
(P<0.001), but no interaction between the treatments. MBC was greatest without
biochar addition (145 mg C kg-1) but decreased significantly with 25 t ha-1 biochar
addition (116 mg C kg-1; Fig. 2.2a). MBC was also significantly lower in the organic
N (135 mg C kg-1) and inorganic N (93 mg C kg-1) treatments compared to the control
(159 mg C kg-1) (Fig. 2.2a). MBN did not change with either biochar or N addition
and there was no interaction between the variables.
The initial microbial biomass C:N ratio was 8:1. This was changed by the addition of
N (P=0.005), but not biochar addition and there was no interaction. The microbial
C:N ratio was significantly decreased by the inorganic N treatment to 4.5:1 (Fig.
2.2c).
Biochar decreased N mineralisation Chapter 2
57
Mic
rob
ial B
iom
ass
C
(mg
kg-1
)
Mic
rob
ial b
iom
ass
N
(mg
kg-1
)
M
icro
bia
l Bio
mas
s 44
C:N
rat
io0
50
100
150
200
250 (a)
0
10
20
30 (b)
0
2
4
6
8
10
0 5 25 0 5 25 0 5 25
Control Compost Inorganic N
(c)
Fig. 2.2: Microbial biomass carbon (a), microbial biomass nitrogen (b) and
microbial biomass C:N ratio (c) results for the control, organic N (compost) and
inorganic N treatments at three added rates of biochar (0, 5 and 25 t ha-1). Error
bars represent standard errors (n=4).
Biochar decreased N mineralisation Chapter 2
58
2.4.4 Microbial community function (CLPP) and structural diversity (amoA T-
RFLP)
Multivariate analysis by PERMANOVA showed that addition of 25 t ha-1 biochar
significantly changed the CLPP (P<0.01), but addition of 5 t ha-1 biochar did not. The
N treatments had no effect on CLPP and there was no interaction between the two
factors (Table 2.4). Analysis of T-RFLP data revealed a significant effect of biochar
(P<0.001) but no significant effect of N (P<0.167) on ammonia oxidiser community
structure. There was a significant interaction (P<0.001) between biochar and N
treatments: without N addition (either compost or inorganic N), there was no effect of
biochar on the ammonia oxidiser community structure. However, in the presence of
compost and inorganic N there was a significant effect of biochar addition (Table
2.4).
The effect of biochar addition on CLPP diversity was only observed in the compost
or inorganic N treatments (P=0.002) where both 1/D and H’ significantly increased
with biochar addition (data not shown).
The amoA T-RFLP richness (S) averaged 64 and evenness, J’, averaged 0.67 across
all treatments; neither were significantly affected by biochar rate or N treatment.
However, in terms of evenness, there was a significant interaction between treatments
(P=0.03); when organic N (compost) was added there was a significant effect on
evenness; this was not the case with inorganic N addition or in the control treatment.
Biochar decreased N mineralisation Chapter 2
59
Diversity averaged 2.78 (H’) and 9.99 (1/D) with no significant effect of biochar rate
or N-treatment (data not shown).
Table 2.4: Significance of biochar and nitrogen treatments on terminal restriction
fragment length polymorphism (T-RFLP) and community level physiological profiles
(CLPP). NS, *, **, and *** denote not significant (P > 0.10), P < 0.10, P < 0.05,
and P < 0.005 respectively.
T-RFLP CLPP
Overall effects
Biochar *** ***
Nitrogen treatment NS NS
Biochar x Nitrogen treatment *** NS
Multiple comparisons within nitrogen treatment for TRFLP
Biochar rate (t ha-1) 0 5
Compost 5 **
25 * *
Nitrogen 5 *
25 * *
No treatment 5 NS
25 NS NS
Biochar decreased N mineralisation Chapter 2
60
2.4.5 Carbon dioxide evolution and nitrogen mineralisation
There was no interaction between biochar and N treatments but both biochar and N
had significant effects on CO2 evolution (P=0.003 and 0.001, respectively).
Cumulative CO2 evolution from soil was significantly lower where biochar was
added at 5 t ha-1 (101 mg CO2-C kg-1); with no difference between the 0 and 25 t ha-1
of biochar addition (Fig. 2.3). The addition of compost did not change soil CO2
evolution while the addition of inorganic N caused a significant decrease (data not
shown).
CO
2 ev
olu
tion
(mg
CO 2
-C k
g -1 )
Incubation period (weeks)
0
50
100
150
0 2 4 6 8 10
0 t ha5 t ha25 t ha
-1
-1
-1
Fig. 2.3: Soil cumulative CO2 evolution in the control treatment for biochar amended
at 0 (crosses), 5 t ha-1 (open triangles) or 25 t ha-1 (solid circles). Error bars
represent standard error (n=4).
Biochar decreased N mineralisation Chapter 2
61
Inorganic N in the incubated soil declined significantly with increasing biochar rate in
all three N treatments (P<0.001; Fig. 2.4). On average across N treatments the
inorganic N concentration was 11 mg kg-1, 7 mg kg-1 and 1 mg kg-1 where biochar
was added at 0, 5, and 25 t ha-1, respectively, after 10 weeks of incubation (Fig. 2.4).
This equated to significantly lower average net N mineralisation rates of 176, 102 and
15 μg N kg-1 day-1 with biochar addition at 0, 5 and 25 t ha-1, respectively (Table 2.5;
P<0.001). Net nitrification rates also decreased significantly with increasing biochar
addition (P<0.001; Table 2.5). Average net nitrification rates decreased from 178 μg
N kg-1 day-1 without biochar addition, to 103 and 12 μg N kg-1 day-1 with biochar
added at 5 and 25 t ha-1, respectively. At week 10, the proportion of N present as
NH4+ was significantly higher with biochar addition at 25 t ha-1 (average of 56%
across N treatments) than at 5 t ha-1 and the control (an average of 3.2% and 2.1%
across N treatments, respectively; P<0.001; Table 2.5).
Biochar decreased N mineralisation Chapter 2
62
Table 2.5: Final rates of net N mineralisation, net nitrification and the proportion of
NH4+-N at week 10. Values represent means ± standard errors (n=4), letters
represent significant differences (P < 0.05).
Nitrogen
Treatment
Biochar rate
(t ha-1)
Net N mineralisation
rate (μg kg-1 day-1)
Net nitrification rate
(μg kg-1 day-1)
NH4+-N proportion of
net N (%)
Control 0 145.8 ±10.8 cd 144.3 ±9.6 c 2.3 ±0.6
5 78.0 ±12.3 b 76.0 ±13.2 b 3.9 ±1.2
25 29.6 ±12.1 a 25.6 ±11.6 a 33.0 ±16.8
Compost 0 195.6 ±20.4 d 191.2 ±20.8 d 2.9 ±0.5
5 138.0 ±37.9 c 136.2 ±35.0 c 2.1 ±1.2
25 5.3 ±5.1 a 1.8 ±4.6 a 79.6 ±16.7
Inorganic N 0 186.3 ±7.7 cd 198.9 ±7.5 d 1.1 ±0.2
5 90.1 ±7.7 b 98.4 ±8.6 bc 3.5 ±1.2
25 8.7 ±5.3 a 8.6 ±5.6 a 57.2 ±24.3
Biochar decreased N mineralisation Chapter 2
63
Ino
rgan
ic N
1
(
mg
N k
g-1)
Ino
rgan
ic N
1
(
mg
N k
g-1)
In
org
anic
N
1
(m
g N
kg-1)
Incubation period (Weeks)
0
5
10
15
0 t ha5 t ha25 t ha
(A)-1
-1
-1
0
5
10
15 (B)
0
5
10
15
0 2 4 6 8 10
(C)
Fig. 2.4: Total inorganic N extracted from the control (a), organic N (compost) (b),
and inorganic N (c) treatments for biochar added at 0 (crosses), 5 t ha-1 (triangles)
and 25 t ha-1 (closed circles). Error bars represent standard errors (n=4).
Biochar decreased N mineralisation Chapter 2
64
2.4.6 Sorption isotherms
Both NH4+ and NO3
- sorbed to the biochar when mixed in water for 24 h (Fig. 2.5b).
In water, the amount of NH4+ sorbed to biochar ranged from 88% at the lowest initial
NH4+ concentration (0.5 mg N L-1) to 50% at higher concentrations (>25 mg N L-1).
Recovery of NH4+ increased upon extraction with 0.5 M K2SO4, where around 35%
of the added NH4+ was sorbed up to a concentration of 5 mg N L-1 (Fig. 2.5a). At
concentrations of 5 mg N L-1 to 25 mg N L-1, 20% of added N was sorbed, with no
significant sorption at greater concentrations (data not shown). In comparison, NO3-
sorption to biochar was much greater ranging from around 95% at concentrations up
to 2 mg N L-1 to 75% at concentrations greater than 5 mg L-1. With 0.5 M K2SO4 it
was possible to recover only 5% of the applied NO3- (i.e. 95% sorption) up to initial
concentrations of 5 mg N L-1 (Fig 2.5a). Above this, NO3- recovery increased
gradually until 12 mg N L-1 after which approximately 50% of the applied NO3- could
be recovered (data not shown). However, at the low rates of biochar addition to soil
(5 and 25 t ha-1; or 0.45 and 2.27% w/w) there was not statistically significant
sorption of either form of inorganic N (Fig. 2.6).
Biochar decreased N mineralisation Chapter 2
65
N c
on
cen
trat
ion
(m
g k
g-1)
P
ost
incu
bat
ion
so
lutio
n
Initial Solution N concentration (mg kg-1)
0
10
20
30
40
50(a)
0
10
20
30
40
50
0 10 20 30 40 50
(b)
Ammonium
Nitrate
Fig. 2.5: Sorption data for ammonium (diamonds) and nitrate (squares) for biochar
extracted with (a) 0.5 M K2SO4 and (b) water. Error bars represent standard errors
(n=3) and the 45° line represents an unchanged solution concentration.
Biochar decreased N mineralisation Chapter 2
66
N C
on
c. (
mg
kg-1)
P
ost
incu
bat
ion
so
lutio
n
Initial Solution N Conc. (mg kg-1)
0
10
20
30
40
50(a) (b)
0
10
20
30
40
50
0 10 20 30 40 50
(c)
0 10 20 30 40 50
(d)
0 t ha
5 t ha
25 t ha
(A)-1
-1
-1
Fig. 2.6: The solution concentrations of 0.5M K2SO4 extracted (a) NH4+ and (b) NO3
-
or water extracted (c) NH4+ and (d) NO3
- for soil (crosses), biochar added to soil at
either 5 t ha-1 equivalent (0.45% w/w; triangles), or 25 t ha-1 equivalent (2.27% w/w;
circles) after shaking for 24 h. Error bars represent standard errors (n=3) and the
45° line represents an unchanged solution concentration.
Biochar decreased N mineralisation Chapter 2
67
2.5 Discussion
2.5.1 Microbial biomass
Bacteria typically inhabit pores greater than 0.6 μm (Strong et al., 1998) and the
biochar used here had a habitable pore area over 6 times that of the soil; assuming all
pores on the biochar were favourable for microbial habitation (i.e. not hydrophobic).
However, when biochar was mixed with soil the calculated slight increase in
habitable pore space (Table 2.2) did not translate into increased microbial biomass
(Fig. 2.2).
Elevation of soil pH in acidic soils can increase microbial biomass and activity
(Aciego Pietri & Brookes, 2008). The addition of biochar (CaCl2 pH; 7.43) would
have introduced alkaline micro-habitats potentially providing more favourable niche
environments for microbial populations (Lehmann et al., 2011). However, MBC, N
mineralisation and net nitrification decreased in the presence of biochar; thus
indicating that any benefit from the ‘liming effect’ of the biochar was outweighed by
a decrease in the mass and activity of the microbial population. An increase in
alkaline microsites may also change ammonia oxidiser community structure as rates
of nitrification, and in particular ammonia oxidation, are significantly reduced in acid
soils (De Boer & Kowalchuk, 2001). However, T-RFLP of the amoA gene
demonstrated that the ammonia oxidiser community did not change with the addition
of biochar alone to soil. Differences were only observed when biochar was added in
conjunction with inorganic or organic N.
Biochar decreased N mineralisation Chapter 2
68
The decrease in both MBC and shoot biomass with increasing biochar application
rate contrasts with other studies (Chan et al., 2008; Steiner et al., 2008; Kolb et al.,
2009; Lehmann et al., 2011). This may be explained in part by differences in biochar
application rate and type (e.g. biochar feedstock, pyrolysis temperature, etc.) along
with soil type. Methodological differences may also explain microbial biomass
differences between studies. Previous studies have used the SIR method (Anderson &
Domsch, 1978) to measure microbial biomass (Steiner et al., 2008; Kolb et al., 2009)
where CO2 evolution measurements may be confounded by abiotic release (Jones et
al., 2011; Zimmerman, 2010). By contrast, this study used the fumigation-extraction
method (Vance et al., 1987). Previous studies may not have used the fumigation-
extraction method due to the potential for sorption of cell contents to biochar after
chloroform induced cell lysis. It could be expected that this may confound results and
skew MBC values. However, results in Table 2.3 show there was no effect of biochar
on the recovery of 14C labelled microbial biomass or sorption of amino acids during
the fumigation-extraction process. This is in agreement with the findings of
Durenkamp et al. (2010) who also found that biochar did not change the 0.5 M
K2SO4 extraction efficiency of C (to determine MBC) or ninhydrin-N (to determine
MBN) although activated charcoal did.
Volatile compounds present on biochar have the potential to decrease microbial
biomass (Deenik et al., 2010). Girvan et al. (2005) showed that benzene can decrease
the microbial biomass at concentrations of 40 mg kg-1 soil. In this study, all tested
volatile compounds of the biochar were below the detection limits apart from benzene
Biochar decreased N mineralisation Chapter 2
69
(0.5 mg kg-1; data not shown). But at such low concentrations the effect is unlikely to
solely be attributed to this.
2.5.2 Carbon and nitrogen mineralisation
Interestingly a decline in soil CO2 evolution occurred when 5 t ha-1 biochar was added
but there was no difference in CO2 evolution between 25 t ha-1 and the control soil.
At the levels of biochar (5 and 25 t ha-1) applied to soil it was assumed that both
biotic and abiotic processes were rate dependent meaning that a larger response
should be observed at the higher rate of addition. Given that the biochar contained
carbonate (2.7%) and the soil was acidic (pH 4.76) some dissolution was likely to
have occurred resulting in the loss of HCO3- as CO2. The rate of dissolution was not
determined but was likely to depend on soil water content, the physical location of
the carbonate within the structure of the biochar (i.e. diffusion limitations) and
possibly localised pH buffering (liming effect) of the biochar (pH 7.43). This was
likely to be significant at micro-sites given the low buffering capacity of this
predominately quartz (95% sand) and kaolinitic clay (1:1 lattice structure) dominated
soil. However, assuming that all the carbonate-C was converted to CO2 during the
incubation then this would equate to 24.3 and 122.7 mg CO2-C kg-1 soil in the 5 and
25 t ha-1 biochar applications respectively; this is of the same order of magnitude as
the CO2 evolved from the control soil. Results indicate there was no difference
between the CO2 evolved from the 25 t ha-1 biochar treatment and the control soil;
this suggests that any CO2 derived from carbonate had to be offset by a negative
priming effect on soil organic matter respiration. Results for the 5 t ha-1 biochar
Biochar decreased N mineralisation Chapter 2
70
treatment supports the occurrence of a negative priming effect as here addition of
biochar did cause a significant decrease in CO2 evolution compared to the control
soil. This is consistent with previous research showing suppression of biotic CO2
evolution after biochar addition to soil (Jones et al., 2011; Keith et al., 2011).
As C and N mineralisation are closely linked a negative priming effect on the soil
organic matter should also have been expressed in the N mineralisation results.
Indeed, the size of the inorganic N pool was significantly lower in the presence of
biochar (P<0.001). This was associated with significantly lower net N mineralisation
rates as biochar application rate increased (P<0.001). Also the rate of nitrification
significantly decreased with increasing rates of biochar addition in the control and in
the presence of both N (inorganic N treatment) and C and N (compost treatment)
(P<0.001; Table 2.4). However, the ammonia oxidiser community structure remained
unaffected by biochar addition, unless in the presence of an added N source and there
was no change in diversity. Here it is likely that the lower NH4+ level in the presence
of biochar has constrained nitrification through substrate limitation due to the
negative priming effect on the soil organic matter decomposition. As such the
negative priming effect of biochar addition on soil organic matter decomposition was
illustrated when assessed by determination of N mineralisation and partially masked
due to the likely abiotic release of CO2 when assessed by CO2 evolution.
A decline in net N mineralisation could also have been caused by an increase in the
rate of microbial N immobilisation compared to the gross N mineralisation rate.
Biochar decreased N mineralisation Chapter 2
71
Gundale & DeLuca (2007) suggested that the relative dominance of mineralisation
versus immobilisation in the presence of charcoal was determined by the level of
bioavailable C in their incubation (i.e. glycine). These results do not indicate an
increase in the size of the MBC where compost was applied and in addition MBN
remained constant in all treatments, suggesting that increased microbial
immobilisation of N did not occur.
Sorption of nitrogenous compounds onto biochar is another mechanism that could
explain the lower inorganic N traces in the presence of biochar. Microbial activity
oxidises biochar through time, the sorption and desorption characteristics of biochar
are likely to change. Over time surface positive charge is likely to decrease, whilst
negative charge increases (Cheng et al., 2008). A weakness of this sorption study is
that measurements have only occurred at one time point, rather than at the start and
end of the incubation. However, the extent of oxidation is highly correlated with
temperature (Cheng et al., 2006; Cheng et al., 2008) and the study by Cheng et al.
(2006) did not measure any net change in total acidic functional groups after a four
month incubation at 30°C. Given the temperature range of the glasshouse (mean
maximum of 25°C and mean minimum of 13°C) and that the subsequent soil
incubations were conducted at 15°C the biochar was unlikely to have oxidised
sufficiently over the course of the experiment to warrant a time course study of
sorption. These results did indicate NO3- and to a lesser extent NH4
+ sorption to
biochar (Fig. 2.5). However when biochar was mixed with soil, the very minimal
extent of sorption that occurred could not explain the significantly decreased N
Biochar decreased N mineralisation Chapter 2
72
mineralisation (Fig. 2.6). This is attributed to the small proportion of biochar within
the soil matrix (2.27 and 0.45 % w/w in the 25 and 5 t ha-1 treatments respectively)
and the slow rate of diffusion of NH4+ in solution.
The decrease in mineralisation may have been caused by the presence of potentially
toxic volatile organic compounds present on the biochar during addition (Deenik et
al., 2010), or the evolution of ethylene (Spokas et al., 2010). The Jarrah biochar was
assessed for volatile organic compounds, but all assessed compounds were below the
detection limit, aside from benzene (0.5 mg kg-1 of biochar; data not shown). At such
low levels, the decreases in mineralisation cannot solely be attributed to the presence
of benzene on the biochar used in this study.
This study has provided evidence that biochar addition to soil decreased MBC but not
MBN. Associated with this was a coincident decrease in soil organic matter
decomposition as indicated by the decline in CO2 evolution and net N mineralisation
rates. The microbial community functional diversity (as measured by CLPP) changed
with biochar addition; however the diversity of the ammonium oxidiser community
only changed when biochar was added in conjunction with an N source (organic or
inorganic). Thus findings did not support the hypotheses that biochar addition to soil
would increase the mass, activity and structural diversity of the soil microbial
community. This highlights the need to further determine the conditions (i.e. biochar
feed stock, production conditions, soil types) under which biochar additions to soil
Biochar decreased N mineralisation Chapter 2
73
will result in positive or negative effects on the soil microbial community and their
associated functions.
74
Clay and biochar decrease inorganic N leaching Chapter 3
75
Chapter 3
Clay and biochar amendments decreased inorganic nitrogen
leaching but not dissolved organic nitrogen leaching in soil
3.1 Abstract
N leaching from coarse textured soils frequently leads to productivity losses and
negative environmental consequences. Historically, clay amendment has been used
on coarse textured soils to decrease water repellence and nutrient leaching. More
recently biochar has been proposed as an alternative soil amendment to decrease N
leaching while simultaneously storing carbon. As biochar has a greater nutrient
retention capacity, it was hypothesised that Eucalyptus marginata derived biochar
would be a more effective amendment than clay at minimising N leaching. Lysimeter
pots were filled with a coarse-textured agricultural soil and amended with the
following treatments: (1) biochar incorporated homogenously into the 0-10 cm soil
layer, (2) clay incorporated similarly, (3) biochar added as a layer at 10 cm depth, (4)
clay added similarly, or (5) a control. Amendments were added at 25 t/ha and watered
periodically over 21 days and watered with the equivalent to 30 mm. Clay and
biochar amendments significantly decreased cumulative ammonium (NH4+) leaching
by approximately 19% and 14% respectively and NO3- leaching by 16% and 28%
respectively. Biochar decreased nitrate (NO3-) leaching significantly more than clay,
possibly due to decreased nitrification. Dissolved organic N leaching was not
influenced by any treatment. N leaching was unaffected by amendment application
Clay and biochar decrease inorganic N leaching Chapter 3
76
method, therefore to decrease N leaching, land managers should apply the most
readily available of the amendments in the most economic and convenient manner.
3.2 Introduction
Improving soil fertility and productivity, while simultaneously decreasing negative
environmental externalities, is one of the greatest challenges in the management of
agricultural and horticultural production systems. This is typically very difficult to
achieve on coarse-textured soils with poor intrinsic soil structure, high water
repellence, low water holding capacity and low nutrient retention capacity (Harper et
al., 2000; Moore, 2004). These soils dominate the agricultural region within the
Mediterranean-type climate zone of Australia (Rovira, 1992). Typical fertiliser
regimes on such soil types range from 400 kg N ha-1 crop-1 in high value horticultural
systems, where 3 crops may be grown per year (Flavel & Murphy, 2006) to typically
40-80 kg N ha-1 for an expected 2 t ha-1 wheat grain yield (Hoyle et al., 2011).
Nutrient leaching from agricultural and horticultural production systems represents
not only an operational inefficiency, but can also cause environmental problems, such
as generating excess soil acidity (Dolling & Porter, 1994), eutrophication (e.g.
Smolders et al., 2010) and pollution of ground water (Power et al., 2001). The risk of
eutrophication in these ecosystems is particularly problematic as fresh and marine
water ecosystems have adapted to low nutrient environments (ANZECC &
ARMCANZ, 2000). One potential intervention strategy to protect these environments
is to minimise the use of soluble fertilisers (Gentile et al., 2009), however, this may
Clay and biochar decrease inorganic N leaching Chapter 3
77
decrease land productivity (Quiroga-Garza et al., 2001). The risk of leaching is
greatest early in the growing season when N availability is greater than demand,
hence leaching moves N below the rooting zone (Hoyle & Murphy, 2011). Soil
amendments such as biochar and clay may retain N and decrease N leaching, thus
improving N use efficiency while also minimising eutrophication risks.
Biochar is the solid remains of thermally decomposed organic matter, from plant or
animal sources, under limited oxygen supply at temperatures less than 700 °C
(Lehmann & Joseph, 2009). It has the potential to sequester carbon (Lehmann et al.,
2006; Ogawa et al., 2006) and improve soil fertility (Glaser et al., 2002). One of the
potential mechanisms for improved soil fertility is through enhanced nutrient
retention, resulting in decreased leaching losses (Lehmann et al., 2003). However,
significant uncertainty still surrounds its impact on soil N dynamics. While biochar
application may decrease ammonium (NH4+) leaching (Lehmann et al., 2003; Ding et
al., 2010) it may stimulate nitrate (NO3-) leaching (Laird et al., 2010b; Lehmann et
al., 2003).
Duplex or texture contrast soils, where underlying the coarse-textured topsoil is a
subsoil of much greater clay content, comprise 56% or 14.2 m ha of Western
Australian agricultural land (Tennant et al., 1992). On some farms, these coarse-
textured topsoils are being ameliorated with subsoil clay, termed ‘clay’ in this paper.
Field application rates are in excess of 100 t ha-1 (Harper & Gilkes, 2004) and as
such, clay amendments can ameliorate water repellence (McKissock et al., 2000;
Clay and biochar decrease inorganic N leaching Chapter 3
78
McKissock et al., 2002). Furthermore, by increasing the ion retention capacity clay
amendment can increase nutrient holding capacity, hence increase yields (Hall et al.,
2010) and decrease leaching. In a similar manner, biochar can also increase yields
and decrease nutrient leaching (Lehmann et al., 2003). However, the ability of each
amendment to ameliorate these properties will vary between clay and biochar types
and their intrisic properties. For example, published cation exchange capacities for
clay amendments range from 6-10 cmolc kg-1 (Hall et al., 2010) and from 6-25 cmolc
kg-1 for biochar (Lehmann, 2007).
The ability of clay and biochar amendments to ameliorate leaching of inorganic and
DON were tested and compared in lysimeter pots. It was hypothesised that both
amendments would decrease N leaching, due to expected greater ion retention
capacity. Whether the placement of these amendments within the profile, i.e. a
homogenous incorporation versus a horizontal layer, influences amendment
performance was also tested. The hypothesis was that the layer would decrease
leaching to a greater extent due to improved interception of leached ions.
3.3 Materials and Methods
3.3.1 Soil, clay and biochar
Soil was collected from a White Subnatric Sodosol (Isbell, 1996) agricultural soil
under a historical crop (generally wheat (Triticum aestivum))/pasture (cape weed
(Arctotheca calendula), annual ryegrass (Lolium rigidum) and clover (Trifolium
Clay and biochar decrease inorganic N leaching Chapter 3
79
subterraneum) dominant) rotation located 15 km north of Meckering, WA, Australia
(31° 30′ S, 116° 59′ E) in 0-10, 10-20 and 20-35 cm horizons. Soil was sieved to 2
mm to remove buried plant residue (i.e. stubble) as no gravel or stones were present.
Lysimeters were reconstructed using these soil horizons. Amendments were either
added as a layer at 10 cm depth or incorporated homogenously into the 0-10 cm soil.
Subsoil ('clay') was collected from a clay pit 400 m from the soil sampling site.
Particle size analysis, determined using the pipette method (Bowman and Hutka,
2002), found the clay contained 60.4% clay and 11.9 % silt while the 0-10 cm soil
contained 94.2% sand (Table 3.1). The random powder X-ray diffraction (XRD)
pattern of clay, obtained for the range 0-70° 2θ (Philips PW 1830 X-ray
diffractometer), showed it contained kaolinite and quartz.
Jarrah wood (trunks and large branches; Eucalyptus marginata Donn ex Sm.) was
pyrolysed at 600 °C for 24 h (Simcoa Ltd, Bunbury, WA). This biochar has a specific
surface area of 273 m2 g-1, determined using a surface area analyser (Micrometrics
Gemini 2375 instrument with a Vac Prep 061 and N2 as the adsorbate). The XRD
pattern of biochar showed it contained calcite, quartz and amorphous carbon.
Soil, clay, and biochar were analysed for NH4+, NO3
–, and DON by extracting 20 g
with 80 mL 0.5 M K2SO4 and analysing the extracts colourimetrically for NH4+
using
the salicylate-nitroprusside method (Krom, 1980; Searle, 1984) and NO3-
concentration using the hydrazine reduction method (Kamphake et al., 1967;
Kempers and Luft, 1988) on an automated flow injection Skalar AutoAnalyser
Clay and biochar decrease inorganic N leaching Chapter 3
80
(San++, Skalar Analytical, The Netherlands). DON contents of the extracts were
determined after alkaline persulfate digestion (Cabrera and Beare, 1993). CEC of soil,
clay and biochar was determined using the compulsive exchange method, without
pretreatment for soluble salts (Rayment and Higginson, 1992). Total C and N of the
soil, clay and biochar were determined by combustion analysis using an Elementar
analyser (vario Macro CNS; Elementar, Germany). Soil, clay and biochar were
analysed for pH in a 4 g soil and 20 ml 0.5M CaCl2 mixture.
Table 3.1: Basic properties of soil (at various depths), clay and biochar used in this
experiment.
Soil Clay Biochar
0-10 cm 10-20 cm 20-30 cm
NO3- (mg N kg-1) 7.2 3.5 1.0 7.3 <0.1
NH4+ (mg N kg-1) 2.7 1.0 0.4 17.7 0.4
Organic N (mg N kg-1) 18.8 9.9 7.7 20.8 8.7
Total C (%) 1.04 0.47 0.19 0.11 75.0
Total N (%) 0.08 0.03 0.02 0.01 0.3
Cation exchange
capacity (cmolc kg-1)
2.08 1.67 1.59 7.43 10.11
pH (1:5 0.5 M CaCl2) 5.3 4.6 4.3 5.5 7.3
Sand (%) 94.2 92.2 90.9 27.8 -
Silt (%) 0.6 2.1 1.5 11.9 -
Clay (%) 5.2 5.7 7.6 60.4 -
Clay and biochar decrease inorganic N leaching Chapter 3
81
3.3.2 Experimental design and setup
The treatments for this experiment were; 1) biochar incorporated homogenously into
the 0-10 cm soil layer, 2) clay incorporated similarly, 3) biochar added as a layer at
10 cm depth, 4) clay added similarly, or 5) a control. Treatments (replicated five
times) were applied at 25 t ha-1 and soil (3.10 kg) or soil plus amendment (3.085 kg
soil and 0.015 kg amendment) was added to each lysimeter pot (8.75 cm diameter, 36
cm depth). The 10-20 cm and 20-35 cm soil layers were added to a bulk density of
1.5 g cm-3 and the 0-10 cm soil to 1.4 g cm-3 to replicate values measured in the field.
Since this soil naturally compacts to a bulk density between 1.4 and 1.6 g cm-3
biochar and clay application did not cause any difference in the final bulk density of
the pots. Nitrogen fertiliser was added in the form of (NH4)2SO4 at a rate of 40 kg N
ha-1 (equivalent to 7 mg N kg-1 soil). To avoid preferential flow all soil was moistened
to 40% WHC during construction of the soil layers in the lysimeters. Glasshouse
mean maximum and minimum temperatures for the experiment were 31 and 18°C,
respectively.
Lysimeters were watered with the equivalent to 30 mm of rainfall equivalent, daily
for the first 10 days, then on days 13, 15, 17 and 20. The day after watering, leachate
was collected and volume assessed, then filtered (Whatman No. 42) and frozen prior
to analysis. Samples were analysed for NH4+
, NO3- and DON as stated previously.
Clay and biochar decrease inorganic N leaching Chapter 3
82
3.3.3 Nitrogen sorption characteristics
Sub-samples (10 g) of biochar, soil, or clay and 100 mL of inorganic N solution were
added to a 120 mL vial in triplicate. N solutions were added as NH4NO3 at
concentrations of 0, 5, 10, 20, 30, 50 and 100 μg N L-1 (0, 50, 100, 200, 300, 500,
1000 μg N g-1 soil). All mixtures were shaken for 24 h, filtered (Whatman No 42) and
analysed using the automated flow injection method.
3.3.4 Water retention
Topsoil (0-10 cm depth), and topsoil incorporated with biochar or clay at 25 t ha-1
was firmly packed to bulk density of 1.4 g cm-3 into a core of 53.8 mm diameter and
10 mm height and saturated with water at atmospheric pressure and its gravimetric
water content determined. Triplicates of identical saturated samples were transferred
to porous ceramic plates, placed in a pressure chambers and subsequently equilibrated
at matric potentials of -10, -100 and -1500 kPa. Gravimetric water contents were
measured after equilibration and then adjusted to volumetric values using bulk
density.
3.3.5 Statistical analysis
Two analysis methods were used to compare the total quantity of leached NO3-, NH4
+
and DON. An ANOVA was conducted using orthogonal contrasts on total cumulative
leached quantities. Orthogonal contrasts were used to test for differences to the mean
(control vs. each of the four treatments), to compare amendments (biochar vs. clay),
to compare incorporation methods (homogenous incorporation vs. a layer) and to test
Clay and biochar decrease inorganic N leaching Chapter 3
83
for interaction. A repeated measures ANOVA was also used. Statistical analysis was
conducted in Genstat 10th edition (Lawes Agricultural Trust, 2007). Differences in N
sorption and water retention were tested using ANOVA and multiple comparisons
using Tukey’s 95% confidence intervals. Statistical significance was considered at
5% (P<0.05).
3.4 Results
3.4.1 Nitrogen leaching
Over the duration of this experiment 15.0 ± 0.6 mg pot-1 of NH4+-N was leached from
the control treatment (Fig. 3.1a). Leaching of NH4+-N significantly decreased with the
addition of biochar (an average of 12.9 mg pot-1) and clay (an average of 12.1 mg
pot-1) (P=0.001; Fig. 3.1a), but there was no significant difference between
amendment types (P=0.323). There was also no difference between incorporation
methods (P=0.671) and there was no interaction (P=0.321; Fig. 3.1a).
Cumulative leaching of NO3--N significantly decreased from 19.5 mg pot-1 in the
control treatment, to an average of 16.3 mg pot-1 with the addition of clay and to an
average of 14.0 mg pot-1 with biochar amendment (P<0.001; Fig. 3.1b). Biochar
amendment decreased NO3- leaching to a significantly greater extent than the clay
amendment (P=0.034). There was no difference between incorporation method
(P=0.300) and no interaction (P=0.930).
Clay and biochar decrease inorganic N leaching Chapter 3
84
Cu
mu
lativ
e N
H
4+ -N
1
le
ach
ed
(m
g p
ot
-1)
C
um
ula
tive
NO
3- -N
1
l
ea
che
d (
mg
po
t-1
)
Days
0
5
10
15
Control
Clay Homogenous
Clay Layer
Biochar Homogenous
Biochar Layer
(a)
0
5
10
15
20
0 7 14 21
(b)
Fig. 3.1: Cumulative quantity of NH4+ (a) and NO3
- (b) leached over 21 days for the
unamended soil (control; solid squares), clay homogenously incorporated into the top
10 cm of the soil (solid triangle), a clay layer at 10 cm depth (hollow triangle),
biochar incorporated homogenously (solid circle), or a biochar layer at 10 cm depth
(hollow circle). Error bars represent standard errors (n=5).
Clay and biochar decrease inorganic N leaching Chapter 3
85
However, by contrast, no significant difference could be found between any of the
treatments with DON leaching (data not shown). The repeated measures ANOVA
showed significant differences in the amount of NO3- (P=0.004) and NH4
+ (P=0.010)
leached but not DON (P=0.501).
3.4.2 Nitrogen sorption characteristics
At all added N concentrations Tukey’s 95% confidence intervals determined that
biochar sorbed the greatest quantity of both NH4+ and NO3
- (Fig. 3.2a; P<0.001 for all
NH4+ and NO3
- concentrations). Biochar sorbed 75% of the NH4+ in solution when
added at the lowest concentrations (2.5 and 5 μg NH4+-N L-1), decreasing to 54%
when added at the greatest concentration (50 μg NH4+-N L-1; Fig. 3.2a). Tukey’s 95%
confidence intervals showed this was always greater than occurred in the soil or clay.
By comparison, the greatest proportion of NH4+ sorption to the clay (32%) occurred
at a concentration of 20 μg NH4+-N L-1 (Fig. 3.2a).
A much greater difference was seen between the sorption characteristics when the
materials were amended with NO3-. While clay exhibited no capacity to sorb NO3
--,
biochar sorbed 80% of the added NO3--N at lower concentrations (2.5- 5 μg NO3
--N
L-1; Fig. 3.2b) decreasing to 38% at the greatest addition rate (50 μg NO3--N L -1; Fig
3.2b).
Clay and biochar decrease inorganic N leaching Chapter 3
86
NH
4+ So
rptio
n (μ
g N
H 4+ -N
g-1
so
il)
N
O 3- S
orp
tion
(μg
NO
3- -N g
-1 s
oil)
Initial solution concentration (μg N g-1 soil)
0
50
100
150
200
250
0 100 200 300 400 500
Soil
Clay
Biochar
(a)
0
50
100
150
200
250
0 100 200 300 400 500
(b)
Fig. 3.2: Amount of NH4+ (a) and NO3
- (b) sorbed by biochar (solid circle), clay
(solid triangle) or the unamended topsoil (solid squares). Error bars represent
standard errors (n=3).
Clay and biochar decrease inorganic N leaching Chapter 3
87
3.4.3 Water retention capacity
When comparing gravimetric water contents, at atmospheric pressure (0 kPa), both
clay and biochar amendments significantly increased the water holding capacity of
the soil (P=0.003), however, there was no difference between the two amendments
(Table 3.2). At an imposed matric potential of -10 kPa there was no significant
difference in soil water content between the treatments. At an imposed matric
potential of -100 and -1500 kPa, both amendments significantly increased gravimetric
water holding capacity (P<0.001 and P=0.05, respectively). The biochar amended
soil at -1500 kPa contained more water than the same treatment at -100 kPa (Table
3.2). This is attributed to the heterogeneity of the biochar.
The volumetric water retention capacity of the soil increased significantly with
biochar and clay amendment at 0 and -100 kPa (P=0.002; Table 3.3). There was no
significant difference between bulk densities for the top soil, clay amended soil and
biochar amended soil treatments (an average of 1.5 g cm-3).
Clay and biochar decrease inorganic N leaching Chapter 3
88
Table 3.2: Gravimetric water holding capacity for the surface soil layer (0-10 cm)
either unamended or amended with clay or biochar (25 t ha-1) at a range of soil
matric potentials. Values (g cm-3) are represented as ± standard errors (n=3). Within
columns, means followed by the same letter are not significantly different at P = 0.05.
0 kPa -10 kPa -100 kPa -1500 kPa
Soil (0-10) 31.4 ± 0.3 a 10.8 ± 2.7 a 3.4 ± 0.1 a 2.9 ± 0.1 a
Soil + Clay 33.7 ± 0.3 b 14.0 ± 0.8 a 4.4 ± 0.0 b 3.8 ± 0.0 b
Soil + Biochar 34.8 ± 0.1 b 15.0 ± 1.0 a 4.8 ± 0.1 c 6.6 ± 1.3 b
ANOVA P = 0.003 P = 0.355 P < 0.001 P = 0.05
Table 3.3: Volumetric water holding capacity for the surface soil layer (0-10 cm)
either unamended or amended with clay or biochar (25 t ha-1) at a range of soil
matric potentials. Values (g cm-3) are represented as ± standard errors (n=3). Within
columns, means followed by the same letter are not significantly different at P = 0.05.
0 kPa -10 kPa -100 kPa -1500 kPa
Soil (0-10) 19.0 ± 0.1 a 6.7 ± 1.6 2.1 ± 0.1 a 2.2 ± 0.0
Soil + Clay 20.1 ± 0.1 b 8.8 ± 0.4 3.4 ± 0.1 b 2.8 ± 0.0
Soil + Biochar 20.9 ± 0.1 c 9.5 ± 0.5 3.6 ± 0.1 b 5.0 ± 1.0
ANOVA P = 0.002 P = 0.297 P = 0.002 P = 0.059
Clay and biochar decrease inorganic N leaching Chapter 3
89
3.5 Discussion
Chapter 3 results support the hypothesis that both the addition of biochar and clay
decrease N leaching. Previous studies have shown biochar amendments can decrease
NH4+ leaching (Lehmann et al., 2003; Ding et al., 2010). The main mechanisms to
explain this are an increase in the nutrient and water retention capacities of the soil
(Lehmann et al., 2003). Overall, there was not a difference in cumulative NH4+
leaching between the two amendment types despite the large discrepancy in their
NH4+ sorption capacity (Fig. 3.2). As such, improved gravimetric water holding
capacity resulting from the soil amendment is likely to play a comparatively greater
role in decreasing NH4+ leaching than NH4
+ sorption. The improved gravimetric
water holding capacity occurred largely at 0 and -100 kPa suggesting that this
additional water is also plant available. Although there were no plants in these
experiments, this additional water could support greater plant growth which in turn
would lower soil solution N concentrations and indirectly decrease N leaching.
The amount of NO3- leaching also decreased with both clay and biochar addition,
contrary to previous biochar studies, where leaching of this anion increased (Laird et
al., 2010b; Lehmann et al., 2003). In this study, biochar amendment decreased NO3-
leaching significantly more than clay. One reason for the difference is potentially due
to the large difference in anion retention capacity between the two ameliorants. Clay,
lacking NO3- retention capacity, is likely to have decreased leaching simply by
increasing the soil’s inherent water holding capacity, whereas biochar is likely to
have decreased NO3- leaching due to both sorption processes and an increased water
Clay and biochar decrease inorganic N leaching Chapter 3
90
holding capacity. The large anion retention is likely to be due to the positive charge of
biochar (Cheng et al., 2008). Alternatively it may be due to changes in nitrification.
The addition of this biochar to soils is known to decrease N mineralisation and
nitrification (Chapter 2). Changes in nitrification might be the reason for both
decreased nitrate leaching in this experiment, and the increased nitrate leaching seen
in others (Lehmann et al., 2003).
In many agricultural regions significant N losses may also occur in the form of DON
(Jones et al., 2005; Murphy et al., 2000). While much of this DON is not available in
soil it can be subsequently transformed in freshwaters by UV photolysis into
biologically available forms of N and hence contribute to eutrophication. In this
experiment, biochar and clay amendments did not decrease DON leaching. As most
DON carries a net negative charge, the case for decreased NO3- leaching due to
decreased nitrification, rather than sorption differences, is strengthened. However
these results also conflict with the idea that increased WHC should aid decreased N
leaching. Suggesting there may be another mechanism influencing NH4+ leaching.
The hypothesis for this experiment was that a layered application method would
provide a more effective barrier to leaching, due to greater interception of leachable
ions and less chance of bypass flow. Surprisingly, the method of clay or biochar
application made little overall difference to N leaching. This contrasts with the results
of Jones et al. (2011) where horizontal banding of biochar reduced the amount of
herbicide leaching in soil in comparison to when the biochar was homogenously
Clay and biochar decrease inorganic N leaching Chapter 3
91
incorporated into the soil. As such, land managers should apply biochar or clay in the
manner that is both most practical and convenient.
It should be noted, however, that over time, as biochar oxidises the net positive
charge is likely to decrease, developing a predominantly negative charge (Cheng et
al., 2006; Cheng et al., 2008). Similarly, the anion exchange sites may become
occupied with stronger sorbing inorganic or organic ligands (e.g. humic substances,
PO43-) reducing its capacity to retain NO3
- or the surface may become blocked due to
biofilm formation. As such biochar application may not provide the same NO3-
leaching amelioration over the long term, but alternatively less NH4+ leaching may
occur. The long term implications on N leaching of biochar in soil therefore requires
further research, especially considering that NO3- is the more mobile of the ions.
Further research is required to ascertain whether the nutrients retained on the soil
amendments are plant available, and whether this is likely to aid N fertiliser use
efficiency in agricultural and horticultural production.
92
Biochar did not change organic N mineralisation Chapter 4
93
Chapter 4
Organic nitrogen mineralisation in two contrasting agro-
ecosystems is unaffected by biochar addition
4.1 Abstract
Biochar additions to soil have been reported to enhance soil fertility whilst
simultaneously storing C. This experiment tested whether either fresh or field-
conditioned (aged) biochar amendment to two contrasting agricultural soils would
alter the mineralisation of organic N compounds. The mineralisation of 14C-labelled
amino acids and peptides were determined over 20 days within each soil. An
exponential kinetic decay model was subsequently fitted to the mineralisation data.
Overall, statistical analysis revealed significant but small differences between the two
biochar treatments and the unamended control treatment. It is concluded that biochar
has very limited impact on the mineralisation rate of low molecular weight dissolved
organic N compounds in these agro-ecosystems.
4.2 Introduction
One method of rapidly sequestering organic C within agricultural systems is the
application of biochar to soil (Sohi et al., 2010). Biochar is the solid products of
thermal decomposition of organic matter, plant or animal, under limited oxygen at
temperatures less than 700°C (Lehmann & Joseph, 2009). Owing to its highly
aromatic structure (Schmidt & Noack, 2000), biochar can remain stable in soil for
Biochar did not change organic N mineralisation Chapter 4
94
thousands of years (Preston & Schmidt, 2006) and has proved agronomically
advantageous in some situations (e.g. Chan et al., 2008; Van Zwieten et al., 2010).
Biochar can also impact soil fertility by providing a habitat for microbes (Pietikäinen
et al., 2000), changing soil pH, and sorbing compounds such as pesticides (Yu et al.,
2006; Bornemann et al., 2007; Cao et al., 2009; Jones et al., 2010) and nutrients
(Mizuta et al., 2004; Ding et al., 2010). In forest soils, biochar addition can stimulate
mineralisation and nitrification (Berglund et al., 2004; Wardle et al., 2008) as
phenolic compounds may inhibit these processes (DeLuca et al., 2006). In
agricultural soils this may not be the case as biochar additions have induced decreases
in mineralisation of both C and N (Kuzyakov et al., 2009; Jones et al., 2011; Chapter
3). Mineralisation of organic matter supplies as much as 80% of crop N within
Australia (Angus, 2001), although, globally fertilisers supply a larger proportion on
crop N (50%; Jenkinson, 2001). Nonetheless organic matter mineralisation remains
integral for crop N supply. Despite this, very little research has targeted the
interaction between organic N compounds and biochar.
The implications of this interaction are likely to change over time. By altering
enzyme function, sorption mechanisms are also likely to influence changes in DON
mineralisation (Bailey et al., 2011). As biochar slowly oxidises over time, surface
functional groups change as the particle gains greater negative charge, increasing its
hydrophilicity (Cheng et al., 2006; Cheng et al., 2008). Correspondingly, the
behaviour of the dominant DON compounds in soil (e.g. peptides and amino acids)
may be more likely to be influenced by the sorption properties of biochar after aging.
Biochar did not change organic N mineralisation Chapter 4
95
Therefore the hypothesis for this experiment was that in agricultural soils, fresh
biochar addition would not change the mineralisation of small organic N compounds,
but that aged biochar amendment would decrease it.
4.3 Materials and Methods
4.3.1 Experimental design and characterisation of soils and biochars
This experiment used soils from two biochar field experiments contained within two
contrasting agricultural environments. Three soil treatments (control, field-
conditioned “aged” biochar and fresh biochar), and three 14C labelled compound
treatments (an amino acid mix, alanine or the peptide trialanine) were applied. Site 1
was located in Buntine, WA, (30°00’S, 116°21’E) and was where wheat residue
derived biochar (pyrolysed at 450°C for 20 min; Pacific Pyrolysis, Somersby, NSW)
had been applied to an Acidic Yellow Kandosol soil (Isbell, 1996) at a rate of 4 t ha-1
(6 g biochar kg-1 soil; soil bulk density 1.33 g cm-3; 5 cm incorporation depth). This
biochar is comprised of 15% ash, 33% volatile matter, and 44% fixed C and has a
specific surface area of 215 m2 g-1. The replicated field plots (3 × 20 m in size; n = 4)
had biochar applied one year before soil collection. Buntine is located within a
Mediterranean climate zone and the paddock is cropped each winter in a wheat
(Triticum aestivum L.)/wheat/lupins (Lupinus angustifolius L.) rotation, and fallow
over the dry summer. Site 2 was located in Abergwyngregyn, Wales (53°14’N,
4°01’W) where the soil was classified as a Eutric Cambisol (FAO, 1988). Site 2 is a
perennial ryegrass (Lolium perenne L.), crested dogstail (Cynosurus cristatus L.) and
Biochar did not change organic N mineralisation Chapter 4
96
white clover (Trifolium repens L.) dominant pasture within a temperate climate.
Mixed hardwood biochar had been applied to the field plots (3 × 6 m in size; n = 4) at
a rate of 25 t ha-1 2.5 years before soil collection (20 g biochar kg-1 soil; soil bulk
density 1.2 g cm-3; 10 cm incorporation depth). The biochar is described in detail in
Jones et al. (2010) and was pyrolysed at 450°C for 48 h (BioRegional Homegrown®;
BioRegional Charcoal Company Ltd, Wallington, Surrey, UK). This biochar is 3%
ash, 16% volatile matter and 77% fixed C, whilst the specific surface area is 39 m2 g-
1. At both sites, batches of biochar were held in storage since field application and
these constituted the fresh biochar used in the experiments described below, whilst
the soil collected from the field biochar plots constituted the aged treatment. Soil
collected from the individual field plots (0-5 cm depth) constituted the replicates in
the experiments.
Soil and biochar characteristics are presented in Tables 4.1 and 4.2. Total C and N
were determined by a combustion analyser. Cation exchange capacity was also
determined (Rayment and Higginson, 1992). Soils and biochar were extracted (0.5 M
K2SO4) and analysed colorimetrically for NO3- (Miranda et al., 2001) and NH4
+
(Mulvaney, 1996), and flourimetrically for free amino-acids (FAA; Jones et al.,
2002) and peptides (Hill et al., 2011). K2SO4 extractable organic carbon and total N
(TN) were measured in a Shimadzu Total Organic Carbon-V-TN analyser. The EC
and pH of the soils and biochar were determined in a 1:5 (w/v) soil-to-water extract.
Soil solution was collected by centrifugal-drainage (Jones et al., 2002) and
characterised as described above.
Biochar did not change organic N mineralisation Chapter 4
97
As the Australian soil was dry when collected, prior to use, these soil samples were
moistened to 70% of their water holding capacity and incubated for 7 d at 20°C. Field
moist Welsh soil was used for the incubation. For the mineralisation assays, 10 g soil
or soil/biochar mixture was placed in 50 cm3 polypropylene tubes at 20 °C.
Application rates of the fresh biochar to previously unamended soil corresponded
with field rates. Uniformly labelled 14C-labelled organic N compounds (100 μM; 2
kBq ml-1) comprising either a mixture of 16 amino acids, alanine or trialanine were
then added to the soil. To capture respired 14CO2, a 1 M NaOH trap (1 ml) was added
to each polypropylene tube and the traps replaced after 0.5, 1, 3, 6, 12, 24, 48, 120,
180, 240, 360 and 480 h.
4.3.2 Statistical Analysis
A repeated measures ANOVA was conducted on rates of 14CO2 efflux in GENSTAT
10th edition (Lawes Agricultural Trust, 2007). A double exponential decay model was
also fitted to the mineralization data where
S = Y0 + Y1 × exp(-k1 t) + Y2 × exp(-k2 t) (1)
and where S it the % of the 14C-label remaining in the soil, Y0 is the pool of
recalcitrant microbially synthesised 14C from the added substrate (Hill et al., 2011),
Y1 represents the size of the primary mineralisation pool respired by the microbial
biomass, k1 is the exponential rate co-efficient describing Y1, Y2 represents the size of
the pool initially incorporated into the microbial biomass and subsequently respired,
Biochar did not change organic N mineralisation Chapter 4
98
k2 is the exponential rate co-efficient describing the Y2 pool, and t is time. This model
complies with the theory that substrate mineralisation follows a biphasic pattern
(Chotte et al., 1998; Saggar et al., 1999; van Hees et al., 2005; Boddy et al., 2007),
and a certain proportion of added substrate is recalcitrant (Hill et al., 2011). The mean
residence time (MRT) of the labile fraction of the substrate was calculated
numerically where
MRT = (Y1/100)/k1 + (Y2/100)/k2. (2)
Significant differences between model parameters and MRT were assessed by
ANOVA with Tukey’s pairwise comparison with a p < 0.05 threshold for
significance. Due to the contrasting agro-ecosystems, biochar type and rates varied
between soil types; as such statistical comparisons were only made within each soil
type.
Tab
le 4
.1:
Pro
pert
ies
of
soil,
so
il w
ith a
ged
bioc
ha
r am
end
men
t, a
nd
fresh
bio
char
pro
per
ties.
Val
ues
rep
rese
nt
mea
ns ±
SE
M (
n=
4).
S
ite 1
(A
ust
ralia
)
Site
2 (
Wa
les)
S
oil
Age
d b
ioch
ar
am
en
ded
soil
Bio
char
Soi
l A
ged
bio
char
am
en
de
d so
il
Bio
char
Tot
al N
(%
) 0
.08
± 0.
01
0.0
8 ±
0.0
1 2
.32
± 0.
02
0.
26 ±
0.0
1 0
.24
± 0.
01
0.6
9 ±
0.0
1
Tot
al C
(%
) 0
.96
± 0.
07
1.0
3 ±
0.0
8 60
.9 ±
0.4
2.
83 ±
0.4
1 3
.23
± 0.
76
85
.1 ±
0.4
Ext
ract
abl
e C
(m
g C
kg-1)
172
± 1
2 1
75
± 5
40
54 ±
254
70 ±
11
56
± 4
16
53 ±
34
9
Ext
ract
abl
e N
(m
g N
kg-1)
18
.5 ±
1.2
18
.5 ±
0.4
21
1 ±
13
25
.6 ±
14.
1 1
7.5
± 4.
7 8.
5 ±
2.7
Pe
ptid
es (μ
mo
l kg-1
) 5
94
± 1
24
78
5 ±
24
2 2
367
± 5
89
4
12 ±
13
5 7
21 ±
30
7 1
388
± 2
14
Am
ino
aci
ds (
mg
N k
g-1)
1.2
1 ±
0.0
6 1
.31
± 0.
07
1.37
± 0
.12
0.
96 ±
0.0
5 1
.28
± 0.
24
0.1
1 ±
0.0
2
NH
4+ (m
g N
kg-1
) 3
.70
± 0.
38
3.8
7 ±
0.7
6 1
.42
± 0.
21
15
.0 ±
14.
2 5
.16
± 3.
50
0.6
3 ±
0.4
5
NO
3- (m
g N
kg-1
) 2
.02
± 0.
31
1.7
4 ±
0.3
0 1
.05
± 0.
05
26
.8 ±
12.
2 3
0.7
± 1
2.6
0.2
4 ±
0.0
8
pH
(1
:5 H
2O)
6.2
4 ±
0.0
9 6
.55
± 0.
10
9.2
3 ±
0.0
2
6.27
± 0
.13
6.1
4 ±
0.15
8
.41
± 0.
15
EC
(μS
cm
-1)
82
.5 ±
13.
6 8
7.3
± 1
1.6
29
80 ±
27
0
37.4
± 2
.32
65
.1 ±
17.
9 38
8 ±
67.
5
Cat
ion
Exc
ha
nge
Cap
acity
(m
mol
kg
-1)
11
.8 ±
0.3
18
.6 ±
1.0
34.9
± 2
.1
85.
9 ±
2.7
76.
4 ±
5.6
9.5
± 2.
9
So
il re
spir
atio
n (m
g C
O 2-C
kg-1
h-1)
1.8
4 ±
0.3
0 2
.11
± 0.
22
n.d
.
2.55
± 0
.35
3.2
7 ±
0.25
n
.d.
Tab
le 4
.2:
So
il so
lutio
n p
rop
ert
ies
from
th
e s
oil
an
d so
il w
ith
age
d b
ioch
ar
am
end
men
t. V
alu
es r
epre
sent
mean
s ±
SE
M (
n=
4).
Pro
per
ties
Site
1 (
Au
stra
lia)
S
ite 2
(W
ale
s)
S
oil
Bio
char
am
end
ed
soil
S
oil
Bio
char
am
end
ed
soi
l
Dis
solv
ed
org
an
ic C
(m
g C
l-1)
106
± 9
108
± 1
9
82
± 8
98
± 1
3
Tot
al d
isso
lve
d N
(m
g N
l-1)
51
.4 ±
6.8
61
.7 ±
12.
6
21.6
± 1
0.3
19.
7 ±
6.8
Pe
ptid
es (
µm
ol l-1
) 3
6.2
± 1
.4 3
0.6
± 4
.6
42.0
± 1
8.5
11.
2 ±
6.6
Am
ino
aci
ds (
µg
N l-1)
18
3 ±
25
173
± 3
0
102
± 2
4 2
30
± 9
0
NH
4+ (m
g N
l-1)
0.48
± 0
.10
0.4
6 ±
0.1
6
6.59
± 4
.42
1.3
5 ±
0.8
8
NO
3- (m
g N
l-1)
48
.1 ±
6.
3 3
5.8
± 6
.1
7.4
± 4
.5 7
.6 ±
3.9
Biochar did not change organic N mineralisation Chapter 4
101
4.4 Results
Repeated measures ANOVA showed that biochar addition only induced a significant
difference in total 14CO2 evolution in the trialanine treatment in the Australian soil
(Fig. 4.1). Similarly, the kinetic model parameters describing mineralisation also
failed to show consistent treatment differences in C partitioning within the microbial
biomass (Y0, Y1, Y2) or rates of C turnover after microbial assimilation (k1, k2) in the
two soils (Tables 4.3, 4.4, 4.5 and 4.6). When significant differences were observed
they tended to be small. For example, in the Australian soil amended with alanine, Y2
was greater in the aged biochar treatment (22.3%) compared to the control (19.2%)
and fresh biochar (19.0%) treatments (p=0.001; Table 4.3). Y2 did not differ in the
corresponding Welsh soil treatment, however, Y1 did (p=0.006; Table 4.3). In the
Welsh soil amended with amino acids, k1 was slightly lower when fresh biochar was
applied to the soil (p=0.049) while no significant effect was seen in the aged biochar
treatment (Table 4.4). In contrast this was not seen in the Australian soil (Table 4.4).
In the Australian soil with peptide amendment the size of the recalcitrant pool (Y0)
and respired pool (Y1) in the aged-biochar treatment changed relative to the control
(p=0.02), however, no effect was seen in the fresh biochar treatment (Table 4.5). The
MRT of the substrate labile pool did not change with biochar addition in either soil
type with any of the added substrates (Table 4.6).
Biochar did not change organic N mineralisation Chapter 4
102
Fig. 4.1: Effect of biochar treatment on the percentage of 14C evolved as CO2 in soil
over a 20 d incubation period for the Australian (A, C, E) and Welsh (B, D, F) soils
amended with 14C-labelled alanine (A, B), a mix of amino acids (C, D) or trialanine
(E, F). Data points represent means ± standard errors (n=4). Curves represent fits to
the kinetic model.
Tab
le 4
.3:
Su
bstr
ate
ha
lf lif
e a
nd
par
am
ete
rs fro
m t
he m
odels
fit
ted
to
the
deca
y cu
rves
of
14 C
-la
belle
d al
an
ine; s
ign
ifica
nt d
iffere
nce
s
are
indi
cate
d b
y N
S (
no
t si
gn
ifica
nt)
, *
(p<
0.05
),
** (
p<0.
01)
an
d **
* (p
<0
.001
); m
ultip
le c
om
pari
sons r
epr
ese
nted
by le
tters
. Val
ues
repr
ese
nt m
ea
ns
± st
and
ard
err
ors
(n
=4
).
R
eca
lcitr
ant p
oo
l R
esp
ire
d po
ol Im
mob
ilise
d p
ool
Su
bst
rate
hal
f life
Y
0 (%
) Y
1 (%
) k 1
(%
ho
ur-1
) Y
2 (%
) k 2
(%
ho
ur-1
) S
t 1/2 (h
ou
rs)
Aus
tral
ian
Soi
l
Co
ntro
l 5
6.3
± 1.
5 2
4.2
± 1
.2 0
.38
± 0.
04
19
.2 ±
0.4
0 a
0.0
06
7 ±
0.0
006
6.04
± 0
.71
ab
Age
d bi
och
ar 5
3.2
± 0.
7 2
4.3
± 0
.4 0
.39
± 0.
01
22
.3 ±
0.4
3 b
0.0
05
8 ±
0.0
001
6.6
8 ±
0.06
b
Fre
sh b
ioch
ar
56.
9 ±
1.0
24
.2 ±
0.8
0.4
3 ±
0.01
1
9.0
± 0
.36
a 0
.00
65
± 0.
00
03 4
.80
± 0.
13 a
P v
alu
e N
S N
S N
S **
*
NS
NS
Wel
sh S
oil
Co
ntro
l 6
4.2
± 0.
7 2
1.4
± 0
.7 b
1.0
6 ±
0.03
1
4.1
± 0
.71
0.0
09
3 ±
0.0
017
1.6
5 ±
0.11
Age
d bi
och
ar 6
4.1
± 1.
0 2
0.7
± 0
.5 b
0.8
9 ±
0.08
1
4.8
± 0
.70
0.0
07
1 ±
0.0
003
2.2
0 ±
0.30
Fre
sh b
ioch
ar
63.
8 ±
1.3
18
.4 ±
0.5
a 0
.71
± 0.
15
16
.9 ±
1.5
0.0
10
9 ±
0.0
011
5.1
0 ±
2.19
P v
alu
e N
S **
N
S N
S N
S N
S
Tab
le 4
.4:
Su
bstr
ate
ha
lf lif
e a
nd
par
am
ete
rs fro
m t
he m
odels
fit
ted
to
the
deca
y cu
rves
of
14 C
-la
belle
d am
ino
aci
d m
ixtu
re (
n=
4);
sig
nifi
cant
diff
eren
ces
are in
dic
ate
d b
y N
S (
not
si
gn
ifica
nt)
an
d *
(p<
0.05
); m
ulti
ple
com
pariso
ns r
ep
rese
nted
by
lett
ers
. Val
ues
repr
ese
nt m
ea
ns
± st
and
ard
err
ors
(n
=4
).
R
eca
lcitr
ant p
oo
l R
esp
ire
d po
ol
Imm
ob
ilise
d po
ol S
ubs
trat
e h
alf l
ife
Y
0 (%
) Y
1 (%
) k 1
(%
ho
ur-1
) Y
2 (%
) k 2
(%
hou
r-1)
St 1/
2 (h
our
s)
Aus
tral
ian
soil
Co
ntro
l 6
1.0
± 0.
7 1
6.4
± 0
.6
0.46
± 0
.03
21.9
± 0
.3 0
.00
60 ±
0.0
005
22
.9 ±
3.4
a
Age
d bi
och
ar 6
1.6
± 0.
6 1
4.0
± 0
.6 0.
47 ±
0.0
3 23
.5 ±
0.5
0.0
052
± 0
.00
03 4
2.9
± 2
.0 b
Fre
sh b
ioch
ar
60.
6 ±
1.5
15
.9 ±
0.4
0.39
± 0
.02
22.6
± 1
.2 0
.00
53 ±
0.0
003
29
.9 ±
5.5
ab
P v
alu
e N
S N
S N
S N
S N
S *
Wel
sh s
oil
Co
ntro
l 7
3.9
± 0.
9 1
2.7
± 0
.6 0.
93 ±
0.1
3 b
13.0
± 0
.7 0
.00
81 ±
0.0
003
6.3
7 ±
1.6
1 ab
Age
d bi
och
ar 7
3.8
± 0.
1 1
3.5
± 0
.3 0.
66 ±
0.0
5 ab
12.1
± 0
.2 0
.00
71 ±
0.0
005
4.0
5 ±
0.2
2 a
Fre
sh b
ioch
ar
72.
1 ±
1.1
12
.1 ±
0.2
0.53
± 0
.08
a 15
.0 ±
1.4
0.0
090
± 0
.00
08 1
1.8
± 2.
6 b
P v
alu
e N
S N
S *
NS
NS
NS
Tab
le 4
.5:
Su
bstr
ate
ha
lf lif
e a
nd
par
am
ete
rs fro
m t
he m
odels
fit
ted
to
the
deca
y cu
rves
of
14 C
-la
belle
d pe
ptid
es
(tri
alan
ine; n
=4
);
sig
nifi
cant
diff
eren
ces
are in
dic
ate
d b
y N
S (
not
si
gn
ifica
nt)
, *
(p<
0.05
) o
r **
(p
<0
.01;
mul
tiple
co
mpar
iso
ns
repr
ese
nte
d b
y le
tters
.
Va
lues
rep
rese
nt m
ea
ns
± st
and
ard
err
ors
(n
=4
).
R
eca
lcitr
ant p
oo
l R
esp
ired
poo
l Im
mob
ilise
d p
ool
Su
bst
rate
hal
f life
Y
0 (%
) Y
1 (%
) k 1
(%
ho
ur-1)
Y2
(%)
k 2 (
% h
ou
r-1)
St 1/
2 (h
ou
rs)
Aus
tral
ian
Soi
l
Co
ntro
l 4
6.0
± 1.
8 b
43.
4 ±
1.4
a 0
.71
± 0.
06
10
.9 ±
0.6
0.0
05
3 ±
0.0
005
1.4
2 ±
0.14
Age
d bi
och
ar 4
0.5
± 0.
8 a
48.
2 ±
0.5
b 0
.78
± 0.
06
11
.6 ±
0.2
0.0
05
5 ±
0.0
003
1.2
6 ±
0.10
Fre
sh b
ioch
ar
41.
9 ±
1.1
ab
47.
3 ±
1.0
ab
0.7
9 ±
0.0
6 1
0.9
± 0
.4 0
.00
53
± 0.
00
04 1
.22
± 0.
08
P v
alu
e *
* N
S N
S N
S N
S
Wel
sh S
oil
Co
ntro
l 4
6.1
± 1.
1 4
7.1
± 1.
0 1
.57
± 0.
06
6.7
± 0
.2 a
0.0
13
5 ±
0.0
017
ab
0.5
4 ±
0.02
Age
d bi
och
ar 4
7.0
± 1.
9 4
5.1
± 1.
6 1
.49
± 0.
08
7.8
± 0
.7 a
0.0
10
5 ±
0.0
019
a 0
.60
± 0.
04
Fre
sh b
ioch
ar
42.
9 ±
1.3
45.
6 ±
1.6
1.4
5 ±
0.0
5 1
1.3
± 1
.1 b
0.0
24
4 ±
0.0
039
b 0
.67
± 0.
04
P v
alu
e N
S N
S N
S **
N
S N
S
Biochar did not change organic N mineralisation Chapter 4
106
Table 4.6: Mean residence time (hours) of the labile component of 14C labelled
substrate added to the various soil treatments. Values represent means ± SEM
(n=4).
Alanine Amino acid mix Tri-alanine
Australian Soil
Control 34.1 ± 5.1 37.6 ± 3.2 22.1 ± 3.5
Aged biochar 38.9 ± 1.4 45.7 ± 3.2 21.8 ± 1.5
Fresh biochar 30.0 ± 1.7 43.2 ± 4.2 21.6 ± 1.7
P value NS NS NS
Welsh Soil
Control 20.7 ± 2.8 16.4 ± 1.2 5.5 ± 0.7
Aged biochar 21.2 ± 1.4 17.5 ± 1.2 9.7 ± 3.7
Fresh biochar 16.2 ± 2.6 16.9 ± 0.3 5.2 ± 0.7
P value NS NS NS
Biochar did not change organic N mineralisation Chapter 4
107
4.5 Discussion
The hypothesis for this experiment was that the addition of fresh biochar would not
change the rate of decomposition of organic N compounds, but that aged biochar
would decrease it. The results from this experiment, however, do not support this
tenet as both short and long term biochar addition failed to reveal major shifts in C
turnover or partitioning within the soil microbial biomass. Based on these results
and those of Kuzyakov et al. (2009), biochar application is unlikely to have a great
effect on mineralisation of organic N compounds. These studies, however, contrast
with studies in boreal forests (Wardle et al., 2008), potentially because these
agricultural soils lack significant quantities of phenolic compounds that suppress N
mineralisation but which can be removed in the presence of biochar (DeLuca et al.,
2006). These results contrast with the findings of Jones et al. (2011), who saw
decreased mineralisation of native organic matter. This is likely to be due to more
recalcitrant nature of the organic matter pool examined and the greater application
rates used. Field aging of biochar also had little effect on mineralisation of N
containing compounds. However, it is known that at least for the aged Welsh
biochar that its chemical properties were very different after being applied 3 years
previously from those of fresh biochar (e.g. pH, nutrient retention; data not
presented). With the large discretion in biochar variables between agro-ecosystems
(e.g. biochar rate and field aging time), differences in DON mineralisation were
expected. For example, the biochar application rate is likely to influence how much
sorption processes will influence DON mineralisation. Furthermore, time may
influence soil-biochar interactions. Initially biochar addition will also introduce
Biochar did not change organic N mineralisation Chapter 4
108
some labile C (Smith et al., 2010) and over time biochar surfaces may become
occluded by soil mineral particles (Van Zwieten et al., 2009) both potentially
influencing OM mineralisation. Despite this, few differences were seen with its
addition. As such mechanisms cannot be isolated from this experiment, but it
provides an important insight into application differences actually likely within
contrasting field based situations and their consequences.
Field application rates of biochar in these agro-ecosystems are unlikely to greatly
influence the turnover of the intrinsic DON reserves in soil.
109
110
Minimal biochar and N fertiliser interaction in the field Chapter 5
111
Chapter 5
Minimal interaction between wheat chaff biochar and N
fertiliser in a broadacre field experiment
5.1 Abstract
Biochar, the remains of pyrolysed organic matter, is an agricultural amendment that
can sequester carbon and has been found to synergistically interact with N fertiliser.
The potential for this amendment to improve wheat (Triticum aestivum L.) yields and
N fertiliser use efficiency via two application methods (banded or spread) in a
broadacre, semi-arid agro-ecosystem was assessed. Wheat residue biochar was
applied (4 t ha-1 banded, 4 t ha-1 spread, or control), in full factorial design with N
fertiliser (0, 20 or 40 kg N ha-1). The hypotheses were that (1) biochar application
would increase wheat yields (2) biochar application would improve N fertiliser use
efficiency, and (3) there would be no difference between application methods. Soil
ammonium (NH4+) and nitrate (NO3
-) to a depth of 90 cm, microbial biomass (0-20
cm), plant N and plant biomass were measured through the year, as was grain N and
yield. At harvest there was no effect of biochar on plant biomass, but there was an
increase in biomass with biochar application at terminal spikelet. Banded biochar
application (1.7 t ha-1) significantly decreased wheat yields from the control (2.1 t ha-
1) but spread application was not significantly different (1.8 t ha-1). Biochar did not
influence soil inorganic N at seeding, re-seeding, anthesis or harvest, and had a small
influence at terminal spikelet sampling. In this experiment biochar had very little
Minimal biochar and N fertiliser interaction in the field Chapter 5
112
impact on N fertiliser and its efficiency of use, possibly because of the low rates
application.
5.2 Introduction
An avenue for mitigating increased atmospheric C levels is through sequestration
within managed soils. The application of biochar is one proposed method to store C
without changing current agricultural practices dramatically (Sohi et al., 2010).
Biochar has been defined as the remains from the pyrolysis of organic matter at
temperatures greater than 250°C (Lehmann & Joseph, 2009). In addition to C
sequestration, biochar has also been shown to improve soil fertility resulting in
improved yields (Glaser et al., 2002; Atkinson et al., 2010), with improved fertiliser
use efficiency in some cropping systems (Van Zwieten et al., 2010a).
Interaction between biochar amendment and N fertiliser additions can occur in some
farming systems (Chan et al., 2007; Chan et al., 2008; Van Zwieten et al., 2010a), but
mechanisms inducing these changes are unclear. Adding a porous, alkaline
amendment that contains a diverse range of minerals and nutrients to acid soils adds
many confounding factors when trying to isolate mechanisms. Apart from a fertiliser
impact through nutrients added in biochar, it may provide a habitat for the microbial
community (Peitikäinen et al., 2000). Increases in rates of N mineralisation and
nitrification in soil with added biochar have been measured in pine forest ecosystems
(Berglund et al., 2004; DeLuca et al., 2006) but changes induced in agricultural and
grassland ecosystems are variable (e.g. DeLuca et al., 2006; Chapter 2; Chapter 4).
Minimal biochar and N fertiliser interaction in the field Chapter 5
113
Biochar addition can also decrease N leaching by increasing water holding capacity
(WHC; Lehmann et al., 2003; Chapter 3), and nutrient retention capacity and cation
exchange capacity through increased electrostatic sorption (Lehmann et al., 2003;
Liang et al., 2006).
In the broadacre agricultural environment in semi-arid environments the application
rate of biochar is likely to be less then 5 t ha-1 (Blackwell et al., 2010). Contextualised
globally, these application rates are considered low, with moderate application rates
5-15 t ha-1 and large rates greater than 15 t ha-1 (Sohi et al., 2010). Experiments
establishing mechanisms inducing N cycling rate changes have been conducted at
larger application rates (e.g. 191 t ha-1, Lehmann et al., 2003; 1% w/w, DeLuca et al.,
2006; >1.1% w/w Van Zwieten et al., 2010a). The applicability and impact of these
mechanisms has yet to have been tested for broadacre agriculture in semi-arid
environments but its impact is likely to be less as contact between biochar particles
and N compounds will be less.
Soil amendments must guarantee yield improvements and require minimal time and
effort to be feasibly applied in broadacre agriculture (Kingwell, 2011); biochar is no
exception. But many studies examining the interaction between biochar and N
fertiliser have not grown crops to maturity, and not assessed crop yield (e.g. Van
Zwieten et al., 2010a; Van Zwieten et al., 2010b). Furthermore, there are not any
studies that assess the best method of application for farmers of the Western
Australian wheatbelt. Within broadacre agro-ecosystems, the most likely application
Minimal biochar and N fertiliser interaction in the field Chapter 5
114
methods are banding or spreading. Banding is the application of the amendment
within the rooting zone of the plant and spreading is the distribution of the
amendment on top of the soil. Previous laboratory and glasshouse studies examining
application method suggest there may be differences in pesticide dynamics (Jones et
al., 2010), but not N leaching (Chapter 3). Both methods contain weaknesses with
their application. Spreading biochar is dusty and prone to loss from wind, while
banding application rates are much lower and biochar is difficult to handle with
current machinery.
It was hypothesised that biochar application would improve N fertiliser use
efficiency, resulting in increased yields. To test these hypotheses, a field experiment
with three biochar and three N rates in full factorial design was designed and
conducted.
5.3 Materials and Methods
5.3.1 Experimental site and design
The field experiment was located on an Acidic Yellow Kandosol (Table 5.1; Isbell,
1996), near Buntine, WA (30°00’S, 116° 21’E). This site had been cropped with
canola (Brassica napus) the previous year and was seeded with wheat (Triticum
aestivum cv. Wyalkatchem) at 75 kg ha-1. This area is located within a mediterranean-
type climate and has an average rainfall of 350 mm, of which 260 mm falls in the
growing season (May - October) (Bureau of Meteorology, 2011). Within the year of
Minimal biochar and N fertiliser interaction in the field Chapter 5
115
this trial, the experimental site received 205 mm of rain (Fig. 5.1). Growing season
mean monthly minimum and maximum temperatures ranged from 13-5 ºC and 26-16
ºC (Fig. 5.1).
The experiment consisted of three biochar treatments and three N fertiliser treatments
in a full factorial design arranged in a randomised block with four replicates. Each
plot was 2 m by 20 m. N fertiliser (urea) treatments were i) control (0 kg N ha-1); ii)
20 kg N ha-1; or iii) 40 kg N ha-1. The three biochar treatments were; 1) the control (0
t ha-1); 2) biochar applied below the surface of the soil at 5-15 cm depth (4 t ha-1;
termed the ‘banded’ treatment); or 3) biochar spread on the surface of the soil (4 t ha-
1; termed the ‘spread’ treatment). Biochar treatments were applied using a cone
seeder one month prior to seeding. The banded treatment was applied by distributing
the biochar through the seeding and fertiliser boots on the tyne of the cone seeder
within a depth range of 50 to 120 mm. For the spread treatment, distribution hoses
were removed from the boots on the tyne, and secured next to the tyne at a height that
ensured biochar was applied to surface. The control treatment was also cultivated in
the same manner, thus ensuring consistent cultivational influences between all three
treatments. All N treatment and basal fertilisers were applied when seeding. All
treatments received 4.5 kg P ha-1, 5 kg K ha-1 and 10 kg Ca ha-1.
The experiment, due to the large ryegrass burden, was sprayed with 3 L ha-1
glyphosate, and re-seeded 3 weeks after initial seeding. No further fertiliser
amendments were applied at this time.
Minimal biochar and N fertiliser interaction in the field Chapter 5
116
Mo
nth
ly r
ain
fall
rece
ived
1
(m
m)
Mea
n m
on
thly
1
1
2
T
emp
erat
ure
(ºC
)
0
10
20
30
40
50
Apr May Jun Jul Aug Sep Oct Nov
0
10
20
30
40
Fig. 5.1: Monthly Rainfall received (mm; histogram) and mean monthly minimum
(triangles) and maximum (circles) temperature (ºC) received over the year of the
trial. Note: January data not available.
The biochar applied was a wheat (Triticum aestivum) chaff biochar pyrolysed at 450
°C for 20 minutes (Pacific Pyrolysis, Somersby, New South Wales). Total C and N
were determined by combustion analysis (Table 5.1). The EC and pH of the biochar
was determined in a 1:5 (w/v) soil-to-water mixture (Table 5.1). Random powder X-
ray diffraction pattern of biochar obtained for the range 0-70° 2θ showed the biochar
contained amorphous carbon, sylvite and quartz. The specific surface area and
micropore volume of the biochar were 214 m2 g-1 and 70 cm3 g-1 respectively,
determined by a surface area analyser using the Brunauer, Emmett and Teller (BET)
method (Brunauer et al., 1938).
Tab
le 5
.1:B
asi
c p
rope
rtie
s of
bio
cha
r a
nd s
oil
bef
ore s
eed
ing
(N
A -
No
t A
ssess
ed).
Va
lues
are
mean
s w
ith s
tand
ar
d err
ors
(n=
4).
So
il D
epth
(cm
) 0
-10
1
0-2
0
20
-30
30
-60
60
-90
B
ioch
ar
pH
6
.24
± 0.
09
NA
N
A
NA
N
A
9.2
3 ±
0.0
2
EC
(μS
cm
-1)
82
.5 ±
13.
6 N
A
NA
N
A
NA
2
980
± 2
70
NH
4+ (m
g N
kg-1
)
1.1
± 0
.1
0.3
± 0
.1
0.3
± 0
.1
0.3
± 0
.1
0.3
± 0
.1
1.4
2 ±
0.2
1
NO
3- (m
g N
kg-1
) 7
.8 ±
0.4
2
.6 ±
0.5
1
.8 ±
0.3
0
.7 ±
0.1
N
D 0
±0
1.
05 ±
0.0
5
Mic
robi
al
Bio
mas
s
(mg
N k
g -1)
7.0
3 ±
1.45
3
.95
± 0.
69
NA
N
A
NA
N
A
Tot
al C
(%
) 1
.05
± 0.
06
0.5
6 ±
0.0
3 0
.23
± 0.
01
0.1
3 ±
0.0
1 0
.11
± 0.
01
60
.9 ±
0.4
4
Tot
al N
(%
) 0
.08
± 0.
004
0.0
4 ±
0.0
05
0.0
2 ±
0.0
02 0
.02
± 0.
00
2 0
.02
± 0.
003
2.3
2 ±
0.0
2
Minimal biochar and N fertiliser interaction in the field Chapter 5
118
5.3.2 Sampling times and analysis
Soils were sampled at the seeding, re-seeding, terminal spikelet, anthesis and harvest
stages of plant growth. Soil was samples were collected at 0-10, 10-20, 20-30, 30-60
and 60-90 cm depth. Soil from the 0-10 and 10-20 cm layers was assessed for MBN
using the fumigation-extraction method (Vance et al., 1987) and the ninhydrin
method (Joergensen & Brookes, 1990) and the flush (fumigated less non-fumigated
values) adjusted by a factor of 6.47 (Sparling et al., 1993). Soil extracts were
analysed colourimetrically for ammonium (NH4+) using the salicylate-nitroprusside
method (Krom, 1980; Searle, 1984) and nitrate (NO3-) concentration using the
hydrazine reduction method (Kamphake et al., 1967; Kempers & Luft, 1988) on an
automated flow injection Skalar Auto-analyser (Skalar San plus).
Plant shoot samples were taken at terminal spikelet, anthesis and harvest. These were
analysed for biomass and N concentration. At harvest, grain yield and grain N
concentration were also determined. All plant N content was assessed by combustion
analysis using an elementar (vario Macro CNS; Elementar, Germany).
5.3.3 Statistical analysis
Statistical analysis was performed using a series of two-way ANOVAs using
GENSTAT 10th Edition (Lawes Agricultural Trust, 2007). Multiple comparisons
were done using Tukey’s 95% confidence intervals. Significance considered at the P
< 0.05 significance level.
Minimal biochar and N fertiliser interaction in the field Chapter 5
119
5.4 Results
5.4.1 Soil microbial biomass
The size of the MBN was 19.2 μg N g-1 soil at terminal spikelet sampling in the 0-10
cm layer. No difference in MBN, due to either biochar or N addition, was detected at
any sampling time or analysed soil depth (0-10 or 10-20 cm).
5.4.2 Soil ammonium and nitrate
There was no effect of biochar application or interaction between biochar application
and N fertiliser at re-seeding. At re-seeding statistical differences in NH4+ at 0-10 cm
and 10-20 cm and NO3- at 0-10 cm depth were caused by N application, where the
greatest concentrations NH4+ and NO3
- could be found within the soils applied with
the most N (Table 5.2).
At terminal spikelet, an interaction was found between the two factors for NH4+ at 10-
20 cm depth, but this was the only instance in all soil samples in which statistical
interaction between the two factors was present. At 20-30 cm depth, in the terminal
spikelet sampling there was a significantly more NH4+ was wheat 40 kg N ha-1 was
applied than 0 kg N ha-1 (Table 5.2). The only significant differences in soil N with
biochar addition were in NO3- at 20-30 cm depth and in NH4
+ at 60-90 cm depth
during terminal spikelet sampling. There was significantly more nitrate detected in
the spread treatment (1.69 mg N kg-1) than in the control (0.77 mg N kg-1) at 20-30
cm depth and less NH4+ detected in both biochar amended treatments (banded - 0.09
Minimal biochar and N fertiliser interaction in the field Chapter 5
120
mg N kg-1; spread - 0.01 mg N kg-1) than the control (0.20 mg N kg-1) at 60-90 cm
depth. At anthesis and harvest significant differences in soil NH4+ and NO3
- were
only due to the application of N fertiliser (Table 5.2).
5.4.3 Plant biomass and nitrogen content
At terminal spikelet plant biomass was affected by both N application (P<0.001) as
well as biochar application (P=0.027), but there was no interaction between
amendments (Table 5.3). Increasing N application significantly increased plant
biomass. Plant biomass was significantly increased with the spread application of
biochar (from 0.60 to 0.72 t ha-1) but not the banded application (Fig. 5.2a).
Interaction between biochar and N applications was present at anthesis sampling
(P=0.015). At this time point, the greatest plant biomass was in the 40 kg N ha-1 and
banded biochar treatment (5.53 t ha-1) and the least plant biomass was in the banded
biochar and 0 kg N ha-1 treatment (3.83 t ha-1; Fig. 5.2b).
At harvest there was no difference in plant biomass due to biochar, and there was no
interaction. N application at 40 kg ha-1 increased plant biomass from 4.70 t ha-1 to
5.69 t ha-1 (P=0.016; Table 3; Fig. 5.2c).
Tab
le 5
.2:
Soi
l N
O 3- a
nd N
H 4+ v
alu
es
(mg
N k
g-1 s
oil)
fo
r sa
mpl
es
influ
enc
ed
by
N a
pplic
atio
n.
Val
ues
rep
rese
nt
mea
ns w
ithin
ea
ch
bio
cha
r tr
ea
tmen
t ±
sta
nda
rd e
rror
s (n
=4)
, le
tter
s re
pre
sent
sig
nifi
cant
diff
ere
nce
s.
Sa
mp
ling
Tim
e R
e-se
edin
g
Re-
seed
ing
R
e-se
edin
g
Ter
min
al
Sp
ikel
et
An
thes
is
An
thes
is
Har
vest
Dep
th (
cm)
0-1
0
0-1
0
10-
20
2
0-3
0
0-1
0
10-
20
0
-10
N t
ype
N
H 4+
NO
3- N
H4+
NH
4+ N
H4+
NH
4+ N
H4+
P v
alu
e
0.0
32
<0
.00
1 0
.009
0
.029
0
.019
0
.014
0
.016
40
kg N
ha-1
0
.35
± 0.
10 a
5
.54
± 0
.50
a
0.3
7 ±
0.3
5 a
0
.71
± 0
.2
0 b
1
.01
± 0
.14
b
0.7
7 ±
0.2
7 b
1
.11
± 0
.10
b
20
kg N
ha-1
5
.36
±1.7
3 a
b
8.5
8 ±
1.4
2 a
1
.37
± 0
.59
a
0.3
2 ±
0.1
0 a
b
0.8
0 ±
0.1
0 a
b
0.5
2 ±
0.0
6 a
b
1.1
0 ±
0.0
8 b
0 k
g N
ha-1
1
4.59
± 6
.41
b
12.
38 ±
1.6
4 b
5
.24
± 1
.87
b
0.1
3 ±
0.0
4 a
0
.60
± 0
.04
a
0.3
0 ±
0.0
5 a
0
.81
± 0
.08
a
Minimal biochar and N fertiliser interaction in the field Chapter 5
122
Table 5.3: Table of significant differences for plant biomass, plant N and grain yield
and N for all measured time points. P values for significant differences due to
biochar (*), N application (^) or interaction (†) are shown; NS represents not
significant.
Terminal Spikelet Anthesis Harvest
Plant Biomass (t ha-1) 0.027 *; <0.001 ^ 0.015 † 0.016 ^
Plant N (%) <0.001 ^ <0.001 ^ <0.001 ^
Grain Yield (t ha-1) - - 0.042 *
Grain N (%) - - <0.001 ^
At all measured time points, there was no significant effect of biochar on plant N
concentration and there was no interaction between biochar and N application.
Increasing N application always increased plant N concentration (P<0.001 at all time
points).
5.4.4 Grain yield and nitrogen content
Grain yield decreased with banded biochar application (P=0.042). The control plot
yielded 2.1 t ha-1, the spread biochar plots yielded 1.8 t/ha and the banded biochar
plots yielded 1.7 t ha-1 (Fig. 5.2d). Yield was not influenced by N application and
there was no interaction between biochar and N fertiliser.
Minimal biochar and N fertiliser interaction in the field Chapter 5
123
P
lan
t Bio
mas
s (t
ha
-1)
Pla
nt B
iom
ass
(t h
a-1)
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nt B
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ass
(t h
a-1)
Gra
in Y
ield
(t h
a-1)
0
0.2
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0.8
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(a)
0
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(b) a
aaaa
bb
bb
bc
ccd d
0
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Con
trol
Ban
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Spr
ead
Con
trol
Ban
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Spr
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trol
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0 kg N ha 20 kg N ha 40 kg N ha
(c)
Time
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2
2.5
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Con
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ead
Con
trol
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0 kg N ha 20 kg N ha 40 kg N ha
(d)
Fig. 5.2: Dry plant biomass at terminal spikelet (a), anthesis (b) and harvest (c) and
grain yield (d). Error bars represent standard errors (n=4).
The percentage of N in the grain was not affected by biochar application, but
increased with increasing N application quantities (P<0.001). There was no
interaction between N fertiliser and biochar application.
Minimal biochar and N fertiliser interaction in the field Chapter 5
124
5.5 Discussion
5.5.1 Interaction between nitrogen fertiliser and biochar
The interaction between biochar and N fertiliser has been suggested to be an
important reason for agronomic improvement resulting in increased yields (Chan et
al., 2007; Chan et al., 2008; Van Zwieten et al., 2010a). It has also been suggested
that biochar can provide nutrients other than N to plants (Chan et al., 2007; Chan et
al., 2008). Phosphorus and potassium, within the biochar, may have been plant
available in the early part of the growing season and thus contributed to the greater
plant biomass seen at terminal spikelet sampling with the spread application of
biochar (Fig. 5.2a). Although the plant samples were not tested for any nutrients other
than N, the decreased yields seen at harvest suggest this is unlikely.
Increased N fertiliser use efficiency has been seen with large application rates of
biochar; greater than 10 t ha-1 for nutrient rich biochars, and even greater rates for
nutrient poor biochars (Chan et al., 2007; Chan et al., 2008; Van Zwieten et al.,
2010a; Van Zwieten et al., 2010b). Given the low bulk density of biochar (0.2 g cm-3
in this study), application rates above 4 t ha-1 would be logistically difficult,
expensive and time consuming. Therefore it is unlikely that significant improvements
in N fertiliser use efficiency as seen in previous research (Chan et al., 2007; Chan et
al., 2008; Van Zwieten et al., 2010a) are to be achieved in broadacre agricultural
environments.
Minimal biochar and N fertiliser interaction in the field Chapter 5
125
5.5.2 Yield influence of biochar
Previous research has suggested that application of biochar frequently increases plant
and/or crop yield (Glaser et al., 2002; Atkinson et al., 2010; Blackwell et al., 2010),
however in this study biochar application resulted in decreased wheat yields
(significant only where biochar was banded). Biochar may have decreased plant yield
by limiting the rate of N mineralisation (Chapter 2). However using soil collected
from this experiment, organic N mineralisation did not change with biochar addition
(Chapter 4). Both that study and the current study did not assess changes in
nitrification rates or urea hydrolysis rates as a result of biochar addition, but they are
unlikely to have changed upon biochar addition analogous to organic N
mineralisation. Furthermore, there was no significant difference in grain N,
suggesting the yield difference maybe due to another factor, potentially a change in
soil nutritional status (Chan et al., 2008; Blackwell et al., 2010).
One mechanism suggested for this is by enhancing plant water supply by increasing
the WHC of the soil (Atkinson et al., 2010; Blackwell et al., 2010). This is likely to
be especially relevant in dry environments with coarse-textured soils; however the
current study suggests this mechanism is unlikely, especially at field applicable
application rates in this agro-ecosystem. This experiment received 195 mm of rainfall
in between February and October of the year, yet moisture content did not change
with biochar addition at any measured time point (data not shown). If WHC of the
biochar is 400% (determined in a small packed core, data not shown), 4 t ha-1 may be
able to hold a maximum of 1.6 mm of rainfall extra at any time. A water use
Minimal biochar and N fertiliser interaction in the field Chapter 5
126
efficiency of 12.7 kg ha-1 grain mm-1 (French & Shultz, 1984) suggests that this could
lead to an increased 20.3 kg ha-1 assuming that all water is plant available and this
increased WHC is only fully utilised once per growing season. As yield decreased
with biochar application (significant decreases with banded biochar application),
biochar is unlikely to have affected this experiment by increasing WHC.
5.5.3 Biochar application method
Biochar application method did influence the effect of biochar in some instances,
although the overall influence of biochar was limited. This was seen with plant
biomass at the terminal spikelet stage where plant biomass was greatest with spread
application of biochar (Fig. 5.2a) and at harvest, where grain yield was lowest with
banded application of biochar (Fig. 5.2d). In both instances, the other biochar
treatment did not significantly change the assessed parameter. This demonstrates that
varying application methods may alter the crop influence of biochar. These results
suggest that application of biochar to the surface of the soil may be more effective
than subsurface application, but further research is required replicate these effects
establish whether the effect is consistent.
5.5.4 Conclusion
In a semi-arid, broadacre environment the application of biochar did not provide
agronomic benefit in the first season of application. Grain yield was decreased with
biochar application. This decrease was significant only when the biochar was banded,
as such spread application appeared less agronomically detrimental than banded
Minimal biochar and N fertiliser interaction in the field Chapter 5
127
application. Interaction between biochar and N fertiliser was sporadic and minimal.
Furthermore, N grain and shoot N content did not change with biochar addition, as
such this biochar did not improve N fertiliser use efficiency.
128
General Discussion Chapter 6
129
Chapter 6
General Discussion
Biochar has previously been preposed to influence soil fertility via a number of
changes to soil physical, chemical and biological properties. By focusing on the
interaction between biochar and the terrestrial N cycle a number of questions have
been addressed in this thesis. These questions revolved around changes to N cycling
rates that are likely to influence on farm productivity, specifically N mineralisation, N
immobilisation and N leaching (Summarised in Table 6.1). The experiments
investigating these questions used biochar types with potential to be used in the
Western Australian wheatbelt. Biochar either decreased or did not change N
mineralisation or the soil microbial biomass. However biochar did decrease N
leaching. This chapter discusses five specific processes that impact (directly or
indirectly) terrestial N cycling, and consequently farm productivity. The specific
processes are: the sorption of LMW N compounds to biochar, the sorption of high
molecular weight (HMW) organic compounds to biochar, the addition of labile C
contained in biochar, and increased WHC resulting from biochar application.
General Discussion Chapter 6
130
Table 6.1: A summary of the questions addressed within each thesis chapter, and the
corresponding answers found in this thesis.
Chapter & Questions Addressed Answers to questions
Chapter 2:
� When added to a coarse textured soil, does
biochar sorb NH4+, NO3
- or amino acids?
� Does biochar influence net N
mineralisation when added to a coarse
textured soil?
� Does biochar addition to a coarse textured
soil induce microbial immobilisation of N?
� No.
� Yes, Jarrah biochar
decreased net N
mineralisation.
� No.
Chapter 3:
� Does biochar addition to a coarse textured
soil decrease N leaching?
� Is sorption the main mechanism
decreasing leaching when added to a
coarse-textured soil?
� Yes, Jarrah biochar did.
� No, not the main
mechanism, maybe due
to increased WHC.
Chapter 4:
� Does organic N mineralisation change
when biochar is added to a coarse textured
soil?
� Does organic N mineralisation change
over time after biochar addition to a coarse
textured soil?
� No, not with wheat
residue biochar.
� No, not with wheat
residue biochar.
General Discussion Chapter 6
131
Chapter 5:
� When added to a coarse textured soil in a
broadacre Western Australian agro-
ecosystem, does biochar interact with N
fertiliser to increase soil N?
� Does biochar influence plant N uptake
when added to a coarse textured soil in a
broadacre Western Australian agro-
ecosystem?
� Generally no, but
interaction between
biochar and N fertiliser
was found during one of
five sampling times.
� No difference was found
in plant N uptake with
wheat residue biochar
added at 4 t ha-1.
6.1 Sorption of low molecular weight nitrogen to biochar
Biochar can sorb nitrate (NO3-; Mizuta et al., 2004; Chapter 2; Chapter 3),
ammonium (NH4+; Hina et al., 2010; Chapter 2; Chapter 3), ammonia (NH3;
Taghizadeh-Toosi et al., 2012) and amino acids (Chapter 2). Consequently sorption
processes have been hypothesised to decrease nitrification, mineralisation and N
leaching (Lehmann et al., 2003; Laird et al., 2010b). In this thesis evidence is
provided to suggest that sorption of LMW N compounds (NO3-, NH4
+; NH3; and
amino acids) to biochar does not influence nitrification (Chapter 2) or mineralisation
(Chapter 2, Chapter 4) and is not the main cause of decreased N leaching with
biochar addition to soil (Chapter 3). The sorption of LMW N has been used to explain
results in previous research (e.g. Lehmann et al., 2003; Laird et al., 2010b) yet prior
to this thesis experiments examining LMW sorption had not been conducted.
Consequently results from other experiments may be interpreted differently with this
General Discussion Chapter 6
132
new finding, provided two important caveats: similar biochar chemistry and structure;
and similar biochar rates of application.
As stated in Section 1.5.1, the sorption affinity of biochar will vary between types
due to variations in surface functional groups (Boehm, 1994) and surface area
(Bornemann et al., 2007). Despite their different pyrolysis temperatures and
feedstocks, the two biochar types applied to coarse textured soils in this thesis, Jarrah
and wheat residue biochars, had a similar pH (9.3 and 9.2, respectively) and hence
may have contained similar surface functional groups and hydrophobic properties. It
could be valid to conclude that most biochar types will influence other experiments
similarly if biochars with contrasting characteristics (i.e. lower pH and more acidic
surface functional groups) to the biochar types used in this thesis had similar
influence on the sorption of LMW N compounds. Thus, further research is required to
assess whether the findings of this thesis can be validly extrapolated to contrasting
biochar types, especially with varying pH and acidic functional groups.
The application rate of biochar is likely to influence N sorption, due to the number of
functional groups introduced to soil, increasing the likelihood for sorption. Practical
application rates in broadacre agriculture in WA are likely to be less than 5 t ha-1
(Blackwell et al., 2010). Consequently, in the drier field environment, changes in
leaching with lower rates of biochar application (4 t ha-1) to coarse-textured soil may
more closely reflect the negligible changes in soil N movement down the soil profile
in Chapter 5 than the significant differences in inorganic N leaching demonstrated in
General Discussion Chapter 6
133
a well watered lysimeter experiment with 25 t ha-1 biochar addition (Chapter 3). In
other agro-ecosystems, rates of application may exceed 25 t ha-1 (e.g. Jones et al.,
2010; Sohi et al., 2010); the sorption of N to biochar with greater application rates
may increase its effect on N leaching and mineralisation.
6.1.1 Applying findings to other experiments
With these two caveats in mind, it is possible to use this new information to re-
interprete the results from some previous experiments. Lehmann et al., 2003 used an
application rate of 20% w/w biochar/soil or 191 t ha-1. Re-interpreting the results of
Lehmann et al. (2003) with this very large rate of application is unwise because if
20% of the soil was biochar; biochar is much more likely to intercept and sorb
nutrients and other compounds at this rate of application. Unfortunately only one
experiment could be found which applied biochar at similar rates and assessed LMW
N leaching. Ding et al., (2010) when applying bamboo biochar at 0.5% w/w, or
approximately 5 t ha-1 (using the conversion rates from chapter 3) state that the
decreased NH4+ leaching in their experiment was due to the sorption of NH4
+ to
biochar; but the findings in this thesis (that the sorption of LMW N to biochar is
unlikely to influence leaching) suggest otherwise. An alternate explanation that is
consistent with their findings (Ding et al., 2010) is that nitrification may have
increased, which would have also increased NO3- leaching. Although this data was
measured, it was not presented. Nitrification increases have been implied in other
agricultural eco-types (e.g. Van Zwieten et al., 2010a), although this is contrary to the
General Discussion Chapter 6
134
findings of this thesis where biochar decreased nitrification (Chapter 2), it could
explain lower NH4+ leaching.
6.1.2 Implications of the minimal impact of sorption of low molecular weight
nitrogen to biochar
In this thesis sorption of LMW N to biochar added to coarse-textured soil did not
influence nitrification and mineralisation and was not the main mechanism decreasing
N leaching, raising further research questions. Three such topics for further
investigation are: the main mechanisms decreasing inorganic N leaching with biochar
addition to a coarse textured soil; the consistency of this LMW sorption over time;
and the scale of measurement.
More research is required to pinpoint other mechanisms causing decreased inorganic
N leaching with biochar addition (discussed in Chapter 3). Increased WHC may
decrease NH4+ and NO3
- leaching by providing refuges for ions to avoid movement
by mass flow. Increased WHC should also have decreased DON leaching similarly,
but no difference in DON leaching with soil amendment was found. Thus, research
investigating other possible mechanisms is that decrease N leaching is required.
Although aging of biochar did not influence organic N mineralisation in this thesis
(Chapter 4), whether more oxidation could change the results or whether the results
from the tested agro-ecosystem are representative remains unclear. Over time the
pores of biochar may become blocked with SOM (Zimmerman et al., 2011), termed
General Discussion Chapter 6
135
protective sorption (Kaiser & Guggenberger, 2000), potentially lessening the
increased WHC with biochar addition and associated decrease in inorganic N
leaching.
This thesis measured changes in bulk soil properties, but differences may be more
likely to be measured at the soil-biochar interface, termed the ‘charosphere’ (Clough
& Condron, 2010). The concept of localisation of NO3- within rhizosphere biochar,
caused by mass flow of NO3- towards the root and sorption to rhizosphere biochar
upon interception (Prendergast-Miller et al., 2011), concurs with the findings of this
thesis. Prendergast-Miller et al. (2011) also found that biochar sorbed N,
demonstrated by greater N content in rhizosphere biochar compared to biochar in
bulk soil, but this did not translate into increased plant N uptake. Similarly, Chapter 2
demonstrated that biochar sorbed N, but the sorption was not detected in the bulk soil
when biochar was applied at paddock applicable rates. Thus sorption of LMW N to
biochar may influence N cycling processes such as N mineralisation, nitrification, N
leaching, and plant N uptake within the ‘charosphere,’ but the influence of biochar on
N cycling within the bulk soil is likely to be minimal, especially at applications rates
likely to be applied in broadacre agriculture in WA.
6.2 Sorption of high molecular weight organic nitrogen compounds to biochar
The negative priming that occurs with biochar addition to soil (Cross & Sohi, 2011;
Keith et al., 2011; Jones et al., 2011; Zimmerman et al., 2011) may be caused by the
sorption of HMW organic N or organic C to biochar. One apparent trend in
General Discussion Chapter 6
136
mineralisation with biochar addition over longer periods of time (greater than 2
weeks) is that increasing pyrolysis temperature of biochar increases its the negative
priming effect when added to soil (Cross & Sohi, 2011; Zimmerman et al., 2011).
Another trend is that the negative priming effect is more apparent in soils with high
organic matter contents (Cross & Sohi, 2011; Keith et al., 2011; Zimmerman et al.,
2011). Negative priming over the long term is thought to be due to the sorption of
organic matter (Zimmerman et al., 2011), in particular HMW organic compounds.
Biochar has been shown to strongly sorb pesticides such as simazine, atrazine and
trifluralin, decreasing pesticide leaching and efficacy (Bornemann et al., 2007; Jones
et al., 2010; Nag et al., 2011). There are multiple mechanisms that can explain
sorption of xenobiotics to biochar (Zhou et al., 2010) but common differences
between LMW N and xenobiotics are the presence of halides and polarity (Fig. 6.1).
Halides are unlikely to cause the sorption differences, because although fluorine is the
most electronegative element, oxygen is the second most electronegative element
(Allred & Rochow, 1958). Therefore oxygen (in leucine and alanine, Fig. 6.1) forms
stronger electron donor interactions than chlorine (in simazine and atrazine, Fig. 6.1).
Thus, the non-polarity of some xenobiotics caused by symmetry surrounding a
benzene or triazine ring must induce hydrophobic based interactions resulting in
greater sorptive affinity of non-polar hydrophobic molecules than polar hydrophilic
molecules. Scaled particle theory suggests that non-polar molecules aggregate to
reduce solute cavity surface area (Jackson & Sternberg, 1994) and increase molecular
entropy (Kleber & Johnson, 2010). Where Zimmerman et al. (2011) hypothesised
General Discussion Chapter 6
137
that the negative priming effect of biochar was caused by organic matter sorption; I
suggest that it is due to the sorption of HMW non-polar organic matter. Although it is
clear that organic matter sorption varies between organic matter molecules (Kasozi et
al., 2010) and with addition to soil (Cornelissen & Gustafsson, 2004), the relationship
between organic matter sorption to biochar and the molecular weight and polarity of
the molecule sorbed requires further investigation. It is likely that the greater the non-
polarity of the organic matter the greater the sorption affinity for biochar and that
correlates with the molecular size of the organic matter. Further, it is unclear whether
the sorption of the hydrophobic tail of amphiphilic organic matter molecules change
the net effect of biochar on mineralisation. It is also unclear whether similar sorption-
desorption hysteresis seen with pesticides (i.e. Yang et al., 2004), is also present in
amphiphilic or hydrophobic organic matter.
HMW compounds have been shown to sorb more strongly to biochar produced under
high pyrolysis temperatures (Kasozi et al., 2010), explaining the trend of greater
negative priming with increased pyrolysis temperature shown by Cross & Sohi
(2011) and Zimmerman et al. (2011). High pyrolysis temperature can decrease the
oxygen content of the biochar, increasing its hydrophobicity, and the accessible area
for adsorption of hydrophobic pesticides (Yang et al., 2004). Sorption of xenobiotics
may also be related to pore area of the biochar, in particular micropores providing
sites for high energy sorption (Zhou et al., 2010), where adsorption energies for high
energy sorption sites may decrease with increasing pyrolysis temperature (Pikaar et
al., 2006). Similarly, the sorption of hydrophobic organic matter compounds, like
General Discussion Chapter 6
138
catechol, increase with increasing pyrolysis temperatures (Kasozi et al., 2010)
explaining the link between higher peak pyrolysis temperatures and the negative
priming effect demonstrated by Cross & Sohi (2011) and Zimmerman et al. (2011).
Fig. 6.1: Chemistries of selected compounds studied for sorption interactions with
biochar. Simazine, trifluralin and atrazine are hydrophobic; alanine and leucine are
hydrophilic. The degree of hydrophobicity is likely to influence their sorption affinity
to biochar.
6.2.1 Implications of the sorption of high molecular weight compounds to biochar
There are a variety of implications of the sorption of hydrophobic HMW organic
matter to biochar in soil. The most important of these is decreased organic matter
mineralisation. Although decreased organic matter mineralisation will increase SOM
(Cross & Sohi, 2011), it will also decrease nutrient availability for plant N uptake.
L-alanine L-leucine
Simazine Atrazine Trifluralin
General Discussion Chapter 6
139
The rate limiting step of N mineralisation is the decomposition of HMW organic N to
LWM organic N (Jones et al., 2004). As mineralised N constitutes a large portion (up
to 90%) of total crop N uptake (Angus, 2001; Jenkinson, 2001), sorption of
hydrophobic HMW N to biochar could potentially decrease crop yields. Decreased
mineralisation causes decreased NH4+ content, which will decrease nitrification and
leaching of inorganic N. Biochar also has a varied influence on enzymatic activity,
decreasing β-glucosidase activity (Bailey et al., 2011; Lammirato et al., 2011) and
increasing β-N-glucoseaminidase activity (Bailey et al., 2011). Although enzymes
must be hydrophilic for mobility within solution, there may be areas with contrasting
electronegativity, and hence varying hydrophilicity, amphiphilicity and
hydrophobicity between enzymes which is likely to alter their sorption affinity to
biochar.
6.3 The addition of labile carbon contained in biochar
To help predict the impact of biochar on microbial abundance and activity, greater
characterisation and quantification of labile C in biochar must be conducted.
Generally, the addition of labile C in biochar increases microbial biomass (Lehmann
et al., 2011) and activity (Steiner et al., 2008a) and increasing addition of biochar and
labile C induces larger increases in microbial abundance (Kolb et al., 2009).
However, labile C is highly variable and can range from being readily microbially
available, such as amino acids and sugars (e.g. Jones & Murphy, 2007) to microbially
inhibitive, such as benzene (e.g. Girvan et al., 2004). Despite potential diversity of
labile C present on biochar, it is commonly documented as VM or volatile content;
General Discussion Chapter 6
140
ranging in concentration from 16-400 g VM kg-1 biochar (Table 1.1). A more
thorough characterisation of labile C in pine biochar has demonstrated the presence of
VOCs such as acetaldehyde, α-pinene and β-pinene (Clough et al., 2010). α-pinene
can decrease N mineralisation and MBN (Uusitalo et al., 2008). Yet, when the pine
biochar was added to soil, α-pinene did not influence N mineralisation and microbial
biomass (Clough et al., 2010) probably because of the small quantity of the α-pinene
added. Benzene has also been detected in low concentrations on biochar (Chapter 2),
although the detected concentrations were lower than previously researched known
toxic concentrations (e.g. Girvan et al., 2004). Thus, the quantity and types of labile
C present on biochar are likely to be a primary cause of the differences in N
immobilisation demonstrated with different biochar types (e.g. Gundale & DeLuca,
2007). Varying labile C through changing pyrolysis temperatures (Baldock &
Smernick, 2002; Mukherjee et al., 2011) is also likely to be partially responsible for
changes in N immobilisation with varying biochar types. It is unclear how the
abundance of specific labile compounds change with other factors of production.
Thus, greater assessment and quantification of specific labile C compounds is
required to predict the net influence of biochar on N immobilisation, net N
mineralisation and the longevity of such changes induced by biochar amendment.
General Discussion Chapter 6
141
6.4 Increasing the supply of phosphorus and potassium in biochar
Previous research has demonstrated that biochar can interact with N fertiliser,
enhancing N fertiliser use efficiency (Chan et al., 2007; Chan et al., 2008; Van
Zwieten et al., 2010a). This is likely to have been caused by biochar addition
simultaneously increasing the supply of other nutrients such as P and K (Chan et al.,
2007; Chan et al., 2008). Changes in P and K availability will vary with biochar
nutrient content and application rate. Other studies demonstrating increases in N
fertiliser use efficiency added biochar at large application rates (50 or 100 t ha-1) for
nutrient poor biochars (e.g. greenwaste; Chan et al., 2007) or lower rates (10 t ha-1)
for nutrient rich biochar (eg. poultry manure biochar; Chan et al., 2008). The nutrient
poor greenwaste biochar contained 400 mg Colwell P kg-1 (Chan et al., 2007), thus
addition at 50 t ha-1 would apply 20 kg Colwell P ha-1. The two poultry manure
biochars contained 11600 and 1800 mg Colwell P kg-1 (Chan et al., 2008);
application of 10 t ha-1 would apply 116 kg Colwell P ha-1 and 18 kg Colwell P ha-1,
respectively. If biochar is to be applied in WA, the two most likely biomass sources
are wheat chaff or Eucalyptus wood. The Jarrah biochar contained 64 mg total P kg-1
and the wheat residue biochar contained 4150 mg total P kg-1 (data not shown). Jarrah
biochar application at 25 and 5 t ha-1 applied 1.6 and 0.3 kg total P ha-1 and wheat
residue biochar application at 4 t ha-1 applied 16.6 kg total P ha-1, although the
proportion of total P that is bio-available is unknown. In WA broadacre agro-
ecosystems application rates greater than 5 t ha-1 are unlikely to be applied, because
of the low bulk density of biochar and thus higher transport and application costs (as
stated in Chapter 5). It is unlikely that increases in N fertiliser use efficiency will
General Discussion Chapter 6
142
occur in WA agro-ecosystems because not enough biochar can be applied to supply P
and K in large enough quantities to induce such a response. Results may be different
if a nutrient rich manure biochar source becomes available or application rates of
biochar can be increased.
To increase biochar application rates, and hence P and K supply, in broadacre WA
agro-ecosystems the bulk density of biochar must be increased. The low bulk density
of biochar causes logistical problems and great expense. For example, a road train,
which can carry 60 t of wheat (bulk density of 0.8 g cm-3) will carry 15 t of biochar in
the same volume, assuming a bulk density of 0.2 g cm-3 (as in Chapter 5). Thus, at an
application rate of 4 t ha-1, this volume of biochar would be applied to only 3.75 ha.
Hence achieving the high application rates that would increase the supply of P and K,
in the broadacre agricultural environment of WA would be expensive and logistically
challenging.
6.5 Water supply and water holding capacity
Adding biochar to soil has been hypothesised to increase WHC and enhancing crop
water supply in sandy soils and dry environments (Atkinson et al., 2010; Blackwell et
al., 2010). Yet when calculated, the amount of water stored appears unlikely to
greatly enhance crop yield at potential application rates in broadacre agriculture in
WA (Chapter 5). Further, in the dry field environment of the WA broadacre agro-
ecosystem movement of inorganic N through the soil profile did not change with
biochar addition (Chapter 5), but in high rainfall environments, such as Amazonia
General Discussion Chapter 6
143
(Lehmann et al., 2003; Steiner et al., 2007) and glasshouse experiments (Chapter 3),
biochar has been shown to decrease inorganic N leaching. This is likely to be due to
the mass flow of water accentuating any changes in soil properties (e.g. refuges and
pores) created by biochar addition. Higher rainfall environments also allow
dissolution and diffusion of nutrients, such as P and K. Evidence of this may have
been provided at terminal spikelet sampling of the field experiment (Chapter 5),
where spread application of biochar significantly increased plant biomass; this
coincided with the wettest period of the growing season. Thus in drier environments
the impact of biochar may be sporadic and highly influenced by rainfall. This
explains the minimal of effect of biochar seen in studies assessing its agronomic
influence in south west Australia (Blackwell et al., 2010; Solaiman et al., 2010).
6.6 No effect of biochar
Previous glasshouse studies have suggested that biochar addition to soil can both
increase N fertiliser use efficiency and plant N uptake (Chan et al., 2007; Chan et al.,
2008; Van Zweiten et al., 2010a) and decrease plant N uptake and N fertiliser use
efficiency (Lehmann et al., 2003; Deenik et al., 2010). In high rainfall field
experiments plant N uptake can also increase (Steiner et al., 2007; Major et al.,
2010), but in a semi-arid environment with wheat residue biochar application at 4 t
ha-1 no influence was apparent (Chapter 5).
Field studies in WA and South Australia have demonstrated only sporadic increases
in shoot N uptake with biochar addition (Blackwell et al., 2010; Solaiman et al.,
General Discussion Chapter 6
144
2010). Shoot N uptake with Jarrah biochar addition at 1 t ha-1 increased in one of nine
treatments in one field experiment (Blackwell et al., 2010). In a second experiment,
35 year old Jarrah and Wandoo (Eucalyptus wandoo Blakely) biochar added at rates
up to 3.3 t ha-1 did not influence shoot N uptake (Blackwell et al., 2010). On a soil
type of greater clay content (sandy clay loam), also in south-western Australia, Oil
Mallee (Eucalyptus sp.) biochar application (6, 3, 1.5 or 0 t ha-1) combined with three
fertiliser application rates (30 or 55 kg ha-1 mono-ammonium phosphate, or 100 kg
ha-1 slow release composite fertiliser) only increased shoot N uptake in one treatment
(6 t biochar ha-1, 30 kg mono-ammonium phosphate ha-1; Solaiman et al., 2010).
Combined, the results of Blackwell et al. (2010), Solaiman et al. (2010) and Chapter
5 suggest that biochar is unlikely to increase plant N uptake in the drier areas of
broadacre agriculture within WA.
For agricultural research, there is a need for biochar based experiments to grow plants
to maturity to properly assess the impact of biochar on plant yield. Within WA and
South Australia, although some yield increases (Blackwell et al., 2010; Solaiman et
al., 2010) and yield decreases (Chapter 5) have been demonstrated, the majority of
the results from field experiments show no effect of biochar addition (Blackwell et
al., 2010; Solaiman et al., 2010; Yield results from year 2 of the field experiment in
Chapter 5 - data not shown). Thus biochar is unlikely to influence wheat yield in the
semi-arid broadacre field environment of WA.
General Discussion Chapter 6
145
6.7 Conclusion
This thesis tested the hypothesis that biochar addition would influence agronomically
important soil N cycling processes. Limited response in N leaching, N mineralisation,
nitrification, N immobilisation, and plant N uptake is likely to occur with
economically/agronomically viable application rates of biochar to coarse-textured
soils used for broadacre agriculture in WA. This is because sorption of LMW N
compounds to biochar added to soil is unlikely to influence N cycling possibly due to
the low application rates. Low application rates also ensure that the addition of P and
K in biochar is also unlikely to increase plant N uptake or nutrient use efficiency.
Where greater application rates can be applied, a high sorption affinity for
hydrophobic HMW organic matter may retard organic matter mineralisation,
decreasing nutrient, especially N, availability to crop plants. Findings reported within
this thesis suggest that biochar is unlikely to influence N cycling for the improvement
of crop productivity on coarse-textured soils in broadacre agriculture of WA.
146
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