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The effect of biochar loading rates on soil fertility, soil biomass, potential
nitrification, and soil community metabolic profiles in three different soils

Article in Journal of Soils and Sediments · September 2016

DOI: 10.1007/s11368-016-1411-8

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J Soils Sediments
DOI 10.1007/s11368-016-1411-8

SOILS, SEC 2 • GLOBAL CHANGE, ENVIRON RISK ASSESS, SUSTAINABLE LAND USE • RESEARCH
ARTICLE

The effect of biochar loading rates on soil fertility, soil biomass, potential
nitrification, and soil community metabolic profiles
in three different soils

Ibrahim S. Abujabhah1 & Richard Doyle1 & Sally A. Bound1 & John P. Bowman1

Received: 1 December 2015 /Accepted: 18 March 2016

# Springer-Verlag Berlin Heidelberg 2016

Abstract
Purpose Biochar is increasingly being used as a soil amend- ment to both increase
soil carbon storage and improve soil chemical and biological properties. To better
understand the shorter-term (10 months) impacts of biochar on selected soil
parameters and biological process in three different textured soils, a wide range
of loading rates was applied.
Materials and methods Biochar derived from eucalypt green
waste was mixed at 0, 2.5, 5, 10 % (wt/wt) with a reactive black clay loam (BCL), a
non-reactive red loam (RL) and a brown sandy loam (BSL) and placed in pots exposed
to the natural elements. After 10 months of incubation, analysis was performed to
determine the impacts of the biochar rates on the different soil types. Also,
microbial biomass was estimated by the total viable counts (TVC) and DNA
extraction. Moreover, potential nitrification rate and community metabolic profiles
were assayed to evaluate microbial function and biological process in biochar-
amended soils.
Results and discussion The results showed that biochar addi-
tions had a significant impact on NH4 and NO3, total C and N, pH, EC, and soil
moisture content in both a soil type and loading-dependent manner. In the heavier
and reactive BCL, no significant impact was observed on the available P and K
levels, or the total exchangeable base cations (TEB) and CEC. However, in the other
lighter soils, biochar addition had a significant effect on the exchangeable Al,
Ca, Mg, and Na

Responsible editor: Hailong Wang

* Ibrahim S. Abujabhah
Ibrahim.Abujabhah@utas.edu.au

1 Tasmanian Institute of Agriculture, University of Tasmania, Private

Bag 98, Hobart, Tasmania 7001, Australia
levels and CEC. There was a relatively limited effect on mi- crobial biomass in
amended soils; however, biochar additions and its interactions with different soils
reduced the potential nitrification at the higher biochar rate in the two lighter
soils. Community metabolic profile results showed that the effect of biochar on
carbon substrate utilization was both soil type and loading dependent. The BCL and
BSL showed reduced rates of substrate utilization as biochar loading levels
increased while the opposite occurred for the RL.
Conclusions This research shows that biochar can improve
soil carbon levels and raise pH but varies with soil type. High biochar loading
rates may also influence nitrification and the function and activity of microbial
community in ligh- ter soils.

Keywords Biochar . Community metabolic profiles . Nitrification . Soil biomass .

Soil fertility

1 Introduction

Biochar has emerged as a commercially available amendment for improvement of soil

health via potentially enhanced soil physical and chemical fertility. Biochar
consists of porous aromatic stable carbon structures which are highly resistant to
chemical and microbial degradation (Glaser et al. 2001). As such, they are capable
of sequestering carbon in soil (Rondon et al. 2005). As biochar amendment can
affect the soil microbial population and its activity, the biochar loading rates
and the soil characteristics themselves needs to be stud- ied to monitor responses
in the biochar-amended soils (Lehmann et al. 2011). While biochar has a high carbon
to nitrogen ratio (C/N ratio), making it a poor source of labile nutrients, it does
have a high surface area that can increase cation exchangeable capacity (CEC), pH,
nutrient retention,
J Soils Sediments

and water holding capacity, most especially in sandy textured soils (Lehmann et al.
2006).
Biochar addition can have a significant impact on the chem- ical and physical
properties of soil which then affect the bio- chemical processes and microbial
functions in soil. However, these relationships between microbial activity,
biological pro- cesses, and changes in chemical and physical properties are still
not well understood. Because biochar may react differently in soils having
different physiochemical properties, varying the loading rate ought to show how
various soil physiochemical and biological properties are subsequently impacted.
Thus, our aim was to evaluate the effect of different loading rates of bio- char in
three very different soils. Biochar can potentially change soil physical properties
such as soil structure, field texture, po- rosity, and density, and these affect
soil aeration, water holding capacity, and hence, microbial activity (Atkinson et
al. 2010).
Biochar can improve the habitat for soil microorganisms and plant roots (Atkinson
et al. 2010) as its application can also enhance moisture retention in lighter
soils and water in- filtration and drainage in heavier soils. Thus, improving the
physical characteristics in soil can enhance chemical interac- tions that support
microbial activity. Because biochar is resis- tant to microbial degradation, these
improvements can persist for a long time. Many studies also indicate that better
under- standing of biochar effects in different soil types would assist in using it
for more effective management of different soil properties and so gain the maximum
benefit from its applica- tion (Sohi et al. 2010).
Biochar can alter the biological processes in soil such as N mineralization and
nitrification by affecting bacterial commu- nities involved in these processes as
well as providing a suit- able environment for overall increased microbial activity
(Berglund et al. 2004). It has been documented that biochar constitutes a large
percentage of organic carbon in various soils but the exact nature of this
component is still not well understood (Zimmerman 2010). Kolb et al. (2009) studied
the effect of biochar addition on microbial biomass and activity; biochar was added
to four different soils: a Mollisol, Alfisol, Entisol, and Spodosol at five
application rates from 0 to
0.1 kg/kg biochar–soil. The results showed a significant in- crease in both
microbial biomass and activity with increasing application rates. The study also
showed similar patterns of biochar impact on microbial biomass, microbial activity,
and nutrient availability in all four soils but the microbial response was diverse,
dependent on the differences of nutrient avail- ability in each soil (Kolb et al.
2009). Also, Butnan et al. (2015) investigated the additions of two types of
eucalyptus wood-derived biochars at four rates (0, 1, 2, and 4 % w/w) added to a
sandy Ultisol and a clayey Oxisol. The results indicated that liming was the most
beneficial aspect of biochar additions in both soils, especially in sandy Ultisols,
which have low fertility. The most beneficial loading rate observed was between 1
and 2 % w/w (Butnan et al. 2015).
The capacity of biochar to amend soil will depend on the type and rate of biochar
and the impact it has in a given timescale (Unger et al. 2011). Biochar amendment
and its high surface area is often correlated with CEC enhancement which may
increase the availability and use efficiency of plant nutri- ents in some soils
depending on biochar specification. Thus, low CEC soils ought to be those most
impacted. Also, biochar can potentially increase pH in soil, which subsequently
influ- ences many of the nutrient transformations and their availabil- ity to
plants, particularly in acidic soils (Fowles 2007). However, soil pH may either
increase or decrease depending on the inherent pH and lime content of the biochar
itself (Lehmann et al. 2011). In general, biochar and other organic amendments
added to soil should increase plant available nu- trient storage in the rhizosphere
(Steiner et al. 2007). Biochar has been shown to significantly enhance crop yield
and qual- ity via improved nutrient supply (Steiner et al. 2007, Unger et al.
2011). Although there is much evidence highlighting advantages in using biochar as
a soil amendment, the interac- tion between biochar in different soil types needs
to be ex- plored to understand the specific interactions and processes.
Therefore, understanding the interaction between biochar loading rates and the
microbial community therein is needed to better model the effect of biochar and its
implications on soil function. This is also needed in a wide range of soil types as
prior works suggest sandier and poorer soils are those that are most improved by
biochar additions (Atkinson et al.
2010).

2 Materials and methods

2.1 Soil collection and processing

Three different topsoils (0–20 cm depth) were collected for this experiment. Red
loam Dermosol topsoil (clay 21.61 %, silt 22.81 %, and sand 55.58 %) was collected
from a farm near Cambridge, Tasmania (42° 48.11.77′ S 147° 26.22.03′ E). Brown
sandy loam Kurosol topsoil (clay 10.43 %, silt
9.43 %, and sand 80.14 %) was collected from the headland in an apple orchard at
Mountain River, located in the Huon Valley region of southern eastern Tasmania (42°
57.2.91′ S
147° 55.2.13′ E). Black clay loam Vertosol (clay 28.67 %, silt
24.35 %, and sand 46.98 %) topsoil was collected from the forested slopes of Mt
Nelson near Bend 3 of Mt Nelson Road, located near the University of Tasmania in
Hobart, Tasmania (42° 54. 25.92′ S 147° 19.22.35′ E). All soils were placed in
plastic containers and taken to the laboratory for immediate water content
measurements. Subsamples of the soils were kept frozen at −20 °C for biological
analysis. The remaining soils were prepared by removing gravel before being mixed
with biochar for the subsequent pot experiment.
J Soils Sediments

2.2 Biochar specifications

Biochar used in this experiment was sourced from eucalypt green waste (Black Earth
Products, Qld, Australia). The bio- char was produced in an updraft rotary hearth
gasifier operat- ing with a peak temperature of 650–750 °C (feedstock depen- dant)
with oxygen-limited atmosphere and residence times not longer than 3 min (most
typically around 100 s). A typical chemical profile of this biochar is shown in
Table 1. Biochar analysis was conducted by Diagnostic and Analytical Services (DAS)
in the Department of Primary Industries, Wollongbar NSW 2477 Australia.

2.3 Experimental design and biochar addition

Biochar was added to the three soils described above at 0, 2.5,

5, 10 % (wt/wt), specifically 0, 32.5, 65, and 130 g of biochar to a total of 1300
g of soil, thoroughly mixed and filled into
1.5-l pots. Each biochar–soil combination was replicated four

Table 1 Chemical analysis illustrated the specification of biochar produced at

Black Earth Products, QLD Australia

Parameters
Biochar

EC (dS/m)
0.27 pH (CaCl2)
7.3
Total nitrogen (%)
0.26
Total carbon (%)
79
KCl extractable ammonium-N (mg/kg) <0.3
KCl extractable nitrate-N (mg/kg)
0.41
CaCO3 (%)
2.4
Total phosphorus (mg/kg)
390
Water soluble phosphorus (%) 64
Citrate insoluble phosphorus (mg/kg) 230
Citrate soluble phosphorus (mg/kg) 90
Available phosphorus (mg/kg) 150
Aluminium (meq/100g)
<0.1
Calcium (meq/100g)
9.2
Potassium (meq/100g)
2.9
Magnesium (meq/100g)
1.8
Sodium (meq/100g)
1.1
CEC (meq/100g)
15
Calcium/magnesium ratio
5.2
Boron (mg/kg)
6.1
Chromium (mg/kg)
450
Copper (mg/kg)
12
Iron (%)
0.71
Manganese (mg/kg)
180
Sulfur (mg/kg)
80
Zinc (mg/kg)
200
times to give a total of 48 pots. Pots were arranged randomly and left exposed to
the natural elements for 10 months outside the teaching glasshouse complex at the
School of Land and Food at UTAS. The annual mean maximum and minimum temperature is
19.9 and 8.3 °C, respectively, while the mean annual rainfall is 613.3 mm (Hobart
Ellerslie Road Weather Station, Lower Derwent Tasmania).

2.4 Chemical and physical analysis

After 10 months of incubation, soil samples were taken from the pots for
gravimetric water content measurements and chemical analysis. Soil chemical
analysis was conducted by CSBP Laboratories, Western Australia. Properties analyzed
included organic carbon (Walkley-Black), total nitrogen, available phosphorous
(Colwell), available potassium (Colwell), sulfur (KCl 40), ammonium nitrogen,
nitrate nitro- gen, electrical conductivity, pH (H2O) 1:5, pH (CaCl2) 1:5,
micronutrients (DTPA: copper, zinc, manganese, iron) and boron (Hot CaCl2),
exchangeable cations (calcium, magne- sium, sodium, potassium, aluminum), and
particle size (pi- pette and sieving method). Chemical and physical data were
statistically analyzed by multivariate analysis of variance (MANOVA) using SPSS v22
to assess the effect of biochar addition on soil properties compared to the
unamended con- trol treatments.

2.5 Soil biomass assessment

Soil bacterial numbers as total viable counts (TVCs) were estimated from colony
forming units (CFUs) on 10 % tryptone soy agar plates incubated at 25 °C for 21
days (Juhnke et al.
1987). Soil biomass was estimated from extracted DNA (n =4 replicates for each
treatment) with quantities estimated via the Nanodrop spectrophotometer instrument
(NanoDrop 8000
Spectrophotometer, Thermo Fisher Scientific Inc., Wilmington, DE, 19810, USA) which
was used as an alterna- tive method to estimate microbial biomass (Marstorp et al.
2000; Bouzaiane et al. 2007).

2.6 Potential nitrification assay

The potential nitrification was assayed by using ammonium sulfate as a substrate as

described by Kandeler (1995); soil samples from each treatment replicate were
incubated at
25 °C for 5 h. After incubation, the nitrite released during
the incubation was extracted with potassium chloride and de- termined using
spectrophotometry at 520 nm using a SPECTRO star Nano plate reader (BMG Labtech,
Mornington, VIC, Australia).

2.7 Soil community level metabolic profiles

Microbial metabolic capacity of the soil communities was determined by examining

the ability to utilize a variety of carbon sources. This was done using Biolog
EcoPlates as described by Garland and Mills (1991) and Frąc et al. (2012). Eco
Plates with 96 wells containing 31 triplicated carbon sources and a control
(water) were prepared by suspending 1-g soil in 99-ml sterilized water, and 120 μl
of each soil suspension then aliquoted into each well. Plates were incubated at 25
°C for 7 days. Absorbance at 590 nm from all
96 wells was measured daily using a SPECTROstar Nano plate reader. The data was
compiled and analyzed in the pro- gram PRIMER 6 + PERMANOVA (Primer-E Ltd, Plymouth
UK) using analysis of similarity (ANOSIM), canonical anal- ysis of principal
coordinates (CAP), and permutation analysis of variance (PERMANOVA). For this the
data was square root transformed and converted to Bray-Curtis similarity values.
ANOSIM was used for a priori analysis of factors (biochar loading, soil type, time
of incubation) followed by PERMANOVA analysis using unrestricted permutation of the
data with 9999 permutations assuming a type III (unbalanced) design. The rates and
patterns of the overall microbial carbon source utilization were expressed by the
av- erage well-color development (AWCD), richness (R), and Shannon–Weaver index (H)
(Garland 1997, Gomez et al.
2004). To reduce the complexity of interpreting the data, car- bon substrates were
divided into five groups: (1) carbohy- drates; (2) carboxylic and acetic acids; (3)
amino acids; (4) polymers; and (5) amines and amides according to Weber and Legge
(2009) and presented as a percentage of the total absor- bance values for each
treatment.

3 Results

3.1 Chemical and physical properties

The addition of different rates of biochar to different soils had a significant

impact on selected chemical and physical prop- erties in soil. These differences
were dependent on the soil type and the amount of biochar added to that soil.

3.1.1 Ammonium and nitrate nitrogen in soil

The interaction between soil type and biochar loading rate had a significant
negative impact on the exchangeable ammonium (p = 0.02) while positive impact on
soluble nitrate (p = 0.012) contents. In the black clay loam (BCL), the
exchangeable ammonium decreased by 32, 53, and 61 % respectively, with increasing
biochar loading rates (2.5, 5, and 10 %) compared to the untreated control as shown
in Fig. 1a. While on the other hand, the NO3–N content increased dramatically by
103, 110,
J Soils Sediments

A
35
30
25
20
Black Clay Loam
15
Red Loam
10
Brown Sandy Loam
5
0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)

B
35

30

25
20
Black Clay Loam
15
Red Loam
10
Brown Sandy Loam

0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)
Fig. 1 a Ammonium nitrogen (mg/Kg) and b nitrate nitrogen (mg/Kg) in a black clay
loam (BCL), red loam (RL), and brown sandy loam (BSL) soil containing different
biochar loading rates (0 to 10 % wt/wt). Errors bars are standard deviation from
four replicate soil pots

and 207 % with increasing biochar loading rates (Fig. 1b). The same trends but to a
lesser extent was observed with the brown sandy loam (BSL) and red loam (RL) soils,
especially when the ammonium levels were higher as in the 10 % biochar
applications. However, no significant differences in soluble nitrate contents were
observed between the biochar treatments in the BSL or RL topsoils.

3.1.2 Total carbon and total nitrogen

Biochar loading rates had a very significant effect on the total carbon in all
three soils (p < 0.001). The total carbon increased with increasing amount of
biochar added to each soil as shown in Fig. 2a. There was no significant effect on
the total nitrogen in the BCL or the BSL after the biochar applications, whereas
the results illustrated that the additions of different loading rates of biochar to
the RL significantly reduced the total nitro- gen (p = 0.007) content after 10
months (Fig. 2b).

3.1.3 Colwell phosphorous and potassium

There was no significant impact of biochar loading rates on the Colwell phosphorous
and potassium in the RL; however, the biochar additions did affect the phosphorous
in the BSL (p = 0.041) and potassium in the BCL (p = 0.028) (Fig. 3). In
J Soils Sediments

A
14

12

10

Black Clay Loam

Red Loam
Brown Sandy Loam

treatment compared to the untreated control (Fig. 3a). The Colwell potassium
content in the BCL increased gradually from 174 mg/kg in the control treatment
(biochar 0 %) to reach 204 mg/kg at the highest level of biochar loading (10 %).
3.1.4 Soil pH and electric conductivity

B
0.6

0.5

0.4

0.3

0.2

0.1

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Black Clay loam

Red Loam
Brown Sandy Loam

Soil pH (Fig. 4) was significantly increased in the BSL from pH (CaCl2) 4.50 in the
control treatment to 4.60, 4.72, and
4.83 at the 2.5, 5, and 10 % biochar loading rates respectively
(p = 0.001). The same pattern was found in the RL where the pH level was 4.60,
4.67, 4.90, and 4.98 at the 0, 2.5, 5 and
10 % biochar rates, respectively (p < 0.001). In the BCL, the effect of biochar
loading rate was less significant (p = 0.037) compared to the BSL and RL soils. The
pH level increased from 4.90 at the 0 % biochar treatment to 5.00 at the higher
loading rates 10 % biochar. No significant differences were observed in EC in the
BCL or the BSL while a slight decrease was observed in the RL within the biochar
treatments (p = 0.049) as shown in Fig. 5.
Fig. 2 a Total carbon (%) and b total nitrogen (%) in a black clay loam (BCL), red
loam (RL), and brown sandy loam (BSL) soil containing different biochar loading
rates (0 to 10 % wt/wt). Errors bars are standard deviation from four replicate
soil pots

the BSL, Colwell phosphorous decreased with the 2.5 and 5 %

biochar treatments but increased with the 10 % biochar

A
20
18
16
14

3.1.5 Soil moisture content

There was a significant impact of different biochar levels on water content in the
all three soils (p = 0.016), especially at
10 % biochar treatments in the RL and BSL (Fig. 6). Soil water content was
increased by 10 % in the BCL at the higher rate of biochar, while no significant
increase was observed at
2.5 and 5 % biochar. The BSL and RL showed the same trend where the water content
was increased by 22 % in the BSL and by 19 % in the RL at 10 % biochar compared to
the
12
10
8
6
4
2
0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)
Black Clay Loam
Red Loam
Brown Sandy Loam
corresponding control.

3.1.6 Exchangeable cations and CEC

The results in Table 2 show no significant effects were mea- sured in the level of
exchangeable cations in the BCL except

B
800
700
600
500
400
300
200
100
0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)

Black Clay Loam

Red Loam
Brown Sandy Loam

5.1
5
4.9
4.8
4.7
4.6
4.5
4.4
4.3
4.2
4.1

y = 0.0325x + 4.85
R² = 0.786

y = 0.135x + 4.45
R² = 0.9529
y = 0.11x + 4.3875
R² = 0.9979

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Black Clay Loam

Red Loam
Brown Sandy Loam
Fig. 3 a Colwell phosphorus and b Colwell potassium (mg/Kg) in a black clay loam
(BCL), red loam (RL), and brown sandy loam (BSL) soil containing different biochar
loading rates (0 to 10 % wt/wt). Errors bars are standard deviation from four
replicate soil pots
Fig. 4 Soil pH (CaCl2) in ab clay loam (BCL), red loam (RL), and brown sandy loam
(BSL) soil containing different biochar loading rates (0 to
10 % wt/wt). Errors bars are standard deviation from four replicate soil pots
J Soils Sediments

0.1

0.08

0.06

0.04

0.02

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Black Clay Loam

Red Loam
Brown Sandy Loam

Biochar addition had no significant impact on manganese in the RL. The biochar
levels significantly increased zinc in both the black clay and sandy loams (p <
0.001), but no differences were observed in the RL. Copper and iron contents in
soils varied dependent on the soil type and biochar level treatments. In the BCL
topsoil, no significant impact was found on the copper content while iron was
significantly different between
Fig. 5 Electric conductivity (ds/m) in a black clay loam (BCL), red loam (RL) and
brown sandy loam (BSL) soil containing different biochar loading rates (0 to 10 %
wt/wt). Errors bars are standard deviation from four replicate soil pots

for potassium which increased significantly by 8 and 16 % at 5 and 10 % biochar

application levels (p = 0.001). On the other hand, exchangeable aluminum, calcium,
and sodium were significantly affected by the addition of different biochar levels
in both the BSL and the RL. Exchangeable calcium and sodium were increased by 19
and 28 % in the BSL and by 9 and 14 % in the RL, respectively, at 10 % biochar
appli- cation levels, whereas the exchangeable aluminum decreased by 68 % in the
BSL and by 66 % in RL at 10 % biochar rates compared to untreated control.
Exchangeable magnesium in- creased slightly (p = 0.02) in the RL soil with the
higher bio- char loading rates, but no significant impact was found in either the
BCL or the BSL. The results in Table 2 also indi- cated that biochar addition had a
potential impact on the CEC. The main differences were found in the BSL where the
CEC increased by 4 % at 2.5 % biochar treatment, 7 % at 5 % biochar, and 14 % at 10
% biochar loading rate. CEC was slightly increased in the RL mainly in the 10 %
biochar treat- ment while no differences were found between biochar appli- cation
rates in the BCL.
3.1.7 Micronutrients

The manganese level (Table 3) was decreased by biochar ap- plication in BCL (p =
0.044) and in the BSL (p < 0.001).

45
40
35
30
25
biochar treatments as shown in Table 3. Iron increased by 6 % in the 2.5 % biochar
treatment and decreased by 13 % at 10 % biochar rates (Table 3). Significant
differences were found for copper (p = 0.019) and iron (p = 0.002) content in the
BSL; copper content decreased by 16 % while iron decreased by
19 % at 10 % biochar level compared to the control. No sig- nificant effect was
found on the iron content in the RL; how- ever, slight differences in copper were
found (p = 0.038) be- tween biochar treatments, with copper content reduced by
10 % in the RL at 10 % biochar treatment compared to un- treated control (Table 3).
Biochar loading rate had a highly significant impact (p < 0.001) on the boron
content in the BSL but no differences were found in the other two soils. The
results in Table 3 show that boron content increased by
19 % at 2.5 biochar, 32 % at 5 % biochar, and by 40 % at
10 % biochar treatments in the BSL.

3.2 Microbial biomass

The TVC results (Fig. 7) showed no significant effect of bio- char loading rate for
the BCL. Furthermore, no effect was observed on TVCs in BSL at the two lower
biochar rates but the addition of biochar at 10 % reduced TVCs compared to
controls. On the other hand, biochar addition enhanced TVCs at 2.5 and 5 % but not
at the 10 % biochar levels in the RL compared to the untreated control. The higher
rate of biochar reduced TVC in the BSL soil.
Soil microbial biomass was also estimated from the amount of DNA extracted at the
beginning of the experiment and after
10 months of incubation with different levels of biochar
(Fig. 8). The amount of DNA extracted from the BCL and BSL was higher in the 10 %
biochar (approximately 21 ng/ μl for both soils) compared to zero time (17.6 ng/μl
from BCL and 13.0 ng/μl from BSL) and 0 % biochar treatments (19.2 ng/μl from BCL
and 16.32 ng/μl from BSL). The amount of DNA extracted from the RL soil was
significantly higher compared to the BCL and BSL; the higher quantity of
DNA was observed in soil taken from the 0 % biochar
20
15
10
5
0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loadin rates (%)
Black Clay Loam
Red Loam
Brown Sandy Loam
(25.0 ng/μl) to 5 % biochar (26.2 ng/μl) compared to initial samples.

3.3 Potential nitrification

Fig. 6 The effect of biochar loading rates (0 to 10 % wt/wt) on moisture content
(%) in a black clay loam (BCL), red loam (RL), and brown sandy loam (BSL) soil.
Errors bars are standard deviation from four replicate soil pots
Biochar and its interaction with the different soils had a sig- nificant impact on
potential nitrification, which was used to estimate the abundance and activity of
the ammonium
J Soils Sediments

Table 2 The effect of biochar loading rates (0 to 10 % wt/wt) on exchangeable

cations (Ca, Mg, Na, K, Al) and cation exchangeable capacity (CEC) in a black clay
loam (BCL), red loam (RL), and brown sandy loam (BSL) soil

Soil type Biochar loading rates (%)

Exc. Al
(meq/100g)
Exc. Ca
(meq/100g)
Exc. Mg
(meq/100g)
Exc. K (meq/100g)
Exc. Na
(meq/100g)
CEC (meq/100g)

BCL 0 % 0.119
21.01 9.43 0.42
0.29 31.14
2.5 % 0.108 20.71
9.29 0.42 0.26
30.67
5 % 0.111 20.67
9.27 0.45* 0.25
30.65
10 % 0.097 21.11
9.29 0.48** 0.26
31.14
RL 0 % 0.158
8.99 2.68 1.51
0.14 13.33
2.5 % 0.085** 9.42
2.74 1.48 0.14
13.77
5 % 0.089** 9.44
2.80** 1.53 0.15
13.93*
10 % 0.054** 9.81**
2.77* 1.51 0.16*
14.24** BSL 0 % 0.186
2.91 1.34 0.21
0.07 4.54
2.5 % 0.134** 3.05
1.37 0.23 0.08
4.73
5 % 0.091** 3.19**
1.37 0.23 0.09**
4.88**
10 % 0.060** 3.48**
1.39 0.23 0.09**
5.19**

**High significant between means (p < 0.01); *Significant between means (p < 0.05)
oxidizer bacteria and archaea (p = 0.011). Generally, the BSL showed the highest
nitrification rates compared to BCL and RL except at the 10 % biochar treatment
level (Fig. 9). Potential nitrification seemed to have the same patterns in the RL
and BSL where nitrification rates increased at the
2.5 % biochar treatments compared to control (0 % biochar). However, no significant
differences were observed between different biochar loading rates in the BCL.

3.4 Community level metabolic profiles

Biolog EcoPlates were used to evaluate whether biochar load- ing rate and soil type
influence microbial communities in the
utilization rates of different carbon substrates. PERMANOVA analysis revealed
significant differences between substrate uti- lization in relation to biochar
loading rates in a soil type- dependent manner. Both biochar loading rate and soil
type strongly interacted (p = 0.0001, F = 5.59) while no interaction occurred with
time of incubation (p > 0.13). The BCL and BSL showed reduced rates of substrate
utilization as biochar loading levels increased while the opposite occurred for the
RL. In the latter case, there was pronounced increase in the utilization of plant
decomposition substrates and lipid analogs, including α-cyclodextrin, cellobiose,
xylose, methyl-β-gluco- side, Tween 40, and Tween 80. The AWCD (Fig. 10b) and R
(Fig. 10c) showed the same patterns where the C substrate

Table 3 The effect of biochar loading rates (0 to 10 % wt/wt) on soil

micronutrients (Mn, Zn, Cu, Fe, B) in a black clay loam (BCL), red loam (RL), and
brown sandy loam (BSL) soil

Soil type Biochar loading rates (%)

DTPA
Mn (mg/kg)

DTPA
Zn (mg/kg)

DTPA
Cu (mg/kg)

DTPA
Fe (mg/kg)

Hot CaCl2
B (meq/100g)

BCL 0 % 58.07
20.93 2.71 191.09
1.05
2.5 % 45.51 21.98
2.66 201.88 0.97
5 % 40.71 23.21
3.01 191.93 1.06
10 % 34.12* 25.81**
3.08 167.18* 1.05
RL 0 % 37.06
6.23 3.19 197.78
0.97
2.5 % 35.43 11.84
3.23 204.54 0.96
5 % 34.27 9.45
3.08 212.79 0.95
10 % 32.23 18.04
2.86* 190.81 1.01
BSL 0 % 9.43
2.68 2.62 108.39
0.29
2.5 % 7.41* 3.74
2.26 100.47 0.34*
5 % 6.49** 4.26*
2.21* 98.15 0.38*
10 % 5.43** 6.38**
2.20* 87.37* 0.40**

**High significant between means (p < 0.01), *Significant between means (p < 0.05)
J Soils Sediments

Fig. 7 Microbial enumeration of bacteria in a black clay loam (BCL), red loam
(RL), and brown sandy loam (BSL) soil containing different biochar loading rates (0
to 10 % wt/wt). Counts are from plates assessed after 1 week and after 2 weeks.
Errors bars are from counts from three replicate soil pots
9.5
9
8.5
8
7.5
7
6.5
6
5.5
5

Black Clay Loam

Red Loam
Brown Sandy Loam

Biochar level in soil (plate count incuba on me)

utilization declined with increasing biochar loading rates in the BCL and BSL
compared to the control; however, C utili- zation in the RL increased with
increasing rate of biochar compared to control. No significant differences were
observed between treatments in terms of numbers of substrates utilized estimated
from the Shannon–Weaver index (H) in all the trial soils (Fig. 10a); however, there
was strong correlation be- tween H, AWCD, and R in the BCL (r = 0.65–0.92). Also,
there was strong correlation between AWCD and R in the RL (r= 0.91) and BSL (r=
0.98). A comparison between the five major carbon substrate groups as shown in Fig.
11 indi- cated that the carbon utilization had the same patterns among treatments
in all soils, suggesting that there are no large chang- es occurring in the
microbial community. However, there is difference in carbon utilization especially
in the amines and
4.1 Soil chemical parameters

Biochar application had a significant impact on soluble am- monium and nitrate
content in the three soils as measured at the end of the 10-month experiment. The
ammonium de- creased while nitrate increased with increasing biochar rate. This
might be explained by improved soil conditions in the
10 % biochar loading, including measured improvements in soil pH and soil moisture
content, might have meant that there was little mineralizable ammonium present
after 10 months as it was all converted to nitrate. While a potential nitrification
test showed that the highest biochar rates had significantly lower nitrification
rates in the two lighter soils, nitrification was otherwise unaffected or increased
with biochar. Biochar produced by higher temperature (>600 °C) has the potential to
amides in BCL (Fig. 11a) and polymers in the RL (Fig. 11b)
adsorb both NH4+ and NO3
ions due to net negative and
compared to the control which may indicate that biochar ad- dition changes the
functional diversity among treatments.
positive surface charges on biochar (Dempster et al. 2012b). However, it is
commonly reported that most biochars have a
+
dominantly negative surface charge and hence NH4
retention

4 Discussion

Although only one biochar type was examined in this study, the results have shown
that biochar loading rate can affect soil physical, chemical, and biological
properties, but it is also influenced by soil type.
capacity (Lehmann et al. 2011). The ability of biochar to ad- sorb elements will
more likely depend on the nutrient and biochar properties (Yao et al. 2012).
The increase in total carbon with increasing application
rates in all soils was predictable as a result of the high C input with biochar
application having a C/N ratio of only 303. According to Lehmann et al. (2011) and
Nelson et al. (2011), addition of biochar tends to rapidly increase

35
30
25
20
15
10
5
0
Zero me Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)

Black Clay Loam

Red Loam
Brown Sandy Loam

5000
4500
4000
3500
3000
2500
2000
1500
1000
500
0

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Black Clay Loam

Red Loam
Brown Sandy Laom
Fig. 8 Soil microbial biomass as DNA extracted from a black clay loam (BCL), red
loam (RL), and brown sandy loam (BSL) soil containing different biochar loading
rates (0 to 10 % wt/wt) with quantities estimated via the Nanodrop
spectrophotometer. Errors bars are standard deviation from four replicate soil
pots
Biochar loading rates (%)
Fig. 9 Potential nitrification rates (ng NO2-N/g Soil/5 h) in a black clay loam
(BCL), red loam (RL), and brown sandy loam (BSL) soil containing different biochar
loading rates (0 to 10 % wt/wt). Errors bars are standard deviation from four
replicate soil pots
J Soils Sediments

A
7
6
5
4
3
2
1
0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)

Black Clay Loam

Red Loam
Brown Sandy Loam
A
100

80

60

40
20

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Amines & amides

Amino acids
Carboxylic & ace c acids
Carbohydrates
Polymers

B
0.025

0.02

0.015

0.01

0.005

C
35
30
25
20
15
10
5
0
Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%
Biochar loading rates (%)

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Black Clay Loam

Red Loam
Brown Sandy Loam

Black Clay Loam

Red Loam
Brown Sandy Loam
B
100

80

60

40

20

C
100

80

60
40

20

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Biochar 0% Biochar 2.5% Biochar 5% Biochar 10%

Biochar loading rates (%)

Amines & amides

Amino acids
Carboxylic & ace c acids
Carbohydrates
Polymers

Amines & amides

Amino acids
Carboxylic & ace c acids
Carbohydrates
Polymers
Fig. 10 The effect of biochar loading rates (0 to 10 % wt/wt) on carbon source
utilization presented as a Shannon–Weaver index (H), b average well-color
development (AWCD), and c richness (R) in a black clay loam (BCL), red loam (RL),
and brown sandy loam (BSL) soil. Errors bars are standard deviation from three
replicates
Fig. 11 Comparison of total carbon source utilisation between amines and amides,
amino acids, carboxylic and acetic acids, carbohydrates and polymers in a black
clay loam (BCL), b red loam (RL), and c brown sandy loam (BSL) soil containing
different levels of biochar (0 to 10 % wt/wt)

decomposition rates of labile soil carbon, which requires utilisation of soil N;

this may be the reason for the observed reduction in total N in the RL. There was
also a significant decrease in the organic carbon, as measured by the Walkley-
Black method, values in the BC at all rates of biochar.
The results also showed an improvement in CEC after bio- char addition in the two
lighter soils (RL and BSL) which has been reported in the literature (Uzoma et al.
2011). However, there was no consistent improvement in available soil phos- phorous
and potassium with biochar amendment. The effect of biochar on nutrient
availability is influenced by biochar types (feedstock and pyrolysis time and
temperature) and its interaction with different soils. An experiment conducted by
Uzoma et al. (2011) to investigate the impact of cow manure biochar on maize yield,
nutrient uptake and physiochemical properties of a dryland sandy soil showed that
cow manure biochar significantly increased soil pH, total C, total N, Oslen-
P, exchangeable cations, and CEC in the soil. The lack of any consistent positive
impacts of biochar loading rate on avail- able P and K levels observed in our study
probably relates to the initial P and K content in this soil and the low P and K
levels in the biochar.
It has been widely documented that biochar amendment increases soil pH in soil
(Kimetu et al. 2008; Chintala et al.
2014). The results of this study indicated that biochar addi-
tions increased soil pH with increased loading rates, especially in the RL and BSL
soils. This increase in soil pH with increas- ing biochar application rates was
expected due to the alkalin- ity of the biochar used in this study (Chintala et al.
2014). Biochar amendment had no impact on EC except for the strongly structured RL
where EC was consistently reduced. This supports our suggestion of greater leaching
potential in the stronger structure soils, i.e., ammonium leaching.
The impact of biochar and its interactions depend on bio- char types and reactions
in different soils (Kookana et al.
J Soils Sediments

2011; Lehmann et al. 2011) and its capacity to adsorb selective nutrients (Novak et
al. 2009). The biochar additions may af- fect micronutrient content in soil due to
their sensitivity to changes in soil pH, soil porosity, and aeration. Soil aggrega-
tion may benefit from biochar additions to clayey soils while sandy soils are also
improved by the surface area increases associated with biochar (Basso et al. 2013;
Ulyett et al. 2014).
Our data showed that available Fe, Mn, and Cu decreased in the soils with
increasing biochar applications but this was generally only significant at the
highest loading rates. These declines in micronutrient availability probably relate
to the increases in soil pH and the associated calcareous nature of the applied
biochar. Kumar and Babel (2011) indicated that micronutrients correlated negatively
with CaCO3 and soil pH increases. Zinc availability was increased in all soils at
all rates but this was generally only significant at the higher rates. However,
zinc levels were 200 mg/kg in the biochar and this might well explain these broad
ranging and consistent in- creases as compared to the other micronutrients. We
interpret the mixed story of some increases and some decreases in micronutrients as
relating to the relative impacts of biochar- induced changes in particular soil pH
increases with loading rates as well as the significant levels of some
micronutrients in the biochar itself.

4.2 Microbial biomass

Previous studies demonstrated that biochar addition to soil enhances microbial

abundance and activity (O’Neill et al.
2009; Bamminger et al. 2014; Gomez et al. 2014). However, our results showed
limited impact of biochar on microbial biomass at least in the short term. Noyce et
al. (2015) studied soil microbial responses over 1 and 2 years following biochar
addition to a temperate forest soil; their results indicated that biochar had
inconsequential impact on bacterial and fungal community composition,
fungi/bacteria ratios, and microbial biomass. The increase of soil pH after biochar
addition nor- mally is expected to enhance microbial abundance in soil (Lehmann et
al. 2011); however, biochar addition at high ap- plication rates seemed to reduce
microbial biomass which may be explained by the changes in organic matter decompo-
sition and N availability (Dempster et al. 2012a). The finding of this experiment
showed a reduction in N content with in- creasing biochar loading rates in RL soil
which might be as a result of N consumption by microorganisms during organic matter
decomposition (Lehmann et al. 2011; Nelson et al.
2011). The initial decomposition might be increased after bio- char addition to
soil which required more N by soil microor- ganism. While there is no fertilizer
input, the nutrient content might be reduced in soil and affect microbial abundance
and activity. Many factors involved in the impact of biochar on microbial abundance
and activity in addition to biochar C in amended soil (Gomez et al. 2014). Carbon
input following
biochar application proportionally affects microbial growth and activity, but the
degree of change is likely to be dependent on the initial soil nutrient content and
C levels. This would explain the difference on biochar effects observed in the dif-
ferent soils studied.

4.3 Potential nitrification

It has been well documented that biochar amendment to soil has the potential to
improve nitrification and consequently increase N availability to plants (Berglund
et al. 2004; Ball et al. 2010; Prommer et al. 2014). Biochar can also reduce N
losses due to its physiochemical properties such as cation and anion exchange
capacity (Dempster et al. 2012b; Yao et al.
2012). Our findings support increased nitrification in biochar- amended soils,
especially in the BSL soil at the 2.5 and 5 % biochar loading rates. The
nitrification enhancement and the biochar sorption capacity are likely to have
contributed to the reduction in NH4 and increase in NO3 with increasing biochar
loading rates. While nitrification was inhibited at the highest level of biochar
amendment, this may relate to the high C/N ratio in the amended soil (Bengtsson et
al. 2003) which gen- erally leads to N immobilization and reduction of ammonium
oxidation activity (Song et al. 2014). The impact of biochar on the C and N pools
in soil may vary depending on the biochar properties. As stated by Zhang et al.
(2015), biochar produced at high temperature (>400 °C), such as used in this study,
has the potential to add more stable C to the soil thus decreasing CO2 emission
and increasing NH4 sorption capacity. However, biochar produced at lower
temperatures provides labile C useable by microorganism and that may temporary
enhance biological N transformation. Biochar produced at high temperature may
improve N usage efficiency, nutrient retention, and alter N and C transformations
(Zhang et al.
2015).

4.4 Community level metabolic profiles

It has been well documented that carbon availability plays an important role in
microbial growth and activity (Alden et al.
2001; Yoshitake et al. 2007). Therefore, application of organic
amendments not just affects the soil chemical and physical properties, it also
enhances microbial function and activity related to nutrient cycling (Ros et al.
2006). Biochar is a very stable form of carbon which is resistant to microbial
degrada- tion, thus the available carbon used by soil microorganisms is more likely
to be from different sources. A study conducted by Dempster et al. (2012a) showed
that the addition of 25 t ha−1 biochar altered the community metabolic profiles;
however, the change in the ammonia oxidizing bacterial community occurred only when
a source of N was combined with biochar application. The results here showed a
decrease in microbial activity through the reduction of organic matter
J Soils Sediments

decomposition and N mineralization in soil, which might be due to the decrease in

microbial biomass. This may explain the decline in the C source utilization in the
BCL and sandy loam soils. However, metabolic profile in the RL increased with
increasing biochar rates, possibly due to the presence of other complex carbon
sources present in this soil. The native grass and root residues in the RL soil
might explain the enhancement in C substrate utilization. Baudoin et al. (2003)
stated that the root exudates stimulate bacterial growth and increase microbial
community. Different plant species and residues as organic compounds in soil may
increase the po- tential metabolic diversity (Baudoin et al. 2003).

5 Conclusions

The results of this study indicate that the interaction between different soil
type and biochar loading rates had a significant impact on the NH4–N and NO3–N
contents especially in BCL soil. A highly significant effect was observed on
the total C in all three soils with no impact observed on the total N
except in the RL soil. Soil pH was significantly increased with in- creasing
biochar loading rates. No significant impact was observed on the P and K
availability, the total exchangeable base cations (TEB) and CEC in BCL.
However, biochar addition had a significant effect on the exchangeable Al,
Ca, Mg, and Na in both the BSL and the RL soils. Biochar addition had a
limited effect on the microbial biomass; however, the interac- tion between
biochar and different soils significantly affected the potential nitrification
especially in RL and BSL soils. There were significant differences in the C
substrate utilization among biochar treatments in the three different soils. The
BCL and BSL showed reduc- tion in substrate utilization rates as biochar
levels in- creased while the opposite occurred for the RL soil, which
indicated that biochar may influence the function and activity of microbial
community. These results show that biochar can improve soil carbon content and in-
crease pH but varies in different soils. High biochar loading rates may
also influence nitrification and the function and activity of microbial community
in lighter soils. More studies are required for better understanding of the
interaction between biochar application and soil fertility under different
conditions and variables includ- ing biochar types and application rates,
different soils, fertilizer inputs, and long-term application.

Acknowledgements We would like to express our appreciation to all colleagues who

provided help during the sample collection. Also, we gratefully thank Mr. Stephen
Paterson for technical assistance.
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