RESEARCH PAPER

https://doi.org/10.1071/SR21162

Responses of soil nutrients and microbial activity to the

mill-mud application in a compaction-affected sugarcane field
Xiangyu Liu A, Rob Milla B, Terry Granshaw B, Lukas Van Zwieten A,C , Mehran Rezaei Rashti A,
Maryam Esfandbod A and Chengrong Chen A,*

ABSTRACT
For full list of author affiliations and
declarations see end of paper
Context. Compaction removal and organic amendment application are commonly used to mitigate
the compaction-induced declines in crop yield, soil carbon (C) and soil health. However, the
*Correspondence to:
Chengrong Chen
response of microbial activities and nutrient pools to the combination of mill-mud amendments
Australian Rivers Institute and School of and decompaction in the soil profile are not fully understood. Aims. A field trial was conducted
Environment and Science, Griffith at Burdekin, Australia, to investigate the effects of different decompaction managements on soil
University, Brisbane, Qld 4111, Australia
nutrient cycling, associated biological activities and sugarcane yield. Methods. This experiment
Email: c.chen@griffith.edu.au
included four treatments: control (CK, without mill-mud), mill-mud shallow furrow (MS), deep
Handling Editor: trenching without mill-mud (DT) and deep trenching mill-mud application (MD). Key results.
Nathan Basiliko The MD treatment increased concentrations of hot water extractable organic C by 30–70% and
hot water extractable total nitrogen (N) by 30–90% at the application depth. Soil microbial
biomass C and N were also higher in mill-mud applied layers. Mill-mud applied treatments
increased plant cane yield by 7% (MS treatment) and 14% (MD treatment) compared to CK.
The DT treatment also increased plant cane yield by 11% compared to CK. Conclusion. The
MD treatment increased the supply of organic C and nutrients to the microbial community
within the entire soil profile, enhanced nutrient cycling processes, improved soil environmental
conditions and soil health for sugarcane growth and thus increased sugarcane productivity.
Implications. Further research should focus on microbial community composition shifts to
further explore the mechanisms responsible for soil microorganism regulation of nutrient
cycling in sugarcane farming systems.

Keywords: Colwell P, compaction removal, deep trench, enzyme activities, labile organic C and N,
microbial biomass, mineral N, sugarcane yield.

Received: 15 June 2021

Introduction
Accepted: 25 October 2021
Published: 29 November 2021 Compaction, a type of soil disturbance that commonly occurs with over use of heavy
machinery in fields, leads to severe crop yield loss and soil health decline worldwide
Cite this: (Shah et al. 2017). Compaction may substantially impact soil functions, such as restricting
Liu X et al. (2022) root growth and limiting soil water and nutrient availability. For example, it has been
Soil Research, 60(4), 385–398. reported that 20% of sugarcane yield losses were due to compaction in Bundaberg,
doi:10.1071/SR21162
Australia (Bell et al. 2002), 12–23% wheat grain yield decline due to compaction was
reported in a field study conducted in Morocco (Oussible et al. 1992) and an average of
© 2022 The Author(s) (or their
16% sugarbeet yield decline was reported in Hungary by Lipiec et al. (2003). The effect
employer(s)). Published by
CSIRO Publishing. of compaction on crop productivity, soil carbon (C) and nutrient pools and dynamics has
This is an open access article distributed been assessed in a range of field studies (George et al. 2011; Colombi and Keller 2019;
under the Creative Commons Attribution- Shukla et al. 2020). It has been reported that compaction, excessive tillage and the
NonCommercial-NoDerivatives 4.0 depletion of soil organic matter are the main contributors to sugarcane (Saccharum
International License (CC BY-NC-ND).
officinarum) (Garside et al. 2005), maize (Zea mays) (Chen and Weil 2011) and spring
barley (Hordeum vulgare) (Czyż 2004) yield decline. However, the response of soil
OPEN ACCESS
microbial activity to compaction removal practices has been less explored. Such studies
X. Liu et al. Soil Research

could provide some insight into the underlying mechanisms application of mill-mud significantly increased sugarcane
and improve the knowledge of field managers to enhance yield via boosting soil nutrient concentrations in a Florida
their decision-making in applying appropriate field manage- sandy soil. Several studies worldwide have indicated that
ment practices to minimise the effects of compaction. mill-mud application substantially increased soil organic C
Adequate soil bulk density, organic C and nutrient pools and nutrient transformation by providing substrates for
support soil microbial performance and further determine microbial utilisation (Fauci and Dick 1994; Lima et al. 2009;
soil health conditions (Magdoff and Weil 2004; Flavel and Tan 2009). It has been suggested that the mill-mud can also
Murphy 2006). High soil bulk density (>1.47 g cm−3 for clay modify soil microbial pool size and microbial C use
soil and >1.8 g cm−3 for sandy soil) affects soil health efficiency in a poorly structured soil (Fang et al. 2020).
condition by reducing soil porosity, which decreases soil Hence, mill-mud application is considered one of the most
microbial activity and restricts root growth (Li et al. 2002). suitable field practices to ameliorate or remove compaction
Labile C, which is largely produced by organic matter impacts. Based on the above discussion, more attention
decomposition processes, is considered the main food source should be paid to the response of microbial activities and
of soil microbes (Valenzuela-Solano and Crohn 2006). Labile changes in soil nutrient pools to organic amendments used
C limitation or organic matter depletion may result in soil for removing compaction. This will enable better predictions
health decline (Liu et al. 2018). While the importance of of soil health condition in the context of boosting crop yield
mineral nutrients for crop growth has been recognised, organic and developing sustainable agriculture.
nutrients are also strongly related to crop yield and soil health This study aimed to investigate and compare the responses
condition (e.g. Liu et al. 2018). Brackin et al. (2015) of soil microbial activity and nutrient pools to different
highlighted that the organic form of N was more suitable for compaction removal practices, such as deep trenching mill-
plant root uptake, while inorganic nitrogen (N) concen- mud application and shallow furrow application, in sugar-
tration in soil was, to some extent, overestimated for plant cane fields. The following hypotheses were tested: (a) the
nutrition due to its extraction method. Organic phosphorus application of mill-mud would increase soil nutrient pools
(P) may not be directly utilised by plants; however, enhancing including total and labile pools, (b) different application
microbial activities would increase P mineralisation, thus methods (shallow furrow application and deep trench
enhancing P availability (Gilbert et al. 2008). In addition to application) would lead to different distribution patterns of
other physical (e.g. soil bulk density) and chemical indicators organic C and nutrients at different depths in the soil profile
(e.g. soil nutrient pools and availability), soil microbial and (c) mill-mud application and soil compaction stress
biomass C and N, microbial activities and the bacterial- removal would increase the size of the microbial community
to-fungal ratio are also considered important and sensitive and microbial activity, which would further increase soil
soil health indicators because these parameters can change health condition and sugarcane yield.
rapidly in response to shifts in environmental conditions
(Bhandari et al. 2018; Bünemann et al. 2018). As a key driver
of nutrient cycling, the soil microbial community plays a vital Materials and methods
role in soil C mineralisation (Nicolardot et al. 1994). A measure
of overall microbial activity was suggested to be an important Site description, field treatments and cane yield
indicator of the impacts of field management practices on soil
measurement
health (Schloter et al. 2003; Arias et al. 2005).
In recent decades, sugarcane yield decline has occurred in The experimental site was located in a sugarcane producing
many sugarcane growing regions in Australia due to com- area, Burdekin (19°30 0 S, 147°20 0 E), Queensland, Australia.
paction and inappropriate field practices. Depletion of soil The soil is a Mesonatric Brown Sodosol in Australia (Isbell
organic matter and overuse of heavy machinery in the field 2016) which is also classified as a Solonchak based on the
are reportedly responsible for declines in soil structure and FAO world reference base (WRB) for soil resources (IUSS
sugarcane yield, particularly in monoculture sugarcane- Working Group WRB 2014). The mean annual temperature
growing areas (Pankhurst et al. 2003; Garside et al. 2005). is 29.2°C and mean annual precipitation is 741.0 mm
To prevent further yield loss and soil health decline, the (Australia Bureau of Meteorology 2021). The experimental
application of organic amendments such as mill-mud field had been cropped with sugarcane for the past 15 years.
(a primary by-product from sugar production) or compost As a common practice, fine agricultural gypsum
has been suggested to increase soil organic matter, reduce was applied to a depth of ~10 cm using the broadcasting
soil bulk density and improve soil structure (Qureshi et al. method. Gypsum was applied to improve soil structure and
2007; Pattison et al. 2011; Ishak and Brown 2018). Mill-mud calcium (Ca) concentration (5 t ha−1). Soil at this field site
application is an efficient way to increase sugarcane yield by has been affected by compaction due to routine use of heavy
improving soil physical, chemical and biological properties machinery (e.g. harvester) for management and harvesting.
(Naidu and Syers 1992; Morris et al. 2007; Gilbert et al. Mean values of surface soil properties from six composite
2008). McCray et al. (2015) reported that broadcast samples (>30 soil cores) taken prior to the study across the

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paddock (c. 50 ha) include pH (6.01), EC (29.1 μS cm−1), clay of soil to distilled water (Rayment and Lyons 2011). Soil
(4.5%), silt (37.3%), magnesium (Mg; 477 mg kg−1), lead (Pb; mineral N (NH4+-N and NO3−-N) was extracted by 2 M KCl
7.7 mg kg−1), copper (Cu; 9.5 mg kg−1), nickel (Ni; at a 1:5 ratio of soil to extractant using an end-over-end
4.7 mg kg−1), titanium (Ti; 398 mg kg−1), manganese shaker for 1 h, filtered by a Whatman 42 filter paper (Rayment
(369 mg kg−1) and iron (Fe; 5.9 g kg−1) (Supplementary and Lyons 2011) with concentrations of NH4+-N and NO3−-N
Table S1a, b). Overall variations among these composite determined by a SEAL AA3 Continuous Segmented Flow
samples were relatively small with coefficients of variation Analyser (SEAL Analytical Limited, USA). Soil Colwell P was
<15% for most parameters measured (e.g. pH, EC, particle extracted by 0.5 M NaHCO3 at a 1:5 ratio of soil to
size, Mg, Pb, Cu, Ni, Ti and Fe) at various soil depths (Table extractant using an end-over-end shaker for 16 h, filtered by
S1a, b). This provided a basis for comparing the field a Whatman 42 filter paper (Rayment and Lyons 2011) with
treatments. In this study, there were four treatments: concentrations of Colwell P measured by a SEAL AA3
(a) control treatment (CK, without application of mill-mud), Continuous Segmented Flow Analyser. Soil total C (TC) and
(b) mill-mud shallow furrow (c. 20 cm depth) addition treat- N (TN) contents were measured by the combustion method
ment (MS, mill-mud shallow furrow), (c) deep trench using a LECO CNS-2000 analyser (LECO Corporation, MI,
treatment (DT, deep trench without mill-mud) and (d) deep USA). Soil total P was measured using an inductively coupled
trench mill-mud application (c. 40 cm depth) treatment (MD, plasma optical emission spectrometer after digestion (ICP-OES;
deep trench with mill-mud application). Mill-mud was Perkin Elmer; Optima 8300). Hot water extractable organic C
applied in January 2018 at a rate of 35 t ha−1 (dry weight (HWEOC) and hot water extractable total N (HWETN) were
based) and sugarcane (S. officinarum) harvested in July 2019. measured using the method described by Chen et al. (2000).
Amendments were applied to the targeted subsoil levels Briefly, 4.0 g (oven-dry equivalent) of fresh soil was incubated
(c. 20 and 40 cm depths) in each trench 2 weeks before with 20 mL of water in a capped Falcon tube at 70°C for 18 h.
mung bean (Vigna radiata (L.) Wilczek) planting using a After incubation, the tubes were shaken on an end-over-end
large trencher and conveyer spreader to drop mill-mud into shaker for 5 min and filtered through a Whatman 42 filter
trenched slots in the area where the cane row would be paper, followed by a 0.45-μm filter membrane. Concentrations
planted. Mill-mud materials (a mixture of mill-mud and mill- of dissolved organic C and total extractable N in the filtrate
ash) were slightly acidic (pH 6.4) and mill-mud nutrients were determined using a SHIMADZU TOC-VCPH (Shimadzu,
included C (9.1% of dry matter), N (0.42% of dry matter), Kyoto, Japan) TOCN analyser. All results were expressed on
P (0.73% of dry matter) and potassium (0.61% of dry an oven-dry basis.
matter). Soil sampling took place in July 2019 with six
replicates from each treatment. Adjacent CK (26.05 ha), MS Soil biological analysis
(26.05 ha), DT (8 ha) and MD (8 ha) treatments were
divided into six subplots. Further description of soil sampling Soil microbial biomass C (MBC) and N (MBN) contents were
is shown in Fig. S1. The soil cores (five) were randomly measured by fumigation-extraction method using an Ec
sampled with a 5-cm diameter auger and divided into four conversion factor of 2.64 (Vance et al. 1987) and an En
layers of 0–10, 10–20, 20–40 and 40–60 cm depth in each conversion factor of 2.22 (Wilson 1988). Concentrations of
subplot and bulked together to make a composite sample. soluble C and N of the fumigated and nonfumigated soil
Fresh soil samples were sieved (<2 mm) and stored at 4°C samples were determined using a SHIMADZU TOC-VCSH/
prior to chemical and biochemical analyses (within 1 week). CSN TOCN analyser. Fluorescein diacetate hydrolysis, widely
Sugarcane yield was measured by harvesting the entire plot accepted as an accurate and simple method for measuring
of four treatments, and yield was calculated based on weight of the overall microbial activity in a range of environmental
harvested cane divided by its entire treatment area (t ha−1). samples, was used to measure soil microbial activity in this
Therefore, unfortunately, there was no replicate of the study (e.g. Green et al. 2006). The activities of soil
sugarcane yield result. This has been the common practice in β-glucosidase (hydrolysing cellulose to glucose) at pH 6.0
the sugarcane farming due to the short window for harvesting and soil phosphatase (hydrolysing phosphomonoesters) at
in Burdekin. It was acknowledged that the yield data (without pH 6.5 were measured according to methods described by
statistical analysis) could only be used as indication of impacts Tabatabai (1994).
of mill-mud amendments.
Statistical analysis
Soil physicochemical analysis
Univariate ANOVA was used for soil properties data using the
The soil sand, silt and clay contents were measured using a IBM SPSS Statistics 23 software package (IBM Corp., Armonk,
Maxing sizer (Malvern Panalytical, UK). Soil bulk density NY, USA). Differences at P ≤ 0.05 between treatments were
was estimated by soil weight divided by its volume using the considered significant and all variables were tested for
method described by Maynard and Curran (2007). Soil pH normality of distribution using the Kolmogrov–Smirnov test.
and EC values were measured in a 1:5 volumetric suspension Pearson linear correlations were used to describe the

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relationships between soil properties. In addition, data on all higher than DT and MD (P < 0.05). In addition, the
soil properties were analysed using principal component treatments that received mill-mud (MS and MD) inputs had
analysis (PCA) to distinguish the effects of mill-mud addition higher pH than treatments without mill-mud (CK and DT)
and deep trench application on soil. for deeper soil profiles (20–60 cm). Total C and N contents
in the MS treatment were generally higher than for other
treatments at 0–20 cm depth. Additionally, the MD treatment
Results had significantly higher TC and TN concentrations than other
treatments at depth of 20–40 cm. Interestingly, at 40–60 cm
Soil physical and chemical properties depth, DT had the highest TC content (0.18%) and MD had
the highest TN content (0.058%) (Table 1).
Soil contained around 5% clay, 38% silt and 57% sand in
topsoil (0–10 cm), and clay content increased with soil
Labile organic C and N pool
depth. Soil bulk density in MS was significantly (P < 0.05)
lower than other treatments at 0–10 cm (1.07 g cm−3) and There were no significant differences in concentrations of
10–20 cm depths (1.38 g cm−3), while there were no HWEOC among all treatments for surface soil (0–10 cm),
significant differences in bulk density among CK, DT and MD despite MS having relatively higher HWEOC concentration
treatments at these two depths (1.47–1.60 g cm−3 at (Table 1). For the 10–20 cm depth, the DT and MD
0–10 cm and 1.54–1.71 g cm−3 at 10–20 cm) (Table 1). At treatments had significantly greater HWEOC than MS and
20–40 cm depth, MD bulk density (1.49 g cm−3) was CK, with the lowest HWEOC in CK. There were no
significantly lower than MS (1.78 g cm−3) (Table 1). The CK significant differences in HWEOC between the two deep
had the highest bulk density (2.32 g cm−3) among all trench treatments (DT and MD). At 20–40 cm depth, the
treatments at 40–60 cm depth, while DT had the lowest bulk MD treatment had the highest HWEOC concentration
density (1.67 g cm−3) (Table 1). All soil samples were (196.9 mg kg−1, P < 0.05), followed by DT and MS
slightly acidic. Soil pH values in CK (5.72) and MS (5.92) treatments, with the lowest value in CK (Table 1). A similar
were significantly (P < 0.05) higher than MD (5.09) at trend was found for the 40–60 cm depth (Table 1).
0–10 cm depth (Table 1). A similar trend in soil pH was There were no significant differences in HWETN
observed at 10–20 cm depth (Table 1). For the deeper soil concentrations among all treatments for surface soil
profiles (20–60 cm), soil pH in CK and MS was significantly (0–10 cm). At 10–20 cm depth, HWETN concentrations

Table 1. Selected physicochemical properties of sugarcane field soil under different field mill-mud applications.
Depth BD Clay Silt Sand pH C (%) N (%) HWEOC HWETN NH4+-N NO3−-N
(cm) (g cm−3) (%) (%) (%) (mg kg−1) (mg kg−1) (mg kg−1) (mg kg−1)
0–10 CK 1.46a 4.8b 40.7a 54.5c 5.72a 0.75ab 0.051a 192.6a 6.05a 3.77b 1.01a
MS 1.07b 3.8c 39.8a 56.4bc 5.92a 0.93a 0.071a 217.8a 7.04a 4.12a 1.12a
DT 1.52a 5.0b 35.9b 59.1a 5.4ab 0.56bc 0.053a 199.9a 6.36a 4.00a 0.99a
MD 1.47a 6.6a 39.8a 53.6c 5.09b 0.34c 0.067a 197.2a 6.13a 4.11a 1.04a
10–20 CK 1.69a 5.1a 37.3a 57.6a 6.2a 0.58a 0.044b 137.9c 4.98b 3.73c 0.95b
MS 1.38b 5.8a 38.8a 55.5ab 6.40a 0.68a 0.061a 154.8b 5.34b 4.18a 1.10a
DT 1.71a 7.8a 38.4a 53.8b 5.15b 0.38b 0.037b 175.4a 5.04b 4.04b 0.90bc
MD 1.65a 5.6a 37.7a 56.8a 4.75c 0.35b 0.072a 187.1a 6.62a 4.01b 0.82c
20–40 CK 1.82a 6.5b 41.9a 51.6b 6.35a 0.22b 0.033b 114.2c 2.83b 3.69b 0.78c
MS 1.78a 8.6a 41.3a 50.1b 6.70a 0.13b 0.029b 133.6b 4.78a 4.59a 1.37a
DT 1.61ab 7.7a 36.7b 55.5a 5.13b 0.22b 0.028b 140.3b 2.29b 2.96c 0.81c
MD 1.49b 7.8a 40.4a 51.8b 5.43b 0.38a 0.068a 196.9a 5.67a 4.55a 1.01b
40–60 CK 1.88a 8.0b 42.4a 49.6a 6.47a 0.09b 0.022c 75.2c 1.65b 2.57c 0.77b
MS 1.88a 10.0a 40.1a 50.0a 6.75a 0.08b 0.029b 84.5b 3.39a 3.24b 0.79b
DT 1.73b 10.2a 38.3a 51.5a 5.35b 0.18a 0.029b 89.3b 0.66c 2.64c 0.81b
MD 1.91a 8.2ab 38.5a 53.4a 5.64b 0.12b 0.058a 130.7a 2.18b 4.1a 0.95a

Note: the reported data are the means of six replicates. Means within a column for each depth followed by the same letter are not significantly different at P < 0.05.
BD, bulk density; HWEOC, hot water extractable organic C; HWETN, hot water extractable total N; CK, control treatment (without mill-mud); MS, mill-mud shallow
furrow (c. 20 cm) treatment; DT, deep trench without mill-mud treatment; MD, deep trench with mill-mud (c. 40 cm) treatment.

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were significantly greater for the MD than the other three 20–40 cm depth. At 40–60 cm depth, MD had the highest
treatments, while there were no significant differences NO3−-N concentration (0.95 mg kg−1, P < 0.05), while there
among CK, MS and DT treatments (Table 1). At 20–40 cm were no significant differences among the CK, MS and DT
depth, as expected, HWETN concentration in the treatments treatments.
receiving mill-mud (MS and MD) were at least 70% higher
(P < 0.05) than the two treatments without mill-mud. At P pools
the 40–60 cm depth, mill-mud application significantly
increased concentrations of HWETN in MS and MD Total P concentrations in MS and MD significantly increased
treatments (3.39 and 2.18 mg kg−1, respectively), (P < 0.05) after mill-mud application at 10–20 cm
compared to CK and DT (1.65 and 0.66 mg kg−1, (380.8 mg kg−1 in MS) and 20–40 cm depths (294.5 mg kg−1
respectively). Interestingly, surface applied mill-mud did in MD) compared with CK and DT at the same soil depth
not increase labile C store in the entire soil profile (0– (Fig. 1a). As expected, MS treatment increased total P
60 cm) while deep trench applied mill-mud significantly concentration at the targeted soil level (10–20 cm) but also
increased labile C store in the entire soil profile (Table 1). increased total P for the surfac soil (0–10 cm) (Fig. 1). The
MD treatment had the highest soil total P for the deep layers
Inorganic N pools (20–40 and 40–60 cm) (Fig. 1). However, total P concentration
decreased with soil depth (20–60 cm) in the CK treatment
Concentrations of soil NH4+-N were in the range of (Fig. 1a).
4.00–4.12 mg kg−1 among MS, DT and MD treatments, being Colwell P concentration decreased with soil depth (20–60
significantly (P < 0.05) higher than in CK (3.77 mg kg−1) cm) in the treatments without mill-mud application (CK and
(Table 1) for the surface soil (0–10 cm). However, there DT) (Fig. 1b). The mill-mud application treatments (MS and
were no significant differences in NH4+-N concentrations MD) exhibited significantly greater amounts of Colwell P in
among MS, DT and MD. At the 10–20 cm depth, the MS treat- soil compared with CK and DT. The MS treatment increased
ment increased NH4+-N concentrations (4.18 mg kg−1) while Colwell P concentration in the surface soil (0–20 cm) but
CK had the lowest NH4+-N concentrations (3.73 mg kg−1). had no impact on the deeper soil (20–60 cm) (Fig. 1b).
Concentrations of NH4+-N in MS and MD were significantly However, the MD treatment significantly increased Colwell P
higher (P < 0.05) than in the other treatments at 20–40 cm levels in deeper soil (20–60 cm) but had no effects on levels
depth. In addition, we found MD had the highest (P < 0.05) in surface soil (0–20 cm) (Fig. 1b).
NH4+-N concentration (4.10 mg kg−1) at 40–60 cm depth,
followed by MS (3.24 mg kg−1). No significant differences in
Microbial C and N pools
soil NO3−-N concentration were found in the 0–10 cm depth
among all treatments. The MS had significantly higher Mill-mud application significantly (P < 0.05) increased levels
(P < 0.05) NO3−-N concentration (1.10 mg kg−1) than the of MBC in the MS (0–20 cm) and MD (0–60 cm) treatments,
other treatments at 10–20 cm depth. The treatments receiving compared with CK and DT treatments (Fig. 2a). Notably, the
mill-mud (MS and MD) had significantly (P < 0.05) higher DT treatment increased the concentration of MBC in the lower
NO3−-N concentration than the other treatments at soil profile (20–60 cm) compared to CK. Increases in MBC

(a) (b) CK
500 a MS
190
Concentrations of Colwell P (mg kg–1)

DT
Concentrations of total P (mg kg–1)

a MD
a 180
400
170
a a
300
160
a
b
200 b b 30
b a
c c a c
d b b b 20
b c
100 d c c d
d b
10 c c b b
c
0 0
0−10 10−20 20−40 40−60 0−10 10−20 20−40 40−60
Depth (cm) Depth (cm)

Fig. 1. Responses of soil total P (a) and Colwell P (b) to different mill-mud application in a compaction-affected sugarcane field (n = 96).
Control treatment (CK, without mill-mud), mill-mud shallow furrow (c. 20 cm) addition treatment (MS, mill-mud shallow furrow), deep
trench treatment (DT, deep trench without mill-mud) and deep mill-mud application (c. 40 cm) treatment (MD, deep trench with mill-mud
application). Columns for treatments with the same letter above are not significantly different at P < 0.05 within each soil depth.

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X. Liu et al. Soil Research

(a) (b) (c)

300 CK 50 16
a

Concentrations of MBN (mg kg–1)

MS a a
Concentrations of MBC (mg kg–1)

a DT
250 a 40 a
MD
a 12
a

Microbial C:N ratio

200 b b a a
a b
b 30
c a
150 c a 8
d b b d a a b
c
b 20 b b b b
b c
100
c c c c
d b bc b
c c c c
d 4 c
10
50

0 0 0
0−10 10−20 20−40 40−60 0−10 10−20 20−40 40−60 0−10 10−20 20−40 40−60
Depth (cm) Depth (cm) Depth (cm)

Fig. 2. Responses of soil microbial biomass C (MBC) (a) and N (MBN) (b) and microbial C:N ratio (c) to different mill-mud application in a
compaction-affected sugarcane field (n = 96). Control treatment (CK, without mill-mud), mill-mud shallow furrow (c. 20 cm) addition
treatment (MS, mill-mud shallow furrow), deep trench treatment (DT, deep trench without mill-mud) and deep mill-mud application
(c. 40 cm) treatment (MD, deep trench with mill-mud application). Columns for treatments with the same letter above are not significantly
different at P < 0.05 within each soil depth.

were mainly in the upper layers (0–20 cm) for the MS Cane and sugar yield
treatment (Fig. 2a). As expected, increases in MBC were in
Fig. 4 shows clearly that field management of MS, DT, and MD
deeper layers (20–60 cm) for the MD treatment (Fig. 2a). In
obviously increased plant cane yield. It is noteworthy that the
terms of MBN, the MS treatment increased the concentration
DT treatment increased plant cane yield (157.4 t ha−1) more
of MBN, similar to the pattern for MBC (Fig. 2b). However, the
than for the MS treatment (152.1 t ha−1). As expected, field
MD treatment had the lowest MBN level in nearly all layers
management also increased sugar yield; however, there was
(Fig. 2b). In general, MBC levels decreased with soil depth,
only a slight increase for the MS treatment while DT and
but this trend was not clear for MBN. Interestingly, mill-
mud application (MS and MD) significantly (P < 0.05) MD treatments boosted sugar yield.
increased the microbial C:N ratio in comparison to CK,
although this pattern became unclear in the MS treatment Relationships between soil physiochemical and
in the 40–60 cm layer (Fig. 2c). Notably, DT treatment had microbial properties and PCA
no impact on the microbial C:N ratio in the 0–40 cm soil
Soil bulk density was significantly negatively correlated with
profile, but significantly increased microbial C:N ratio at
many biological properties such as microbial activity,
40–60 cm depth (Fig. 2c).
enzyme activity, MBC and MBN (r = 0.557–0.768, P < 0.01)
(Table 2). Soil pH was also negatively correlated with
Microbial and enzyme activities microbial activity, phosphatase activity, MBC and microbial
Overall, microbial activity for CK, MS and DT treatments C:N ratio (r = 0.308–0.519, P < 0.05) although there was no
decreased with soil depth, but MD tended to have increased significant correlation between pH and β-glucosidase activity
microbial activity down to 40 cm and then declined in the (Table 2). In addition, TC, TN and total P were positively
40–60 cm layer. It is notable that the DT treatment increased correlated with microbial activity, enzyme activities, MBC
microbial activity in the deeper soil profile (20–60 cm) and MBN (r = 0.285–0.880, P < 0.05). As expected, soil
compared to CK, which was confirmed by the MBC result. Colwell P was positively correlated with microbial activity,
The MS treatment had the highest microbial activity in the enzyme activities, MBC and MBN (r = 0.505–0.696,
surface soil (0–20 cm) while the MD treatment had the P < 0.01). Soil NH4+-N was positively correlated with
highest MBC at 20–40 cm depth (Fig. 3a). microbial activity, enzyme activities, MBC and microbial C:N
Activities of β-glucosidase and phosphatase enzymes were ratio (r = 0.334–0.566, P < 0.05) while NO3−-N only
highest at 10–20 cm depth for the MS treatment, and highest showed a positive correlation with enzyme activities
at the 20–40 cm depth for MD, which corresponded well (r = 0.354–0.542, P < 0.05). Notably, soil labile pools also
with the target depth of mill-mud application for the MS and showed positive correlations with microbial activity, enzyme
MD treatments. In the CK treatment, activities of both activities, MBC, MBN and microbial C:N ratio (r = 0.364–
enzymes decreased with soil depth, while in the DT 0.846, P < 0.05), while labile N showed no correlation with
treatment, β-glucosidase activity tended to decline with soil microbial C:N ratio (Table 2).
depth, but phosphatase increased in the 20–40 and The PCA results showed that principal components (PCs)
40–60 cm layers (Fig. 3b, c). PC1 and PC2 explained 57.0% and 12.2% of the data

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(a) 2.0 20–40 and 40–60 cm depths, the MD treatment was

CK
MS separated from the other treatments along PC1, while the
DT DT treatment was separated from the CK and MS along PC2
(μg fluorescein g–1 dry soil h–1)

1.6 a MD
a a
b b
at these depths (Fig. 5). The parameters with the highest
b b
Microbial activity

c c correlation coefficients (>0.85) for PC1 were total C, labile

1.2
b a C, MBC and microbial and β-glucosidase activities; and the
c b parameter showing the highest correlation coefficient
0.8 d
(>0.85) for PC2 was pH (Table S2a, b).
c c

0.4
Discussion
0.0
0−10 10−20 20−40 40−60
In the present study, it was assumed that adjacent soils (under
Depth (cm)
different treatments) within a paddock were similar in their
(b) 100 a
origin and parent materials. This assumption has been
a
generally accepted and applied as the basis of many paired-
(μg p-nitro g–1 dry soil h–1)
β-glucosidase activities

75 a b site studies (e.g. Chen et al. 2004). This has been supported
a a a
by similar results of particle size and elemental analyses
50 b
among composite samples across the paddock prior to the
c c c treatment (Table S1a, b; Materials and methods section). In
addition, the paddock was uniform due to the same cropping,
a
25
d ploughing and management history. We acknowledge the
c b limitation of this study, as for many other paired-site studies,
d
0 conducted using pseudo-replication. Soil compaction and the
0−10 10−20 20−40 40−60
decrease in soil organic matter are key issues that limit
Depth (cm)
the continuous increase of sugarcane yield (Liu et al. 2018).
(c) 800
The compaction is brought about by the overuse of heavy
a
a a machinery in harvesting while lower organic matter results
(μg p-nitro g–1 dry soil h–1)

b b a from long-term monoculture and cultivation. To address

Phosphatase activities

600 a c
b b bc
c c b sugarcane yield decline due to deterioration of soil health
c (Garside et al. 2005), mill-mud has been widely applied to
d
400 the surface soil of sugarcane fields, but this application is
limited to within 20 km of the sugar mill due to the freight
200
cost. Mill-mud application to soil can enhance soil health
and fertility through an increase in soil organic matter and
supply of nutrients (Orndorff et al. 2018), improved soil
0 physical structure and retention of water (Fang et al. 2020),
0−10 10−20 20−40 40−60
increased soil microbial activity (Ishak and Brown 2018) and
Depth (cm)
enhanced soil pH buffering capacity (Medina et al. 2015).
Fig. 3. Responses of soil microbial and enzyme activities to different It has been reported that deep trench application of
mill-mud application in a compaction-affected sugarcane field (n = 96). composted municipal-biosolid to sugarcane fields can help
Control treatment (CK, without mill-mud), mill-mud shallow furrow plant growth and increase sugarcane yield (Viator et al.
(c. 20 cm) addition treatment (MS, mill-mud shallow furrow), deep trench 2002). However, soil nutrient pool and microbial activity
treatment (DT, deep trench without mill-mud) and deep mill-mud responses to compaction removal may vary with different
application (c. 40 cm) treatment (MD, deep trench with mill-mud depths of mill-mud application, soil type and environmental
application). Columns for treatments with the same letter above are conditions such as soil temperature and moisture content.
not significantly different at P < 0.05 within each soil depth. The PCA results showed a clear separation of soil samples
under different treatments (Fig. 5), indicating that overall,
variance, respectively (Fig. 5). Treatments were clearly mill-mud application methods had diverse and significant
separated from each other based on PC1 and PC2. At the impacts on soil properties and function. Changes in the key
0–10 and 10–20 cm depths, the MS treatment was clearly soil parameters (including soil pH, total soil C, labile C,
separated from the other three treatments along PC2. The MBC and microbial and β-glucosidase activities) induced by
CK, DT and MD treatments could be separated from each the different treatments contributed to the separation of
other along PC2 at different soil depths (Fig. 5). At the samples.

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(a) (b)
170 23
22.1
160.3 21.9
22
160 157.4

Sugar yield (t ha–1)

Cane yield (t ha–1)

152.1 21
150
20.1
20 19.8
141.4
140
19

130 18
CK MS DT MD CK MS DT MD

Fig. 4. Responses of cane yield (a) and sugar yield (b) to different mill-mud application in a compaction-affected
sugarcane field. Control treatment (CK, without mill-mud), mill-mud shallow furrow (c. 20 cm) addition treatment
(MS, mill-mud shallow furrow), deep trench treatment (DT, deep trench without mill-mud) and deep mill-mud
application (c. 40 cm) treatment (MD, deep trench with mill-mud application).

PCA of depth 0−10 cm PCA of depth 10−20 cm

MS MS
MS
MS MS
MS

CK
PC2 (12.2%)

PC2 (12.2%)

CK
CK CK
CK
CK
DT
DT
DT DT DT DT
MDMD
MD MD
MD
MD

PC1 (57.0%) PC1 (57.0%)

PCA of depth 20−40 cm PCA of depth 40−60 cm

MS
MS MS
MS CK
MS
MS CK
CK
PC2 (12.2%)

PC2 (12.2%)

CK
CK
CK DT
MD DT
DT MD
MD
DT DT DT MD

MD MD

PC1 (57.0%) PC1 (57.0%)

Fig. 5. Score plot of principal component analysis (PCA) showing the separation of soil samples collected under different mill-mud
application and loading values of the individual soil parameters for PC1 and PC2 for soil samples (n = 96). Control treatment (CK,
without mill-mud), mill-mud shallow furrow (c. 20 cm) addition treatment (MS, mill-mud shallow furrow), deep trench treatment
(DT, deep trench without mill-mud) and deep mill-mud application (c. 40 cm) treatment (MD, deep trench with mill-mud application).

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Table 2. Pearson correlation coefficients (r) among soil physicochemical and biological properties (n = 96).
BD Moisture Clay Silt Sand pH TC TN TP Colwell NH4+-N NO3−-N HWEOC HWETN Microbial β- Phosphatase MBC MBN Microbial
P activity glucosidase C:N

BD 1

Moisture −0.459** 1

Clay 0.527** −0.285* 1

Silt 0.129 0.125 0.295* 1

Sand −0.323* 0.020 −0.654** −0.916** 1

pH 0.206 0.131 0.153 0.356* −0.347* 1

TC −0.714** 0.569** −0.748** −0.082 0.379** −0.082 1

TN −0.574** 0.273 −0.501** −0.160 0.337* −0.395** 0.601** 1

TP −0.608** 0.341* −0.442** −0.003 0.188 0.080 0.666** 0.595** 1

Colwell P −0.593** 0.629** −0.470** −0.004 0.200 0.190 0.697** 0.477** 0.822** 1
+
NH4 -N −0.466** 0.067 −0.363* −0.002 0.154 −0.066 0.365* 0.639** 0.485** 0.321* 1

NO3−-N −0.374** 0.221 −0.194 0.093 0.008 0.209 0.326* 0.307* 0.445** 0.389** 0.710** 1

HWEOC −0.664** 0.329* −0.635** −0.110 0.354* −0.494** 0.718** 0.784** 0.469** 0.371** 0.657** 0.414** 1

HWETN −0.562** 0.318* −0.758** −0.038 0.348* −0.260 0.726** 0.681** 0.475** 0.406** 0.549** 0.260 0.825** 1

Microbial −0.768** 0.287* −0.622** −0.202 0.421** −0.519** 0.793** 0.725** 0.637** 0.517** 0.511** 0.273 0.846** 0.757** 1
activity

β-glucosidase −0.690** 0.164 −0.510** −0.139 0.324* −0.128 0.734** 0.589** 0.764** 0.505** 0.566** 0.542** 0.690** 0.605** 0.775** 1

Phosphatase −0.617** 0.248 −0.331* −0.090 0.210 −0.308* 0.508** 0.539** 0.737** 0.546** 0.334* 0.354* 0.494** 0.364* 0.683** 0.683** 1

MBC −0.557** 0.377** −0.490** −0.089 0.276 −0.409** 0.616** 0.880** 0.737** 0.626** 0.554** 0.254 0.737** 0.629** 0.766** 0.565** 0.663** 1

MBN −0.562** 0.647** −0.522** −0.065 0.271 −0.038 0.812** 0.285* 0.492** 0.696** 0.095 0.169 0.458** 0.486** 0.615** 0.490** 0.409** 0.399** 1

Microbial C:N −0.091 −0.142 −0.058 −0.074 0.083 −0.416** −0.022 0.661** 0.338* 0.066 0.485** 0.115 0.380** 0.214 0.289* 0.172 0.335* 0.687** −0.355* 1

*Significant at P < 0.05 (two-tailed). **Significant at P < 0.01 (two-tailed).

BD, bulk density; TC, total C; TN, total N; TP, total P; HWEOC, hot water extractable organic C; HWETN, hot water extractable total N; microbial C:N ratio, microbial biomass C and N ratio.

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Impact of different compaction removal Mill-mud application significantly increased soil P

approaches on soil physiochemical properties concentration (MS and MD) in the applied layer mainly due
to the substantial amount of P in the mill-mud (c. 0.73% P
Mill-mud application significantly reduced soil bulk density at of dry matter). Increased P availability (Cowell P) following
targeted levels (c. 20 cm soil depth in MS and c. 40 cm soil mill-mud application could also contribute to (i) enhanced
depth in MD) compared to CK. In addition, DT treatment competitive sorption between low molecular-weight
also reduced bulk density down to the 60 cm depth (Table 1). aliphatic acid (via mill-mud application) and P for soil
This indicated that deep trench and mill-mud application sorption sites and (ii) increased metal complexation and
could be considered compaction removal methods to dissolution reactions by organic matter, resulting in
reduce soil compaction stress. Early research conducted on increased release of sorbed P (Bolan et al. 1994; Hue et al.
a clay soil showed that deep trench field practices would 1994; Maurice 1995). Additionally, mill-mud application
reduce soil bulk density down to 102 cm depth to improve may improve soil moisture conditions (Parmar and Sharma
soil water infiltration and cotton root growth (Heilman and 1996) and cause structural shifting of soil microaggregates
Gonzalez 1973). In the present study, deep trenching did not which decrease the number of potential P sorption sites
have a significant impact on soil pH while mill-mud applica- (Wang et al. 2001). Importantly, mill-mud application
tion slightly increased soil pH which concurs with results of increases soil mineralisable C which boosts microbial
other studies (Morris et al. 2007; Liu et al. 2018). Notably, incorporation of P, resulting in increased available
increased soil pH via mill-mud application will provide P (Chen et al. 2000; Guppy et al. 2005). In the present
better environmental conditions for soil nutrient turnover study, we also found that mill-mud application increased
which would further increase crop yield (Morris et al. 2007). soil labile C and available P simultaneously. However, the
Labile pools of soil organic matter are key fractions in terms DT treatment alone had no effect on soil P content.
of soil quality as they are available for microbial consumption
and play an important role in soil nutrient cycling (Hu et al. Impact of different compaction removal
1997). Chen and Xu (2005) suggested that the hot water approaches on soil biological properties
extractable soil organic C and total N measured as soil labile
C and N pools are some of the best indicators of the impact Mill-mud application to sugarcane fields can supply available
of field management practices on soil fertility. It has been organic C and reduce compaction stress for microbial
reported that compost application can increase soil organic activities. Liu et al. (2018) indicated that organic amendment
application might not only increase soil MBC, but also enhance
matter content (Pankhurst et al. 2002; Rahman et al. 2007;
soil MBN content. In the present study, soil MBC contents in
Luo et al. 2010) and provide the bioavailable organic C and
the mill-mud applied layers (0–10 cm in MS and 20–40 and
N for soil microbial growth and for stimulating soil nutrient
40–60 cm in MD) were significantly higher compared with
recycling (Mendham et al. 2003). In this study, deep trench
other treatments. This may be ascribed to the organic C
application of mill-mud significantly increased concentrations
inputs to the targeted and adjacent soil layers in the mill-
of HWEOC and HWETN in the deep soil layer due to the inputs
mud applied treatment, and subsequently more litter, which
of labile organic matter from mill-mud (Table 1). However, DT
may increase MBC contents in the soil profile. Lima et al.
treatment alone also increased HWEOC concentrations
(2009) confirmed that presence of carbohydrates from organic
throughout the soil profile compared with the CK, which
amendment influenced synthesis of humic substances and
might be ascribed to the increased mineralisation of organic biological properties in general. Higher concentrations of
matter because of the disturbance effects induced by deep MBN in MS at the 0–20 cm depth were observed compared
trenching. As expected, mill-mud application substantially with other treatments, which was similar to the trend in
increased soil total C and total N concentrations in the present MBC. However, the DT treatment had the highest MBN for
study due to the presence of high C and N in the mill-mud. This deep layers (20–60 cm), which may contribute to leaching
result indicates that application of mill-mud increases soil C and improved porosity due to decreased bulk density.
and N stock and, with the deep trench application method, Leaching of soluble organic C and N provides an available
mill-mud can significantly increase soil total C and N energy source for utilisation by soil microbial communities,
concentrations in deep soils (40–60 cm depth). Eghball et al. leading to an increase in soil microbial activity (Mendham
(2004) suggested that soil mineral N concentrations increased et al. 2003). This is supported by the positive correlations
following organic amendment application to soil. Our study between soil microbial biomass content and HWEOC and
also showed an increase in concentrations of soil NH4+-N HWETN contents in the present study. Li et al. (2002)
and NO3−-N following mill-mud application; however, this reported that decreased bulk density increased microbial pools
occurred at different depths for the MS and MD treatments. as it is negatively correlated with bulk density. This statement
The MS treatment slightly increased mineral N in the supports our finding that the DT treatment increased MBC and
10–20 cm layer while the MD treatment slightly increased MBN content in deep soil layers (20–60 cm), which suggests
NH4+-N and NO3−-N in the 20–40 cm layer. that compaction removal would increase soil microbial

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pools. In the present study, we found that MD had the highest significantly positively correlated with MBC, MBN, enzyme
MBC concentration of all treatments, indicating that the deep activities and microbial activity. Therefore, the labile organic
trench method could help mill-mud boosting of soil MBC C and nutrients derived from the mill-mud decomposition
content. enhanced soil microbial activity and nutrient cycling and
Field application of soil organic amendments has been ameliorated soil compaction stress for sugarcane growth.
reported to significantly increase soil enzyme activity This subsequently improved soil chemical and physical
(Albiach et al. 2000). In the present study, overall microbial, conditions and increased the plant cane yield. In the present
β-glucosidase and phosphatase activities tended to be higher study, surface applied mill-mud slightly increased cane
in the mill-mud application than the other treatments yields but there was no significant increase in soil labile C
(10–20 cm in MS and 20–40 cm in MD). This was likely due stores and microbial activity in comparison to CK.
to the higher organic inputs from the mill-mud as shown by Interestingly, the DT treatment significantly increased plant
the significant and positive correlations between enzyme cane yield and moderately increased soil labile C store and
activities and soil total C and N as well as soil HWEOC and microbial activity. As there was no organic matter input in
HWETN contents. Phalke et al. (2016) showed that soil the DT treatment, the significantly reduced soil bulk density
organic C content had a significant correlation with soil resulting from this practice increased soil aerobic condition
β-glucosidase activity after organic amendment of a (Drew 1983), availability of nutrients (Jusoff 1991) and
soybean–maize cropping system. In addition, it has been decomposition of native organic matter which may increase
reported that the increase in soil enzyme activities can be nutrient cycling (De Neve and Hofman 2000), labile C store,
related to decreased soil bulk density (Kaiser and Heinemeyer microbial activity (Li et al. 2002) and crop yield (Wallace
1993). In this study, microbial activity showed a significant and Terry 1998). However, this field compaction removal
negative correlation with soil bulk density and thus the practice may not be good for sustainable farming as huge
improved enzyme activities in the mill-mud and deep-trench soil organic C losses would occur in a long run without extra
applied treatments, suggesting a partial alleviation of organic matter input. This study clearly demonstrated that
constraint due to soil compaction removal practices. mill-mud deep trench application would simultaneously
increase plant cane yield and maintain soil health by
Impact of different compaction removal methods removing soil compaction, increasing soil labile C store and
on sugarcane yield boosting microbial activity.
Organic amendment field application can significantly
improve crop yield (Goswami et al. 2017). Tejada and
Gonzalez (2003) argued that the significantly increased grain Conclusions
yield after organic amendment application could be
attributed to increased soil organic matter. Liu et al. (2018) It was clearly demonstrated that application of mill-mud
demonstrated a significantly increased cumulative sugarcane improved soil physiochemical properties including bulk
yield over 4 years in an organic amendment applied treatment, density, nutrient availability and labile organic C and N
which linked improved soil chemical properties to the resulting (HWEOC and HWETN) contents, thereby stimulating nutrient
yield. In the present study, increases in plant cane yield and cycling, and enhancing soil health and crop yield. Deep trench
labile C took place in deep trench and mill-mud applied application of mill-mud stratified nutrients supplied the soil
treatments. This indicated that soil labile C from slow microbial community in all soil layers, enhanced nutrient
decomposition of mill-mud plays a vital role in increasing cycling processes and thus increased soil health and
sugarcane production. A possible explanation could be sugarcane productivity. In contrast, surface applied mill-mud
lignin-like products from decomposition of plant tissue only increased surface nutrients and organic matter content
benefiting the formation of soil humus, which further benefits with a limited crop yield improvement. Surface mill-mud
plant growth (Tan 2009), and carbohydrates released from application would also result in C loss from soil. It is
mill-mud being a very active soil organic component and noteworthy that the DT treatment unexpectedly increased
ready energy source for improving microbial activities sugarcane yield, which was mainly attributed to increased
(Pascual et al. 1999). Ghimire et al. (2017) also suggested mineralisation of native organic matter, increased nutrient
that soil labile C pools played important roles in maintaining availability and subsequent ameliorated soil compaction,
soil fertility. In addition, soil labile C, which is extracted by improved soil structure and redistribution of soil organic C
hot water, is biologically active and highly related to soil through the entire soil profile. Further research should focus
microbial activity (Chen et al. 2004). Mill-mud would also on microbial community composition shifts and functional
increase soil P and mineral N to further improve crop yield genes involved in C and N cycling in response to compaction
by overcoming P deficit (Balemi and Negisho 2012) and removal to further explore the mechanisms responsible for
balancing the soil N:P ratio (Dai et al. 2020). In the present soil microorganism regulation of nutrient cycling in sugarcane
study, labile C, mineral N, total P and Colwell P were farming systems.

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Supplementary material De Neve S, Hofman G (2000) Influence of soil compaction on carbon and
nitrogen mineralization of soil organic matter and crop residues.
Biology and Fertility of Soils 30, 544–549. doi:10.1007/s003740
Supplementary material is available online. 050034
Drew MC (1983) Plant injury and adaptation to oxygen deficiency in the
root environment: a review. Plant and Soil 75, 179–199. doi:10.1007/
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Data availability. The data that support this study will be shared upon reasonable request to the corresponding author.
Conflicts of interest. The corresponding author, Chengrong Chen, is an Associate Editor of Soil Research, but was blinded from the peer review process for
this paper. The other authors declare no conflicts of interest.
Declaration of funding. This work has been supported by the Cooperative Research Centre for High Performance Soils whose activities are funded by the
Australian Government’s Cooperative Research Centre Program. Moreover, it is acknowledged that Xiangyu Liu is financially supported by a Soil CRC Top-up
Scholarship and a Griffith University Postgraduate Research Scholarship.
Acknowledgements. We would like to thank Danielle Skocaj and other staff members of Sugar Research Australia for providing access to the field sampling
site. Also, we would like to give special thanks to staff members of Burdekin Productivity Services Pty Ltd for their assistance in the field sampling. We would also like
to thank Clare Morrison for her English editing throughout the manuscript.

Author affiliations
A
Australian Rivers Institute and School of Environment and Science, Griffith University, Brisbane, Qld 4111, Australia.
B
Burdekin Productivity Services, 210 Old Clare Road, Ayr, Qld 4807, Australia.
C
NSW Department of Primary Industries, Wollongbar Primary Industries Institute, Wollongbar, NSW 2477, Australia.

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