1 Preliminary Proposal

2 PhD Research – How Biodiversity Influences Grassland Carbon Stocks?

3
4 Rivandi Pranandita Putra ([email protected])
5
6 Supervised by Dr. Jonathan Bennett (The Bannett Ecology Lab of the University of Saskatchewan) and Dr. Hongjie Zhang (The
7 Agriculture and Agri Food Canada/AAFC)
8
9
10 I. INTRODUCTION
11
12 Global warming is a global issue to combat. Efforts to sequester atmospheric carbon dioxide (CO2) into soil are paramount to mitigating the
13 increasing CO2 (Cong et al., 2014; Turgut et al., 2015; Garcia-Diaz et al., 2016; Keesstra et al., 2016). This is due to the fact that the total annual
14 anthropogenic CO2 emissions are equal to 4% of total global SOC stocks (Poeplau et al., 2018). Grasslands, one of the most important pools for
15 carbon (C) storage, store roughly 30% of the global terrestrial C stocks (Bardgett & van der Putten, 2014). Grasslands could sequester
16 atmospheric CO2 as stable soil C (Reid et al., 2004), contributing to climate change mitigation (Allard et al., 2007). On the other hand,
17 grasslands could also release CO2, emit methane (CH4) from grazing livestock, and emit dinitrogen oxides (N2O) from soils. Thereby, proper
18 grassland management is needed to preserve and enhance soil C storage and reduce greenhouse gas emissions (Chang et al., 2021).
19
20 Numerous attempts are available to manage grasslands, and one of them is by enhancing biodiversity (plant, microbial, and fungal diversity).
21 Previous field studies have found that biodiversity is linked with more C storage in the soil (Steinbeiss et al., 2008; Lange et al., 2015; Wu et al.,
22 2017; Yang et al., 2019; Prommer et al., 2020; Anacker et al., 2021; Spohn et al., 2023). This SOC storage is due to the alteration of plant carbon
23 inputs and soil microbial metabolisms. However, actions to improve biodiversity could also cause C loss as a trade-off. For instance, sowing new
24 plants with soil tillage is disruptive and may reduce SOC stocks (Elias et al., 2023). Additionally, the results of such field studies may be site-
25 specific, in which the positive or negative impacts and their magnitude may differ in various locations with different climatic conditions. For
26 instance, afforestation increased soil C in dry sites in Argentina and Uruguay up to 1012 kg C x ha(-1) x yr(-1), while soil C decreased (as much
27 as 1294 kg C x ha(-1) x yr(-1)) in wet sites (Berthrong et al., 2012). Another issue is when we intend to improve plant diversity with legumes or
28 high-yielding grasses. The question is: what are potential legumes or high-yielding grasses that could sequester C effectively?
29
30 Aside from the enhancement of grass species biodiversity, many other management options are interlinked with that, for instance, some studies
31 argue that grazing management could increase grass and microbial biodiversity. The question is: in what way could we manage grazing? Should
32 it be rotational grazing, or any other grazing management options? Such things could be a novelty for further studies, including this one. Future
33 studies should focus on exploring the interlinked effects while considering possible synergies and trade-offs for biodiversity conservation, C
34 sequestration, and food production. This PhD research primarily aims to enrich the current knowledge about how biodiversity (with the specific
35 intervention management that will be discussed later with the supervisors) influences grassland C stocks in the temperate climate of Canada.
36
37 II. RESEARCH QUESTIONS

38 The general research questions are as follows:

39  What are possible grassland management interventions to enhance ecosystem services, including soil C storage, in the (specific site) of
40 Canada?

41 The management intervention that will be investigated further could be one or more combinations of some management interventions
42 (interactive effects), as stated in Table 2. For instance, by sowing legume and grass species with high-yielding forage. This will be
43 discussed with the supervisors. This will first be done through a literature review and probably a meta-analysis.

44  How is the impact of grassland management interventions on soil C storage in Canada?

45 The management intervention that will be investigated further could be one or more combinations of some management interventions, as
46 stated in Table 2. The specific site in Canada is to be decided later after being discussed with the supervisors. This is a field trial, but we
47 also use comparative data on the history of soil C storage over time from multiple ongoing large scale surveys and experiments.

48
49 III. LITERATURE REVIEW
50
51 3.1. How biodiversity influences C stocks in grasslands
52
53 Numerous studies have revealed that high biodiversity can promote soil C storage (Table 1) via enhanced productivity, belowground C
54 allocation, and alterations in microbial communities. High biodiversity can also affect ecosystem C accumulation and interact with eCO2 and soil
55 nitrogen supply (Reich et al., 2001; Reich et al., 2024). Typically, grasslands tend to sequester more soil C in environmental conditions that limit
56 decomposition processes (Himmel et al., 2007). The following are the conceptual model explaining the relationship between biodiversity and C
57 storage in soil (Figure 1).
58
59

60
61 Figure 1. Conceptual model illustrating the linkage between biodiversity and C storage in soil (Persiani et al., 2008)
62

63 Bennett et al. (2019) mentioned that high plant diversity causes soils to be dominated by fungi and associated soil biota, make it more
64 conservative and resulting in greater retention of nutrients and increased C storage. On the contrary, soils dominated by bacteria and bacteria-
65 feeding organisms tend to cycle nutrients more rapidly, resulting in systems with more nutrients available to plants, but greater nutrient leaching
66 and increased C fluxes. Cong et al. (2014) argued that greater soil C accumulation in diverse species mixtures was primarily linked to enhanced
67 soil C input from higher root biomass production.

68 Table 1. Some studies reported positive relationship amid plant diversity and grassland C stocks (note: PSR = plant species richness; N/A = not
69 available)
No. Literature Type of research Site Result Conclusion
1 Spohn et al. Field trial 84 grassland sites PSR increased SOC content, PSR enhances soil C sequestration,
(2023) over six continents, and this effect was strongest in especially in warm and arid
i.e., natural and semi- arid and warm grasslands. climates.
natural grasslands
with a diverse
climatic conditions.
No. Literature Type of research Site Result Conclusion
2 Bai et al. (2022) Meta-analysis N/A 2.3 to 7.3 billion tons of CO2e PSR can provide low-cost and/or
year-1 for PSR. high-carbon-gain options for
natural climate solutions in global
grasslands.
3 Anacker et al. Field trial Colorado, the USA Soil C was remarkably and Management for species richness
(2021) positively related to plant provides the co-benefit of soil C
species richness, while native storage.
perennial graminoid species
richness exhibited an even
stronger positive relationship.
4 Wang et al. Meta-analysis N/A SOC was greater in mixtures C sink in was larger in mixtures
(2020) than monocultures, which was than monocultures.
attributed to a greater
aboveground biomass,
belowground biomass, total
biomass, soil respiration (Rs),
and heterotrophic respiration
(Rh) in the mixtures.
5 Prommer et al. Field trial Jena, Germany PSR accelerated microbial PSR favors faster rates of microbial
(2020) growth and turnover and growth and turnover, likely due to
increased microbial biomass greater plant productivity, resulting
and necromass. PSR enhanced in higher amounts of microbial
SOC via its positive influence biomass and necromass that
on microbial biomass C. translate into the observed increase
in SOC.

6 Yang et al. Field trial Minnesota, the USA Increasing PSR in grassland PSR may greatly increase C capture
(2019) from 1 to 2, 4, 8, or 16 species and storage rates on degraded and
following agricultural abandoned agricultural lands.
abandonment enhanced soil C
storage by 60 to 178% due to
 No. Literature Type of research Site Result Conclusion
greater aboveground and root
biomass inputs.
7 Wu et al. (2017) Field trial Gansu, China Legumes increased soil C and The mix of legumes and grass
N due to increased C and N species may greatly enhance
input. Legumes also increased ecosystem functions including soil
soil C and N and alter the soil C and N storages, productivity, and
C: N ratio. diversity in the semi-arid grassland.
8 Lange et al. Long-term data N/A PSR increases rhizosphere C Enhanced C storage with PSR is a
(2015) from a grassland inputs into the microbial direct function of the soil microbial
biodiversity community, leading to community, indicating that the
experiment (The increased microbial activity increase in C storage is mainly
Jena Experiment) and C storage. Increases in soil limited by the integration of new C
and radiocarbon C were related to the enhanced into soil and less by the
(14C) modelling accumulation of recently fixed decomposition of existing soil C.
C in high-diversity plots.
9 Steinbeiss et al. Field trial Jena, Germany C storage increased with sown PSR might lead to higher soil C
(2008) species richness in all depth sequestration in the long-term.
segments. C losses were
significantly smaller with PSR.
70
71 3.2. Identification of management interventions that can enhance grassland ecosystem services, including C storage
72
73 The amount of SOM retained by grassland soils is strongly influenced by management (Jones & Donnelly, 2004). In order to mitigate climate
74 change as well as achieve food security, priority actions to enhance soil C stocks should be concentrated on improved management practices to
75 increase C inputs and retention in soils (Whitehead, 2020). Improved grassland management to enhance soil C stocks could help mitigate
76 greenhouse gas emissions (Smith et al., 2016a), enhance soil fertility (Lal, 2004), and improve the resilience of agriculture to extreme weather
77 events (Pan et al., 2009). According to Post & Kwon (2000), some of the most significant factors that might determine the direction and rate of
78 shift in SOC in response to management are:
79
80  Increasing the input rates of organic matter (OM);
81  Changing the decomposability of OM inputs to increase the light fraction organic C;
82  Placing OM deeper in the soil; and
83  Enhancing physical protection through either intra-aggregate or organomineral complexes.
84
85 Table 2. Previous studies investigating the correlation between grassland management interventions and soil C storage
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
1 Improving plant  Plant diversity As can be seen in N/A Reseeding involving More trial is needed to
diversity by increases soil Table 1 soil tillage is disruptive identify more grass
sowing seeds of microbial activity, and may reduce SOC species and their impact
various grass thereby increasing stocks (Elias et al., on the soil C in
species soil C (Lange et al., 2023). grasslands under
2015). diverse climatic
 High species conditions and their
richness due to interlinkages with soil
climate biodiversity.
favourability (high
precipitation) leads
to higher
belowground
biomass, resulting
in higher SOC
storage (Chen et
al., 2018).
 Other argued that
plant biomass is
not correlated with
plant diversity.
Plant diversity
influences SOC
through increased
OM quality, (not
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
because of its
quantity/biomass)
(Spohn et al.,
2023).
2 Inclusion of  Legumes increase  Rodríguez et al.  Rodríguez et al. Inclusion of legumes More trial is needed to
legumes the primary (2022), with (2022), with increases N2O identify more legume
productivity of legume legume emissions due to higher species and their impact
plant communities proportion of 7– proportions of soil N availability, on the soil C in
through increased 17% (meta- 7–17%. which typically negates grasslands under
N availability, analysis) ±30% of the increased diverse climatic
resulting in  Bai et al. (2022), C sequestration conditions and their
enhanced SOC (Wu with 147 (Henderson et al., interlinkages with soil
et al., 2017). megatons of 2015). biodiversity.
 N inputs through CO2e year-1 for
legumes may sown legumes in
inhibit oxidative pasturelands
enzymes (meta-analysis)
production to
degrade the more
recalcitrant
compounds, leading
to reduced CO2
emissions and C
losses (De Deyn et
al., 2011; Spohn et
al., 2016).
 Increased SOC due
to legume can be
linked to low C/N
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
ratios of legume
residues (i.e., litter,
root exudates, etc.)
more similar to soil
microorganisms
and SOM than
other plant residues
(Jensen et al.,
2012). Substrates
with low C/N ratios
can lower microbial
N acquisition and
enhance their C use
efficiency,
facilitating
humification and
plant residue
decomposition into
SOM (Spohn et al.,
2016).
3 Sowing high- Increased soil C stock  Gregory et al. N/A More trial is needed to
yielding forage is due to higher soil C (2021) in the identify more high-
grasses/cultivars supply in the root UK, with yielding grass type and
biomass under the Lolium perenne their impact on the soil
high-yielding forage L. cv. C in grasslands under
grasses (Gregory et ‘AberMagic’ diverse climatic
al., 2021). and L. perenne conditions and their
L. x Festica interlinkages with soil
pratensis Huds. biodiversity.
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
Cv. ‘Prior’
 Ma et al. (2000)
with switchgrass
(Panicum
virgatum).
 Lal et al. (1998)
with tall fescue
(Festuca
arundinacea)
and smooth
bromegrass
(Bromus
inermis).
4 Afforestation Grassland  Tian et al.  Berthrong et al. Afforestation with trees More research is
with trees afforestation can (2023) (meta- (2012) in can also cause needed to explore of the
sequester large analysis), Argentina and substantial soil C loss impact of various type
amounts of CO2 by recommended Uruguay in wet due to: of trees on the soil C in
increasing plant Lespedeza sites (decreased  Site preparation grasslands under
biomass. Fine root bicolor, Sophor (as much as causes substantial diverse climatic
biomass and deadfall a davidii, 1294 kg C x ha(- soil disturbance, conditions and their
accumulation are and Cotoneaster 1) x yr(-1)) deteriorates soil interlinkages with soil
remarkably and multiflorus as structure and biodiversity.
positively linked with high potential C weakens soil
SOC (Tian et al., sequester. aggregates, thereby
2023).  Güner et al. stimulating SOC
(2021) in decomposition (Guo
Turkey, with & Gifford, 2002;
black pine Turner et al., 2005).
(235.2 t C ha−1)  Trees have longer-
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
and scots pine lived, coarser roots
(206.1 t C ha−1) than grasses, and
than bare soil therefore contribute
(37.4 t C ha−1). less to SOC
 Berthrong et al. formation (Guo &
(2012) in Gifford, 2002).
Argentina and  Replacing high more-
Uruguay in dry mineral associated
sites (increased OM (MAOM)
up to 1012 kg C grassland with low-
x ha(-1) x yr(- MAOM
1)). ectomycorrhizal
forest also reduces
the stable soil pool of
grasslands (Cotrufo
et al., 2019).
 As forests have a
higher surface
albedo, they tend to
absorb more heat
than
grasslands (Bonan,
2008). Planting trees
in grasslands may
thus result in a net
warming effect,
which could reduce
or even offset the
benefits of increased
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
C
sequestration (Bonan,
2008; Bright et al.,
2015).
5 Grazing  Grazing is  Bai et al. (2022),  Hoglund (1985)  Overgrazing threaten  Further study should
management important as with 148 to 699 in New Zealand SOC via their consider different
(rotational exclusion of megatons of due to detrimental effects climatic conditions
grazing and herbivores leads to CO2e year-1 for overgrazing on plant diversity, and grass type
moderate an accumulation improved and that the (C3/C4) in grazing
grazing) of litter, which grazing overgrazing effects management
negatively affects management are particularly (Abdalla et al.,
decomposition (meta-analysis). strong under arid 2018).
(Cox et al., 2001).  Orgill et al. conditions (Liu et  Further study is
 Grazing increases (2018) in al., 2022). needed to assess the
plant productivity, Australia, with  Overgrazing reduced impacts of grazing
C allocation to grazing of native below-ground inputs management
roots, root growth, grassland was to soil through a practices on the
and turnover. better than the decline in C belowground process
 A rotational ungrazed allocation to roots that affects the
grazing strategy treatment (Kong & Six, 2010). formation and
can reduce C loss  Conant et al. decomposition of
in subhumid (2017) with SOM and carbon
pasture by improved stocks (Whitehead,
significantly grazing 2020).
increasing C management
fixation (Oates & practices (meta-
Jackson, 2014). analysis).
 Grazed pastures  Koncz et al.
may sequester (2017) in
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
more C than Hungary, with
grasslands used extensive
for silage or hay grazing
production, due to  Chaplot et al.
the recycling of (2016) in South
OM and nutrients Africa, with
from faeces and high density and
plant residues short duration
(McEvoy, 2019). (HDSD)
grazing.
 Chen et al.
(2015) in China,
with constant
moderate
grazing
intensity.
6 Conversion from The conversion of  Guan et al. Causing conflicts: Further study could be
bare/arable to arable land into (2016) in China,  Arable land for done to compare the
permanent permanent grassland revegetation of cultivation and impact of introducing
grassland increases C storage in bare soil with production of food different plant species
the soil due to the all- alfalfa, bush (economic aspect). (legume or non-
year plant soil cover clover and milk  Preservation of legume) on the soil C in
and reduced tillage. vetch stands. habitat and grasslands under
 Osborn (1993) biodiversity (nature diverse climatic
in the USA, conservation aspect). conditions and their
revegetation of  Grassland for use as interlinkages with soil
highly-erodible pasture. biodiversity.
cropland or
other
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
environmentally
sensitive areas
to resource
conserving
vegetation.
Other grassland management options (which may alter the fungal and microbial biodiversity in soils):
7 Biochar When applied at high  Rafiq et al. Biochar addition to A lot of knowledge
incorporation rates, biochar could (2020) in grassland soil may also gaps as summarized by
into the double the organic C Tibetan plateau cause loss of native soil Brown et al. (2023):
grassland soil in the topsoil of many organic matter (SOM)  Biochar composition
degraded grasslands in short term, through is highly variable as
(Brown et al., 2023). the addition of labile it is being derived
nutrients leading to from different raw
priming (Cross & Sohi, materials and quality
2011; Wang et al., and produced under
2020). different pyrolysis
conditions, so what
effect does these
differences have on
the ability to store C
in grasslands?
 When is the best
time to add biochar
into grasslands, i.e.,
at reseed (one large
loading) or yearly
(repeated lower
loading rates)?
 Since grasslands has
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
already large stores
of SOC, is there a
negative priming
effect due to biochar
addition? Does this
response appears in
the field over long
time periods?
 What is the optimal
size of biochar (i.e.,
chunks/pellets/dust)
and how does this
affect application
technology and
agroecosystem
function?
 The interaction
effects between
biochar, slurry and
other organic
resources applied to
grassland need to be
explored.
 The interaction
impacts of biochar
with other potential
methods to sequester
C in agricultural
systems, such as
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
enhanced silicate
rock weathering and
iron mediated
stabilisation.
 What is the potential
effect on non-
CO2 GHG (direct
N2O, indirect N2O
(NO3− leaching,
NH3emissions) and
CH4 efflux) under
field conditions?
 The incorporation of
biochar to the topsoil
vs subsoil (e.g.
during deep tillage)

According to Zhang et
al. (2015):
 the application rates
of biochar
 the long-term effects
of biochar
amendments on SOC
storage under field
conditions.
 Biochar C stability
may be differ in
various climatic
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
conditions.
8 Fertilization  Fertilization  Poeplau et al.  Potential of GHG Future study could
increases in litter (2018) in the emissions due to N compare different type
inputs as a result of Netherlands and fertilization. and forms (liquid vs
higher plant Germany, with  However, Jasal et al. solid) of fertilizer,
production and to PK and NPK (2018) found that N rate/doses, timing, and
reduced SOM and (Poeplau et al., fertilization its methods of
humus 2018). decreased net GHG application on the soil
decomposition  Conant et al. by 2.28 t CO2 ha−1, C in grasslands under
(Jassal et al., 2008), (2017) (meta- thereby indicating a diverse climatic
thereby increasing analysis). favorable effect of conditions and their
SOC.  Chaplot et al. fertilization despite interlinkages with soil
 N fertilization (2016) in South significant N2O biodiversity.
enhances primary Africa. emissions.
production (Conant
et al., 2001), while
inhibiting soil
microbial
respiration
(Ramirez et al.,
2010), thereby
increasing C
allocation in soils.
 However, Poeplau
et al. (2018) found
that root C stocks
were not affected
by fertilization and
potential litter
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
decompostion was
unchanged,
meaning that
increased SOC
stocks could not be
explained by
altered
belowground C
inputs or
decomposition
rates.
9 C import from  Matsuura et al. Some of the manure Future study could
manure and (2020) in Japan, application treatments compare manure from
dairy effluent with low level resulted in losses in soil different animals and
of farmyard C stocks and changes in methods of application
manure. the distribution of C (traditional, injected,
 Maillard & with depth through the etc) on the soil C in
Angers (2014), soil profile, linked to grasslands under
with manure priming effects (Angers diverse climatic
application et al., 2010). conditions and their
(meta-analysis). interlinkages with soil
 Barkle et al. biodiversity.
(2000) in New
Zealand.

10 Irrigation  Increased plant  Trost et al.  Mudge et al. Further study should
productivity linked (2013) (meta- (2021) in New examine the impacts of
with irrigation has analysis). Zealxwand. irrigation under various
been assumed to methods (sprinkler,
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
enhance SOC open, etc.) and rate (%
(IPCC, 2019, Smith water applied) on the
et al., 2016b). soil C in grasslands
under diverse climatic
conditions and their
interlinkages with soil
biodiversity.
11 Earthworm  Earthworms  Lubbers et al.  Ferlian et al.  Earthworms emit Further study should
effect produce (2017) in the (2020) with CO2 and N2O by 33% examine the impacts of
microaggregates Netherlands, invasive and 42%, different earthworm
from their feeding found that earthworms respectively (Drake (anecic, epigeic) on the
and casting earthworms can (meta- & Horn, 2007; soil C in grasslands
activities, in which simultaneously analysis). Lubbers et al., 2013). under diverse climatic
the increase C  Lubbers et al.  Some species may be conditions and their
macroaggregates incorporation in (2017) in the invasive and alters interlinkages with soil
can mediate C aggregate Netherlands, native species. biodiversity.
stabilization fractions and found that
emit CO2. earthworms
can
simultaneously
increase C
incorporation
in aggregate
fractions and
emit CO2.
12 Liming  Liming removes an  Muñoz et al.  Kowalenko &  Liming adds Coming study should
acid-soil constraint (2013) in Chile Ihnat (2013) in carbonates that break concentrates on the
to plant growth,  Briedis et al. Canada down in acid soil to hypothesis that liming
resulting in greater (2012a,b) in  Moore et al. produce CO2. increases OC inputs
No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
biomass Brazil (2012) in  Liming increases the which stimulate
production, and  Srámek et al. Canada soil biological microbial activity and
thus enhanced (2012) in Czech  Chan & Heenan activity, thus increase microbial
SOC. Republic (1999) in favoring the biomass, thereby
 Increased SOC  Fornara et al. Australia mineralization of supporting the slow
stocks after liming (2011) in the  Persson et al. OM, resulting in CO2 build-up of MAOM and
is due to the UK (1995) in losses and a decrease C accrual by microbial
combined effects  Mijangos et al. Sweden of the SOC stocks transformation of plant-
on OC inputs, (2010) in Spain (Paradelo et al., derived C (Schroeder et
physicochemical  Poulton et al. 2015; Lochon et al., al., 2024).
protection of OM, (2003) in the 2018)
microbial activity, UK
and the microbial  Šimek et al.
metabolic (1999) in Czech
efficiency (direct Republic
and indirect  Sapek &
controls) Burzynska
(Schroeder et al., (1996) in Poland
2024).  Derome (1990)
 Liming ameliorates in Finland
soil structure,
increasing the
stability of clay
assemblages and
clay-OM bonds,
thereby increasing
SOC physical and
physicochemical
protection.
 No. Grassland Underlying (+) result(s) on (-) result(s) on Constraint(s) Research
management mechanism(s) soil C soil C gap/novelty/suggestion
interventions sequestration sequestration for future studies
(Paradelo et al.,
2015)
86 (+): increases SOM and (-): decreases SOM
87
88 Table 3 and Figure 2 illustrates how different management options affects soil C storage through meta-analysis.
89
90 Table 3. Changes in soil C concentration summarized by type of management change implemented (Conant et al., 2017)
Treatment Soil C concentration (%) Change (%)
Initial Final

Conversion: cultivation to grass 0.97 1.35 +39.2

Conversion: native to grass 2.97 2.55 −14.0
Earthworms 2.24 2.89 +28.8
Fertilization 3.44 3.85 +11.8
Fire 1.09 1.20 +10.5
Grazing 2.62 2.89 +9.99
Grass ley 2.50 2.68 +10.3
Reclamation 8.0 15.9 +98.8
91
 92
93 Figure 2. C sequestration from various grassland management (Jordon et al., 2024)
94
95
96 IV. MATERIAL AND METHODS
97
98 4.1. Site description
99
100 The detailed site for the experiment will be discussed in more detail with the supervisors, either in Saskatoon or Swift Current in Canada.
101 Saskatoon has a humid continental climate, no dry season, and a warm summer climate. The average elevation of the city is 491.31 meters above
102 sea level. Saskatoon's annual temperature is 3.01 ºC, which is 0.92% higher than Canada’s averages. Saskatoon receives roughly 12.68 mm of
103 precipitation and has around 26.35 rainy days a year (Weather and Climate, 2024). Meanwhile, the Swift Current climate is typically cold and
104 temperate, with an average elevation of 817 meters above sea level. The average annual temperature and precipitation in Swift Current are 4.7
105 °C and 491 mm, respectively (Climate Data, 2024). Both locations are classified as Dfb according to Köppen and Geiger (Climate Data, 2024;
106 Weather and Climate, 2024).
107
108 4.2. Plot treatments

109 A more detailed set of plot treatments will be decided with the supervisors’ agreement. The management intervention that will be investigated
110 further could be one or more combinations of some management interventions (Table 4). According to Tian et al. (2023), further studies should
111 incorporate more biotic (microbial) and abiotic factors to be able to quantitatively identify the contribution of diverse mechanisms to soil C
112 sequestration. Such a thing will be beneficial to fully understanding the relationship between above- and below-ground components of temperate
113 vegetation. Nevertheless, all of these treatments have to be discussed further with the PhD supervisors.

114 Table 4. The example of treatments to be used in the trial

No. Potential treatment plots
1 Sowing legume with different types of legume.
2 Sowing non-legume grass with a species or various species.
3 Sowing high-yielding plant species. According to Aasen & Bjorge (2009), some examples of high-yielding plant species in Canada
especially Alberta are alfalfa, red clover, sweetclover, smooth bromegrass, tall fescue, meadow foxtail, reed canarygrass, intermediate
wheatgrass, slender wheatgrass, and tall wheatgrass.
4 Grazing management, such as intensive grazing, moderate grazing, rotational grazing, ungrazing, etc.
5 Conversion of bare/arable land to grassland with different types of non-legume species.
6 Addition of biochar, which is made from various raw materials and quality and different pyrolysis conditions, with different size of
biochar (chunks/pellets/dust, etc.), different application rate, and under different application time (i.e., at reseed (one large loading) or
yearly (repeated lower loading rates)).
7 Addition of manure/slurry derived from different animals and various application methods (traditional, injection, etc).
8 Etc.
Combination of more than one points above (the interactive effects of some treatments), for instance:
 Sowing legume x sowing non legume grass
 Sowing legume x grazing management
 Sowing legume x sowing high-yielding forage grass
 Sowing legume x addition of biochar
 Sowing legume x liming
 Sowing legume x addition of manure/slurry derived from different animals and various application methods (traditional, injection, etc).
 No. Potential treatment plots
 Etc.
115
116
117 4.3. Material
118
119 Materials needed for the experiment include a soil sampler, plastics, an oven, a SoliTOC analyzer, stainless steel crubicles, a 2-mm mesh sieve, a
120 ball mill, and an autosampler. The SoliTOC instrument can work either with the classical direct and different methods (according to DIN 15936)
121 or with the temperature ramp method (according to DIN 19539). The latter does not require sample preparation or the use of acids. Overall, the
122 carbon content can be measured simply and reliably using the instrument.
123
124 4.4. Methods
125
126 The soil sampling and soil C measurement will be done before and after the treatments. Soils will be sampled randomly through the treatment
127 plots.
128
129 4.4.1. Soil sampling
130  At each site, plots measuring 5 × 5 m will be established.
131  Stratified soil samples are collected at 0–10, 10–20, and 20–30 cm depth.
132  At each sampling plot, at least five independent samples per plot are taken using a soil sampler after surface litter is removed.
133  All samples of the same soil depth per plot are pooled into one sample, thereby, there are three samples per plot.

134 4.4.2. Soil C measurement

135  Soil samples are firstly air-dried with an oven at 50◦C until constant weight. Next, they are cooled in the air temperature.
136  The soil samples are sieved with a 2-mm mesh sieve, then handpicked to remove fine roots and other coarse debris.
137  The soil samples are ground in a ball mill until they become homogenous powders.
138  Homogenous powdered samples (± 40-100 mg) are weighed in stainless steel crucibles and placed in an autosampler.
139  The analysis was carried out with a “smart combustion” method using SoliTOC (Zethof et al., 2019), involving a three-step heating of the
140 samples to 400, 600, and 900 °C with holding times of 230, 120, and 150 s, respectively, to measure different C fractions, i.e., :
141 (i) the thermally labile organic C (TOC400) is stripped out at temperatures below 400 °C
142 (ii) the residual oxidizable C (ROC) at temperatures of 500–600 °C, and
143 (iii) the total inorganic C (TIC) is derived from the thermal breakdown of carbonate minerals at 650–850 °C.
144  The total C (TC) is calculated using the instrument as the sum of TOC and TIC.
145
146 4.4.3. Measurement of total N and the other parameters
147 Measurement of the total N will be done to quantify the C:N ratio. It is also likely to measure the other soil parameters, such as pH. The detailed
148 method for the measurement of the total N and the other soil parameters will be decided later (adjusting the tools available in the lab).
149
150 4.5. Climatic data
151 Climatic data such as mean annual temperature and mean annual precipitation will be collected from the Canada.ca/weather. When possible, the
152 data on potential evapotranspiration (PET) will also be collected for all the sites.

153 4.6. Statistical analysis

154
155 Regression analyses will be carried out based on the means calculated for each site (and not based on the plot-level data since the observations
156 for different plots of one site are not independent of each other).
157
158 V. POSSIBLE CONSTRAINTS
159
160 The following are the possible drawbacks for this research that are likely impacting the quality, delivery, and overall project “success”.
161  Choosing the treatments may be tricky, as there are a lot of similar studies on the general topic (although there are also many research
162 gaps and novelty), thereby a careful planning in choosing the treatments should be done with a robust literature review and discussion
163 with the supervisors.
164  There might be high variations in soil types and climate conditions between treatment plots that may affect the results, indicating the
165 importance of preliminary soil test and careful selection of the plots.
166
167 VI. TIMETABLE
168
169 The PhD study will last for approximately four years. The following is the initial planned annual timetable (Table 5). The timetable is tentative.
170
171 Table 5. Planned annual timetable
Stage Activity Estimated Deliverable
duration
 Research Finalizing research 1 month Confirmed research problem/questions
design and problem/questions
planning Developing research design Draft research design section for final report
Preparing research proposal Research proposal/ethical approval
submission
Literature Searching, capturing and 1 month Notes and other output from the review
review synthesizing relevant literature process
Preparing literature review draft Draft literature review section for final report
Data collection Sampling plan finalization 10 months Sampling plan
Developing data collection Draft data collection instruments
instrument
Pre-test/pilot data collection Finalized data collection instrument
instrument
Data collection in the field Raw data
Data collection writing Draft data collection section for final report
Data analysis Data preparation for analysis 6 months (can be done Data ready for analysis
in parallel with data
Data analysis collection) Statistical method
Drawing conclusions/ Draft data analysis and findings section final
recommendations report
Writing Final draft for dissertation 6 months (can be done Final draft
Draft review in parallel with data Notes of feedback
Final editing collection and Final application
Submission of the dissertation to Final submission of the dissertation
the supervisors analysis)
Searching for a relevant top-tier Found a suitable journal
journal
Submission to the top-tier journal Final acceptance in the top-tier journal
and revision process
172
173
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