Master's Project – ENV690

Experimenting with Modified Extruded Seed Pellets for Large Scale

Mine Rehabilitation

An example experimental plot at BHP's Mt Whaleback mine. Image: E. Stock

Emma Stock

BSc (Env Science/Env Restoration), Grad Cert Environmental Assessment & Management.

Environmental and Conservation Sciences

College of Science, Health, Engineering and Education

June 2019

Supervisors: Dr Rachel Standish, Dr Todd Erickson, Dr Miriam Muñoz-Rojas, Prof Richard Bell

This document is referenced according to Restoration Ecology journal format

i

Declaration

I declare that this thesis is my own account of my research,

with acknowledgement of external contributions,

and contains as its main content, work which has not been previously

submitted for a degree at any tertiary education institution.

Emma Stock

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Acknowledgements

I would like to thank my team of supervisors: Dr Rachel Standish, Dr Todd Erickson, Dr Miriam

Muñoz-Rojas and Prof Richard Bell for their generous and helpful advice, constant encouragement,

timely feedback and practical support in the field in extreme temperatures. Thanks also to the

Environment and Rehabilitation teams at BHP Mt Whaleback iron ore mine for providing permission,

logistical support, transport, and even isotonic icypoles while I conducted this experiment.

Thank you to the science team at Kings Park Botanic Garden for technical assistance and conducting

laboratory testing, and to the Ramaciotti Centre for Genomics at University of New South Wales for

conducting DNA sequencing. Cheers to fellow students Monte Masarei and Amber Bateman for

discussions and entertainment in the field. Thanks also to the wonderful staff at the Newman

Recreation Centre for my daily de-stress workout and to all the friends who encouraged me as I

navigated the steep curve of learning to use R software.

This research was conducted as part of the Global Innovation Linkages project grant (GIL53873)

titled “Eco-engineering solutions to improve mine-site rehabilitation outcomes” funded by the

Department of Industry, Innovation and Science. Logistical support was also funded by Rosemary

Grigg and Peter Flanigan.

In memory of Larry the Triodia seedling.

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Abstract

Current methods of mine rehabilitation have a high failure rate at the seedling emergence stage due

to the limited availability of topsoil and the low water holding capacity of alternative growth media

such as waste materials and tailings. Extruded seed pellets, formed by extruding soil mixtures and

seeds into pellets, can potentially increase soil water availability through enhanced soil-seed contact

and improve seedling emergence in arid systems. I tested a modified extruded seed pelleting

method in a three-factor field experiment in a purpose-built mine rehabilitation research facility in

the Pilbara region of northwest Western Australia. The aims of the experiment were to assess (i)

native seedling emergence and establishment from modified extruded seed pellets and; ii) the

physico-chemical and microbiological changes that occur with seedling emergence from pellets. I

tested three native plant species and the experimental factors were pellet soil mixtures, with or

without Triodia pungens biomass as an organic amendment and rainfall regimes. Results suggests

trade-offs among responses. Pellets made with a 50:50 blend of topsoil and waste material had the

highest seedling emergence, while 100 % topsoil pellets had lower emergence probably because of

hardsetting. Triodia pungens survived to the end of the experiment but Indigofera monophylla and

Acacia inaequilatera did not. Adding Triodia biomass amendment inhibited Triodia seedling

emergence but increased soil microbial activity and could therefore stimulate recovery of soil

function. Pellets preserved seed viability and topsoil microbial communities during the growing

season and therefore show potential as a means to inoculate rehabilitation sites with both seeds and

microbes. Overall, modified extruded 50:50 seed pellets show promise for rehabilitation of keystone

species Triodia and beneficially, make use of ‘waste’ material. Further research is needed to improve

pelleting methodology for other species, and to test the applicability of the method at scale, in this

and other arid land ecosystems.

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Table of Contents

Declaration ............................................................................................................................................... i

Acknowledgements ................................................................................................................................. ii

Abstract .................................................................................................................................................. iii

List of Figures .......................................................................................................................................... v

List of Tables ........................................................................................................................................... v

1. Introduction ........................................................................................................................................ 1

2. Materials and methods ....................................................................................................................... 4

2.1 Experimental design ...................................................................................................................... 4

2.2. Pellet preparation ........................................................................................................................ 5

2.3 Plot preparation ............................................................................................................................ 6

2.4 Rainfall simulation......................................................................................................................... 8

2.5 Soil analyses .................................................................................................................................. 9

2.6 Plant growth analysis .................................................................................................................. 10

2.7 Statistical analysis ....................................................................................................................... 10

3. Results ............................................................................................................................................... 11

3.1 Seedling emergence. ................................................................................................................... 11

3.2 Seedling survival.......................................................................................................................... 16

3.3 Plant aboveground biomass........................................................................................................ 16

3.4 Soil analysis. ................................................................................................................................ 17

4. Discussion .......................................................................................................................................... 20

4.1 Modified extruded seed pellets for arid land restoration. ......................................................... 20

4.2. Rainfall and pellet type effects on seedling emergence. ........................................................... 23

4.3. Rainfall and pellet type effects on seedling survival. ................................................................. 24

4.4 Microbial activity and community composition ......................................................................... 25

4.5. Implications for large-scale mine rehabilitation. ....................................................................... 25

Conclusion ............................................................................................................................................. 27

Literature Cited ..................................................................................................................................... 28

Appendix ............................................................................................................................................... 34

v

List of Figures

Figure 1: Drydown curve for soil pellets……………………………………………………………………………………………. 6

Figure 2: Experimental design…………………………………………………………………………………………………………… 6

Figure 3: One completed experimental quadrat………………………………………………………………………………… 7

Figure 4: Boxplot showing thickness of pellets………………………………………………………………………………….. 8

Figure 5: Seedling emergence and survival……………………………………………………………………………………… 13

Figure 6: First round and total emergence………………………………………………………………………………………. 14

Figure 7: First round and total survival……………………………………………………………………………………………. 16

Figure 8: Total aboveground biomass……………………………………………………………………………………………… 17

Figure 9: Soil physico-chemical nMDS ordinal matrix………………………………………….………………………….. 17

Figure 10: Microbial activity within pellets……………………………………………………………………………………… 18

Figure 11: Relative abundance of soil microbial communities…………………………………………………………. 19

Figure 12: Soil microbial group nMDS ordinal matrix………………………………………………………………………. 19

Figure 13: Pellet responses to wetting and drying cycles…………………………………………………………………. 22

List of Tables

Table 1: Effect of rainfall amount and pellet type on first round and cumulative emergence………….. 16

Table 2: Effect of rainfall amount and pellet type on first round and total survival of Triodia…………. 16

Table 3: Summary of experimental results………………………………………………………………………………………. 20

Table 4: Physico-chemical properties of soils………………………………………………………………………………….. 33

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1. Introduction

Successful mine rehabilitation requires the design and construction of safe and stable landforms and

the establishment of functional and self-sustaining ecosystems to meet the desired post-operational

land use (Australian Government, 2016). Rehabilitation is therefore an essential post-mining activity

that requires companies to plan, develop, and execute effective landform reconstruction, soil

management, and planting techniques to achieve certain regulatory criteria aligned with positive

ecosystem recovery. Delivered correctly, successful rehabilitation ensures the mining industry’s

ongoing social and environmental licence to operate (Australian Government, 2016). In Australia,

few examples exist of mine rehabilitation that has reached regulator-approved completion metrics

(Lamb et al., 2015; Commonwealth of Australia, 2019), particularly in arid systems where plant

establishment is challenged (Shackelford et al., 2018). This lack of rehabilitation capacity jeopardises

the economic viability of the mining industry as well as the biodiversity and sustainability of the

impacted ecosystems (Cross et al., 2017; Stevens & Dixon, 2017).

The arid Pilbara region of north-west Western Australia, a resource-rich area covering approximately

179,000 km2 (McKenzie et al, 2009), is subject to intensive mining, with a cumulative footprint

comprising at least 3,000 km2 of mined and disturbed land requiring rehabilitation (EPA, 2017).

Although human activity has led to the region being characterised as a biodiversity hotspot (Pepper

et al., 2013; Department of Environment & Energy, 2019), development continues to accelerate

(EPA, 2018). At this scale, there is a critical need to develop cost-effective, repeatable and large-scale

methods to restore functional landscapes and preserve biodiversity (Kildisheva et al., 2016; Muñoz-

Rojas et al., 2017b). Yet, rehabilitation performance still lags behind the need for rehabilitation at

scale. For instance, a recent synthesis of Pilbara mine rehabilitation showed that most sites lack

comparable plant cover, density and species diversity values when compared to un-disturbed

reference plant communities (Shackelford et al., 2018).

Mine rehabilitation in the Pilbara is constrained by multiple abiotic and biotic barriers. Moisture

availability is the primary limitation, with highly variable rainfall associated with unpredictable

thunderstorms and cyclones, and evaporation that exceeds annual rainfall by up to 10-fold across

the region (Charles et al., 2015). Native plant recruitment in such arid environments is spatially and

temporally irregular, with a high degree of climate sensitivity (Audet et al., 2013; Svejcar &

Kildisheva, 2017). Episodic precipitation events in arid ecosystems also control various physico-

chemical and biological soil processes (Schwinning & Sala 2004). For instance, pulses of biological soil

activity, and therefore nutrient cycling, are often limited to periods of high soil moisture conditions

(Austin et al., 2004; Ford et al., 2007; Collins et al., 2008; McIntyre et al., 2009).

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Plant regeneration is further constrained by Australian native seed recruitment physiology which has

evolved complex inherent seed dormancy mechanisms that limit germination, (Lamb et al., 2015;

Erickson et al., 2017) and wild seed production is variable within and between seasons (Ritchie et al.,

2017). Wild-sourced seeds are expensive and the quantities required to cover the affected

disturbance area exceed the capacity of natural seed production and collection (Broadhurst et al.,

2015; Nevill et al., 2016). Therefore, ways to maximise the efficient and responsible use of native

seed resources is required and will also generate cost savings (Merrit & Dixon, 2011; Golos et al.,

2019; Pérez et al., 2019).

One of the biggest challenges in mine rehabilitation is to restore soil function in reconstructed soil

profiles (Muñoz-Rojas et al., 2016a; Cross et al., 2018). Intensive mining activity leaves behind large

amounts of degraded soils and growth media, such as waste materials and tailings, which have

altered properties that are hostile for seedling development: in particular, low organic matter and

nutrients, reduced water holding capacity and low microbial activity (Jasper et al., 1988; Muñoz-

Rojas et al., 2016a; Lamoureux et al., 2016; Bateman et al., 2018). In these altered soils, the

establishment of native plant species can be extremely difficult, with a potential consequential shift

in ecosystem function or loss of biodiversity (Muñoz-Rojas, 2017b; Williams et al., 2017). A

multidisciplinary science-based approach is therefore needed to fill essential knowledge gaps that

can improve seed and growth media management practices and deliver benefits to large-scale mine

rehabilitation programs (Erickson et al., 2017; Miller et al., 2017).

The current best practice method of rehabilitation is to cover re-profiled waste material with

stockpiled topsoil (initially salvaged from the top 10-20cm of the pre-disturbed soil profile), but

topsoil quantities are limited and quality declines over the duration of stockpiling (Cooke &

Johnston, 2002; Golos et al., 2016; Merino-Martin et al., 2017a). The use of waste materials from

mining operations as an alternative growth media is an option, but these substrates may require soil

amendments or accelerated weathering to facilitate the development of pedogenic processes

(Muñoz-Rojas et al., 2016b; Kumaresan et al., 2017; Kneller et al., 2018). Inorganic soil amendments

such as gypsum and urea have been trialled but results vary and can be expensive and impractical at

scale (Bateman et al., 2019). Further, organic amendments including compost, mulch and sewage

have been shown to increase water holding capacity and soil microbial activity (Bastida et al., 2008;

Beningo et al., 2013; You et al., 2016; Kneller et al., 2018), but are also difficult to apply across large

areas.

The native perennial grass genus Triodia, or spinifex, is an underused biomass resource with

beneficial material properties which are being investigated for a range of purposes (Gamage et al,

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2012; Pennells et al., 2018). Triodia biomass is being explored as an organic soil amendment as the

plants are commonly available across aridlands, and sustainable harvesting can be considered as an

alternative to burning for management (Gamage et al., 2014). Experiments show that incorporating

Triodia biomass can increase microbial activity and C mineralisation in mine wastes (Kneller et al.,

2018).

The use of soil microbial inoculum as a potential scalable restoration tool has been investigated

(Bowker, 2007). Soil microbial communities are critical to the functioning of terrestrial ecosystems

and it is increasingly recognised that the manipulation of these communities for restoration can

enhance the rate of recovery after disturbance (Rawls et al., 2003; Harris, 2009; Bardgett & van der

Putten, 2014; Bastida et al., 2015; Wubs et al., 2016; Kumaresan et al., 2017; Bastida et al., 2017).

The inoculation of rehabilitation sites with soil microorganisms such as cyanobacteria, a phylum of

poikilohydric organisms that can potentially restore soil functionality, is a recent avenue of research

(Bowker, 2007; Williams et al., 2017; Muñoz-Rojas et al., 2018a; Muñoz-Rojas et al., 2018b; Román

et al., 2018). Due to the importance of interplay between biotic and abiotic factors in soil

functionality, restoration strategies need to re-establish both plant communities and soil health

(Kildisheva at al., 2016; Kneller et al., 2018)

One promising approach to improving success in seed based restoration programs is the application

of engineering solutions incorporating seed enhancement technologies combined with precision

delivery methods (Kildesheva et al., 2016; Brown et al., 2018; Nevill et al., 2018; Madsen et al.,

2018). Seed coating technologies are commonly used in the agricultural industry and may improve

cost effectiveness in the rehabilitation arena by reducing seed wastage (Madsen et al., 2016a;

Pedrini et al., 2017; Turner et al., 2006). Another technique is to make extruded pellets containing

seeds mixed with beneficial soil materials and amendments which help the seedlings through the

emergence phase. Such seed enhancement technologies with increased matrix material can improve

seed-soil contact and provide more favourable soil water potentials, which are conducive for

seedling survival and enhanced mineralisation through greater microbial activity (Davies et al., 2018;

Lewandrowski, 2017a; Madsen et al., 2016b). Using this approach, seeds are incorporated into

pellets that are formed by squeezing dough-like soil mixtures through modified food processing

equipment. However, early ex-situ seed pelleting trials incorporating treated seeds (i.e. to alleviate

innate seed dormancy) found that Triodia species (a critical keystone grass in arid ecosystems) failed

to germinate. This failure was attributed to the mechanical pressure of the extrusion process

reimposing physiological dormancy (Erickson, pers. comm. July 2018). A novel modified extruded

seed pelleting method, analogous to seed pelleting but with less pressure, was proposed to reduce

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extrusion pressure on the sensitive seeds and therefore alleviate dormancy and related recruitment

issues.

Here, I tested modified extruded seed pelleting in a plot scale experiment as a method of re-

establishing target native plant species and soil microbial processes in the Pilbara region of

northwest Western Australia. The specific objectives of the experiment were to assess (i) native

seedling emergence and establishment from modified extruded seed pellets (ii) the effect of adding

Triodia amendment on the germination and establishment of selected native plant species in

reconstructed mine waste soils and (iii) the physico-chemical and microbiological changes that occur

with seedling emergence from pellets. The implications of my results for achieving scalability in

aridland mine rehabilitation are also discussed.

2. Materials and methods

2.1 Experimental design

A three-factor field experiment was conducted within a purpose-built rehabilitation research facility

at BHP’s Mt Whaleback iron ore mine, near Newman in the Pilbara, Western Australia (-23°21'55.30"

S, 119°40'31.4" E). The facility comprises a rain out shelter covering 64 irrigated, raised beds (400 cm

× 200 cm × 25 cm) filled with five different reconstructed soils sourced from post-mining materials.

These raised beds, hereafter ‘plots’, have been designed to allow the evaluation of seedling

emergence and growth under a range of soil treatments and rainfall conditions to simulate field

settings in mine site rehabilitation (Erickson et al., 2017). Three common native plant species utilised

in Pilbara rehabilitation programs were tested. Triodia pungens, a soft-leaved spinifex, is a keystone

species commonly occurring on the moister downslopes of arid environments and cued to germinate

following fire and periods of high precipitation (Lewandrowski et al., 2017b). Additionally, two

legumes, Acacia inaequilatera and Indigofera monophylla, were selected in part for their reliable

establishment in field conditions.

The experimental design included three fixed factors: soil blend (two levels), organic amendment

(two levels) and rainfall regime (two levels). Specifically, modified extruded pellets were made from

two different soil blends, with or without the addition of an organic soil amendment in the form of

dried T. pungens leaf material, and exposed to two rainfall regimes. Fully factorial treatment

combinations were replicated nine times across six plots (n = 2 × 2 × 2 × 9 × 6 = 216 experimental

units in total). The two soil blends were 100 % stockpiled topsoil (TS) and a 50:50 % blend of

stockpiled topsoil and waste material (TS/WM). The experiment ran for 16 weeks over the summer

season, from October 2018 to February 2019, to coincide with both the optimum germination

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temperature range for selected species, at around 20 – 35 oC (Erickson et al., 2016a; Erickson et al.,

2016b) and when native plants usually recruit (i.e., after summer rainfall). The facility is open on all

sides and so air and soil temperatures in the facility approximated outside temperatures.

2.2. Pellet preparation

Seeds were sourced from either Bushland Native Seeds Supply (Australind, WA) or Nindethana Seed

Service (Albany, WA) in October 2010 for Acacia inaequilatera, September 2014 for Indigofera

monophylla, and March 2015 for Triodia pungens. Seeds were stored at constant temperature and

relative humidity (i.e., 15–20 °C, 30–50 % relative humidity), between their collection and pellet

preparation. Prior to pelleting, T. pungens seeds were removed from the floret to overcome floret-

imposed physiological dormancy (after Erickson et al., 2016a). The two legume species were treated

with hot (90 °C) water for 1–2 minutes to break dormancy (after Erickson et al., 2016b). Seed

germination on water agar after 28 days at constant 25 °C was 86 % ± 2.6 % for T. pungens, 31 % ±

1.9 % for I. monophylla and 69 % ± 1.0 % for A. inaequilatera. A standard mix of 40 T. pungens, 15 I.

monophylla and 10 A. inaequilatera seeds was included in each pellet.

Each pellet was prepared separately to maintain consistency in moisture content and seed

quantities. Soil blend quantities were determined through preliminary experimentation to replicate

a roughly circular ‘cow pat’ shape with a target thickness of 5–10 mm to match a suitable

germination and emergence depth for T. pungens seeds (Erickson et al., 2017). The soils used to

make the extruded pellets were sourced from stockpiled topsoil (TS) or waste material (WM) and

sieved to obtain a < 5 mm fraction for use. Soil was weighed and hand mixed with water to obtain a

slurry consistency. TS pellets contained 101 g of soil and 33 g of water, whereas TS/WM pellets

contained 109 g of soil and 28 g of water. Triodia leaf material was air dried for six weeks and cut

into approximately 5 mm pieces; 0.4 g was added to each pellet. This amount of material

approximated that of the amounts of organic material in topsoil, but then halved to reduce the

priming effect (sensu Kneller et al., 2018) following the principle of minimum effective dosage

(Hueso-González et al., 2018). For ease of reporting, pellet types are labelled with their acronym i.e.,

TS (100% topsoil), TS+TB (100% topsoil with Triodia biomass amendment), TS/WM (50% topsoil

blended with 50% waste material) and TS/WM+TB (as previous with amendment added).

The pellets were air dried for approximately 48 h prior to watering. To ensure that pellets dried

rapidly enough to prevent imbibition and germination prior to experimental watering (i.e., a ‘false

start’), I first tracked pellet weights over time in a preliminary pilot study to obtain a dry-down curve.

Results indicated that pellets were dry within 6 h of extrusion, which is shorter than the time when

6

seed imbibition reaches its maximum at between 8 h and 48 h (Erickson et al., 2016c) (Figure 1).

Thus, experimental pellets likely dried in time to prevent a false start.

Figure 1. Dry-down curve for freshly constructed soil pellets (n=3).

2.3 Plot preparation

The experiment utilised six 4 m × 2 m (8 m2) plots of a uniform waste material (WM) substrate

interspersed throughout the facility. Refer to appendix for soil properties of WM. Three 1 m × 2 m

quadrats were evenly spaced across each plot, and twelve modified extruded pellets were placed

within each quadrat; three pellets each of the four pellet types. To minimize the frequency of

manufacturing errors, pellets were clustered by soil blend type, which was randomised across each

quadrat (Figures 2 and 3).

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Figure 2. Experimental design showing example 4 m × 2 m plot containing substrate waste material soil with three 1 m x 2

m (2 m2) quadrats spaced evenly across the plot. Each quadrat contained twelve pellets; three each of four different pellet

types. To minimize manufacturing errors, pellets were clustered by soil blend type, which was randomised within each

quadrat. Experimental units are individual pellets (i.e. n = 216).

Figure 3. One completed experimental quadrat.

Pellets were delivered via hand extrusion from a 60 ml Terumo syringe (Terumo Corporation,

Philippines) with the end removed to create a 30 mm opening. Soil blends containing 100 % TS were

extruded from a height of 60 cm and those containing a 50:50 % blend of TS/WM were extruded

from 40 cm. The drop heights were determined experimentally as a trade-off between mixture

fluidity, obtaining the desired surface coverage and thickness, the constraints of retaining the

integrity of pellets on impact and the projected operational requirements of large-scale machine

delivery in the future. Pellet thickness was subsampled by measuring the vertical and horizontal

diameter of pellets in the centre quadrat of each plot (Figure 4) indicating that there was a

significant difference in thickness between pellets of the two soil types and there was a small

increase in thickness induced by the addition of Triodia biomass.

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Figure 4. Boxplots (minimum, first quartile, median, third quartile and maximum, n=18) showing thickness of pellets as a

function of pellet type. There was a difference between the topsoil (Pellets 1 & 2) and the blend (Pellets 3 & 4) and a small

increase in thickness introduced by the Triodia biomass (Pellets 2 & 4).

2.4 Rainfall simulation.

Three plots were randomly allocated one of two rainfall regimes to facilitate seed germination and

seedling growth. Rainfall was simulated via Hunter MP1000 Rotator sprinkler heads applied at 10

mm/h (Hunter Industries, San Marcos, CA, USA). Water was sourced from groundwater pumped

from an active mine pit, with indicative water quality parameters of pH 8.1 and EC 1100 µS/cm.

Rainfall amounts of 120 mm and 60 mm were delivered to mimic large rainfall events typical of the

Pilbara wet season (Bureau of Meteorology, 2019), with the duration of simulated rainfall selected

to maximise soil water potentials and therefore the envelope for germination and emergence (after

Lewandrowski et al., 2017a). Adjacent plots were paired and irrigation was applied alternately in 2 ×

15 mm or 2 × 7.5 mm events every second day for 7 d to achieve the rainfall totals (i.e. 4 × 30 mm

over three plots and 4 × 15 mm over three plots). Follow-up rainfall pulses of 10 mm were

subsequently applied across all plots fortnightly to mimic the soil wetting and drying cycles of

sporadic wet season rainfall (Bureau of Meteorology, 2019). A second large rainfall event was

simulated during the 11th week of the trial, consisting of 60 mm applied across all plots in the same

manner as the first event. Volumetric soil water content (EC-5 ECH2O Dielectric Aquameter;

DecagonDevices Inc.) and soil temperature were recorded in two of the plots with Hobo® RX-3000

remote data logger stations (Onset Computer Corporation, Bourne, MA, USA).

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2.5 Soil analyses

A bulked soil sample of 1 kg was taken from the top 5 cm of each of the six plots and the TS, WM and

the 50:50 blend (TS/WM) prior to the field experiment. Samples were air dried, sieved < 2 mm and

divided into two subsamples; one to characterise physico-chemical properties and the other stored

at 4 oC pending analysis of soil microbial activity. In addition, five 3 g samples were randomly taken

from each plot and three from each of the donor soils for soil microbial community composition

analysis. These samples were refrigerated for transportation to the science laboratory at Kings Park

Botanic Garden and stored at -20 oC until analysis. Qualitative observations of pellet morphology

responses were recorded at the end of the first simulated rainfall event.

On completion of the trial, a 50 g sample (sieved < 2mm) was taken from the top 1 cm of plot soil

immediately beneath selected pellets and analysed for microbial activity and microbial community

composition. Within each plot, pellets were sampled according to whether they contained surviving

plants or not and soil type (n=8 per plot). The starting quadrat for each plot was randomly selected,

and the with/without plant samples were deliberately paired in space to reduce the contribution of

spatial (soil) heterogeneity to the difference between them. Subsequent samples were taken

progressively from quadrats from left to right. The pellets themselves were also collected from the

plot (P36) that was visually estimated to contain the most plant biomass.

Soil physico-chemical analyses were performed at the Chem Centre laboratory (Bentley, Western

Australia) following standard methods described in Rayment & Lyons (2011). Soil pH and electrical

conductivity (EC) were measured in 1:5 soil extract in deionised water, total organic carbon (TOC)

was estimated by loss on ignition at 500 °C and total nitrogen by the Kjeldahl method (Bremner &

Mulvaney, 1982). All other soil nutrients were determined by Mehlich Extraction (Mehlich, 1984)

followed by inductively coupled plasma-atomic emission spectroscopic (ICP-AES) analysis. Particle

size analysis was subcontracted to Microanalysis Australia and analysed by laser diffraction using a

Mastersizer 2000 (Malvern Instruments, Malvern, England).

Soil microbial activity was determined with the 1-day CO2 Solvita soil respiration test (Haney et al.,

2008). This method determines soil microbial respiration rate based on the measurement of the CO2

burst produced after moistening dry soil (Haney et al., 2008; Muñoz-Rojas et al., 2016). In brief, soils

were dried at 40 oC and 40 g of each sample were placed into 50 ml plastic beakers inside a 240 ml

glass jar. All soils were wetted by adding deionised water into the jar to achieve 50 % of water filled

pore space wetting, and a CO2 probe was inserted before incubation at 25 oC. Measurements were

taken after 24 h with a digital colour reader, a mini spectrometer specific for the Solvita test.

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Environmental genomic DNA was isolated from 500 mg of soil using the DNeasy PowerSoil Kit

(Qiagen, Venlo, Netherlands) according to the manufacturer’s instructions. The hypervariable

regions V3-V4 of the 16S rRNA gene were targeted using the primer set 341F-785R (Klindworth et al.

2013) tailored with Illumina adaptor overhangs and amplified according to the PCR conditions

described by Nielsen et al., 2017. Amplified products were submitted to the Ramaciotti Center for

Genomics (UNSW, Australia) where they were purified before a second indexing PCR to incorporate

unique Illumina Nextera XT indexes for each sample. Sequencing was performed on an Illumina

MiSeq using a v3 Reagent Kit with a 2 x 300 bp run format.

2.6 Plant growth analysis

Plant growth was monitored over the period of the trial with emergence and survival counts

conducted weekly for two weeks after each simulated 7-day rainfall pattern, and fortnightly

thereafter. After 16 weeks, survivorship, leaf stage and total biomass were assessed. Total above

ground biomass was determined by harvesting plant shoots and oven drying at 60 °C for four days

and weighing with a five-point balance.

2.7 Statistical analysis

Soil and plant variables (seedling emergence and survival) were tested for normality and variance

homogeneity using the Shapiro-Wilk and Levene’s tests. I explored plant growth over time but

focused on emergence and end-of-experiment survival and biomass for the analyses. While the

experimental design was 3-factor, I opted to use 2-factor ANOVA per species to test differences in

plant emergence, survival, biomass and microbial activity between rainfall amount and pellet types

for ease of interpretation. Comparisons between means were performed with the Tukey’s HSD

(honestly significant difference) test (p < 0.05). Variables were transformed where necessary to

normalise data (plotted data are untransformed).

Bioinformatic analyses of soil DNA sequencing data were performed at the Ramaciotti Center for

Genomics. Data were received de-multiplexed via the Illumina cloud-computer BaseSpace and

analysed using the inbuilt 16S Metagenomics application. Briefly, this service performs quality

filtering of sequences before taxonomic classification using the ClassifyReads implementation of the

Ribosomal Database Project Classifier as per Wang et al., 2007, referencing an Illumina-curated

GreenGenes taxonomic database (16S Metagenomics BaseSpace App Guide (version 15055860 A)

performed in April 2019). Stacked bars were created based on the operational taxonomic units

(OTUs) generated and to include only the top 10 most abundant OTUs at order level.

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Non-metric multidimensional scaling (nMDS) was used to investigate differences between soils and

microbial community data using Euclidean distance metric for soils and Bray-Curtis distance metric

for soil microbes. nMDS uses ranked distances to arrange soil types along a number of axes based on

the values for the soil properties within them. The placement of plots and number of axes in an

nMDS ordination were calculated as the solution of 50 randomisations, minimizing the final stress

between the dissimilarity in the original data matrix and that in the reduced ordination matrix

(Clarke & Gorley, 2015). A biplot, based on Pearson’s correlations, was drawn on the ordination to

display the relationship between the explanatory variables and the ordination axes, where the angle

and length of the line indicate the direction and strength of the relationship. All analyses were

performed with R statistical software version 3.5.1 (R Core Team, 2018) and Primer (Clarke & Gorley,

2015).

3. Results

3.1 Seedling emergence.

A large amount of seedlings emerged from the modified extruded seed pellets, with an overall

average of 7.3 plants per pellet (ca. 11 % emergence). However, fewer survived to the experiment

conclusion and establishment success depended on species and the timing of simulated rainfall

patterns (Figure 5). A high proportion of seeds that did not germinate in the first simulated rain

event remained viable, and then germinated and emerged after the second simulated rain event. Of

the three species, Triodia emergence was most successful with 887 seedlings (10 % of seeds) that

emerged in the first simulated rain event and 655 seedlings (8 % of remaining seeds) in the second

rain event. At the end of the experiment, 84 (9 %) and 295 (45 %) of these plants survived their

respective emergence rounds.

Indigofera was less successful than Triodia with 252 seedlings (i.e. 8 % of seeds) in the first round

and 138 seedlings (4 % of remaining seeds) in the second round. However, only one seedling from

the first round and 11 from the second round survived to the end of the experiment. While Acacia

emergence results were initially high (20 %) with 440 seedlings in the first round, the second round

produced only 10 seedlings and no Acacia plants survived the duration of the experiment.

After the first rainfall event, Indigofera seedling emergence was significantly higher (p ≤ 0.001, Table

1) under 120 mm rainfall (ranging from 1.22 ± 0.25 seedlings in TS+TB pellets to 1.78 ± 0.33

seedlings in TS/WM pellets) than under 60 mm rainfall (ranging 0.70 ± 0.18 seedlings in TS pellets to

1.00 ± 0.24 seedlings in TS/WM pellets). The difference between the 60 mm and 120 mm of

simulated rainfall, however, was not significant for the Triodia or Acacia seedlings (Figure 6, a & b).

12

There was no residual effect of the initial difference in rainfalls in the cumulative emergence results,

as all plots received a uniform amount of rainfall in the second simulated event (Table 1).

The effect of pellet type on first round emergence was significant for Triodia (p ≤ 0.001, Table 1) but

not for Indigofera or Acacia. Triodia emergence was highest in TS/WM pellets under both rainfall

regimes (6.52 ± 0.47 seedlings under 60 mm and 7.56 ± 0.48 seedlings under 120 mm) (Figure 6, a &

b). Triodia emergence was lowest in the TS+TB pellets under both rainfall regimes (1.55 ± 0.27

seedlings under 60 mm and 1.40 ± 0.25 seedlings under 120 mm). Triodia emergence under TS

pellets and TS/WM+TB pellets was not significantly different but results were lower for TS/WM+TB

pellets (3.67 ± 0.50 seedlings under 60 mm and 3.48 ±0.42 seedlings under 120 mm) than TS pellets

(4.25 ± 0.45 seedlings under 60 mm and 4.41 ± 0.55 seedlings under 120 mm). In the second round,

seedling emergence did not appear to be strongly influenced by pellet type (pers. obs.). I have not

reported these data it was wholly dependent on the number of seeds depleted by the first round

germination. Instead, I have reported first round and cumulative emergence. Cumulative emergence

results did not differ from the trend of first round emergence (Figure 6, c & d).

13

Figure 5. Seedling emergence (peaks) and survival (troughs) over the duration of the experiment (Day 1 = 2nd November 2018). Soil water content (Y2 axis) obtained from

data logger in Plot 50 and corresponds to the timing of the simulated rainfall events.

14

Figure 6. First round emergence under the two different rainfall regimes (a, b) and cumulative emergence after

the second simulated rainfall event (i.e. 60 mm) that was applied to all plots (c, 60 mm + 60 mm; d, 120 mm +

60 mm). Pellet types: TS – 100 % topsoil, TS+TB – 100 % topsoil with Triodia amendment, TS/WM – 50 % blend

of topsoil and waste material, TS/WM+TB 50 % blended soils with Triodia amendment.

15

Table 1. Effect of rainfall amount and pellet type on first round and cumulative (total) emergence. Statistical

significance levels: N/S = not significant at p = 0.05, *** = significant at p ≤0.001.

Species Factor First round emergence Cumulative emergence

F value P value F value P value

Triodia pungens Rainfall 0.11 N/S 0.87 N/S

Pellet type 53.97 *** 18.08 ***

Rainfall x Pellet type

0.40 N/S 0.66 N/S

Indigofera monophylla Rainfall 12.78 *** 0.00 N/S

Pellet type 1.31 N/S 2.37 N/S

Rainfall x Pellet type

0.07 N/S 0.53 N/S

Acacia inaequilatera Rainfall 1.37 N/S 0.83 N/S

Pellet type 0.05 N/S 0.10 N/S

Rainfall x Pellet type

0.08 N/S 0.09 N/S

Survival of Indigofera was low and no Acacia seedlings survived the duration of the trial (Figure 7) so

these results were discarded from survival analysis. The experimental factors were not significant on

the survival outcome for the first round of Triodia seedlings that emerged, which ranged from 0.15 ±

0.07 seedlings for TS/WM+TB pellets under 120 mm to 0.59 ± 0.14 seedlings for TS pellets under 120

mm. Similarly, rainfall and pellet type did not significantly influence total survival and the only

significant difference in total survival was between TS pellets (2.44 ± 0.33 seedlings) and TS/WM

pellets (1.04 ± 0.22 seedlings) under 180 mm total rainfall (p ≤ 0.05) (Table 2).

Table 2. Effect of rainfall amount and pellet type on first round and total survival of Triodia. Statistical

significance levels: N/S = not significant at p = 0.05, * = significant at p ≤ 0.05,

Factors First round survival Total survival

F value P value F value P value

Rainfall 0.2362 N/S 1.4868 N/S

Pellet type 1.8390 N/S 1.9377 N/S

Rainfall x Pellet type 0.5959 N/S 2.8129 *

16

3.2 Seedling survival.

Figure 7. First round survival under the two different rainfall regimes (a, b) and total survival after the second

simulated rainfall event (i.e. 60 mm) that was applied to all plots (c, 60 mm + 60 mm; d, 120 mm + 60 mm).

Pellet types: TS – 100 % topsoil, TS+TB – 100 % topsoil with Triodia amendment, TS/WM – 50 % blend of

topsoil and waste material, TS/WM+TB 50 % blended soils with Triodia amendment.

3.3 Plant aboveground biomass

Due to low survival of Indigofera and Acacia, above ground biomass data was pooled into a single

metric incorporating data for all three species. None of the experimental factors were significant at

p ≤ 0.05 and there was a large standard error due to a small number of large individual plants that

were evenly distributed across pellet types (Figure 8).

17

Figure 8. Total above ground biomass at the end of the experiment (n = 161, 149 of which were Triodia). Data

are mean ± SE; n = 16 to 22 per treatment combination.

3.4 Soil analysis.

Figure 9. Soil nMDS plot of plots (P41 to P53) and pellet ingredients (TS = Topsoil, WM = Waste Material) based

on Euclidean distance; stress = 0.03. Pearson correlation coefficients for biplot > 0.8. For results of individual

soil properties refer to Table 4 (Appendix). MA CO2 = Microbial Activity (CO2 –CO ppm)

18

Results for initial soil physico-chemical properties split the data into pellets and substrates along the

vertical axis (Axis 2) correlating with sand and silt (Figure 9). The horizontal axis (Axis 1) correlating

with Na, pH and clay further split the pellets into three distinct groups but also separated the plot

substrates, with P41 being different from the rest with higher clay content. The 50:50 blend pellets

were half way between TS and WM, which appeared to be favourable for Triodia emergence.

At the end of the trial, substrate soil microbial activity was slightly higher under the higher total

rainfall, but this was not significant at p ≤ 0.05. There was no significant effect detected between

pellet types or between pellets with or without plants. Pellet microbial activity was significantly

increased with the addition of Triodia biomass amendment, with 24.3 ± 4.79 mg/kg CO2-C in TS+TB

pellets and 32.4 ± 0.75 mg/kg CO2-C in TS/WM+TB pellets (Figure 10).

Figure 10. Microbial activity within pellets at the end of the trial (n = 4, 4, 4 and 2). There were insufficient

samples to analyse different rainfall regimes. Data are means ± SEs.

The pellets maintained the full microbial community that was present in the topsoil for the duration

of the trial (Figure 11). The dominant bacterial groups were Actinobacteria and Proteobacteria which

preferred the topsoil substrate. Cyanobacteria was present in the topsoil and persisted in the pellets

but was not present in the waste material. There was some evidence that Cyanobacteria (yellow,

Figure 11) was beginning to transfer to the soil underneath, as the subpellet soil was positioned

between the pellet and plot soils in the nMDS (Figure 12).

19

Figure 11. Relative abundance of soil microbial communities.

Figure 12. Soil microbial groups nMDS plot (Bray-Curtis dissimilarity matrix). Stress = 0.14.

20

4. Discussion

My experiment demonstrated proof of concept for the efficacy of modified extruded seed pellets as

a medium for establishing Triodia on a waste material blend in an arid zone. A compelling result was

that Triodia pungens emerged from the pellets and survived to the experiment conclusion. Generally

too, pellets prolonged seed persistence in the soil and preserved the range of soil microbial

communities present in topsoil, and therefore shows potential as a means to inoculate rehabilitation

sites with both seeds and microbes. The range of results suggests trade-offs among responses and

that there is no simple prescription for arid land rehabilitation (Table 3). For example, on the one

hand, Triodia biomass increased microbial activity, and on the other, it inhibited Triodia emergence.

Indigofera emergence increased under the higher rainfall regime, which suggests emergence and

perhaps also survival, were water-limited rather than being unsuited to seed pelleting. More

research is needed to confirm this interpretation.

Table 3. Summary of experimental results

Factor Response variable Response measured

Pellet type 50 % TS : 50 % WM

50 % TS : 50 % WM

100 % TS

Highest seedling emergence

Pellet stability

Hard setting

No effect on microbial activity

Amendment Microbial activity

Triodia emergence

Increased microbial activity

Suspected allelopathic effect

Species Emergence

Survival

Triodia>>>Acacia> Indigofera

Triodia>>> Indigofera>Acacia

Rainfall regime

Emergence

Survival

No effect on Triodia and Acacia

120 mm increased Indigofera

No effect on survival

4.1 Modified extruded seed pellets for arid land restoration.

Burial of seeds has been found to increase overall seedling recruitment when compared to the

common mining practice of surface sowing (Erickson et al., 2018; Golos et al., 2019). Surface

exposure is a major cause of the low seedling emergence percentages in aridland restoration

(typically less than 10%) (Merritt & Dixon, 2011; James et al., 2013; Benigno et al., 2013; Larson et

al., 2015; Merino-Martin et al., 2017b). As the seeds in my pellets were randomly mixed throughout

21

the slurry, and the pellets had a high surface to volume ratio, the location of seeds within the pellet

was critical to seedling survival. Experiments using dungpats as a seed dispersal mechanism found

that seedling survival was highest when seeds were located at the periphery and 0.5 cm from the

bottom of pats (Gokbulak, 2009). However, in my experiment many seeds that germinated on the

top or bottom of the pellet were unable to emerge successfully before the pellet became

hardsetting. In this manner, the pellet or dungpat introduces limitations to seedling recruitment,

however, it may be that on balance, seed use efficiency is increased by the percentage of seeds that

emerge from a favourable location compared to broadscale surface seeding which relies on seeds

falling into a favourable environment (Barrett-Lennard et al., 2016; Golos et al., 2019). My results

show that seed pellets can potentially provide a safe site for recruitment.

One of the main differences among pellet types was their physical behaviour as they underwent

alternate periods of wetting and drying (Emma Stock, pers. obs.). At the end of the first rain event,

the range of physical behaviours was especially evident and some had deleterious impacts on

seedlings. The pellets with 100 % topsoil (TS) developed cracks round the edge as they dried. Such

pellets were less integrated with the substrate, as seen in similar studies (Madsen et al., 2018), and

susceptible to movement or even flipping by the wind. Furthermore, up to 13 % of TS pellets were

lifted from the ground as the large-seeded Acacia inaequilatera germinated within and underneath

them (Figure 13). Conversely, almost half the surface of TS/WM pellets were ruptured as the Acacia

seeds germinated. However, the addition of WM also induced stability with up to 39 % of these

pellet types recording no movement with wetting and drying.

Processing component soils through a slurry phase during pellet manufacture appeared to produce a

hardsetting product that stayed hard for the duration of the experiment. Hardsetting soils dry to

form to an apedal mass which increases in strength as it dries. Hard-set soils remain difficult to

cultivate until the profile is rewetted (Chan, 1995; Daniells, 2012). Hardsetting was most visible for

TS pellets although all pellet types formed a persistent thick physical crust throughout the

experiment. Pellet integration with substrate soil did not occur but may have occurred in time.

Indeed, signs of integration were detected for TS pellets, particularly in plots that received higher

rainfall. The fortnightly pulsed rain events were insufficient to overcome detrimental pellet

behaviours but after the second rainfall event, cracking was reduced and pellets became better

integrated with the substrate.

Pellets have been hypothesised to improve the recruitment potential of seeds through increased

soil-seed contact and more favourable moisture conditions (Madsen et al., 2016a; Madsen et al.,

2016b; Davies et al., 2018). In this study, the results did not demonstrate any substantial

22

improvement due to the physical make-up and cracking nature of some pellet types. This

presumably may have led to reduced soil contact and a decrease in moisture retention underneath

the pellet. Madsen et al. (2018) also found that their seed 'pods' provided only moderate

enhancements in the microsite surrounding the seed as the pods disconnected with the soil surface

as they dried. Further research would be required to determine the effect of different rainfall

regimes and recommended timing of irrigation, where available, to minimize disruption to seedlings

and maximise the window of emergence for seedlings. Biochar is a potential addition that has been

trialled as an amendment for hardsetting soils, and found to reduce tensile strength and increase

field capacity (Chan et al., 2007; Gebhardt et al., 2017). Pellet consistency and thickness were other

variables not explored in this experiment that could provide additional refinements. For now, my

results suggest TS/WM pellets are worthy of further research.

The pellet type made from 50 % blend of topsoil and waste produced the highest seedling

emergence. Blended topsoil and waste substrates have been shown to be comparable to topsoil

(Muñoz-Rojas et al., 2016b; Kumaresan et al., 2017; Merino-Martin et al., 2017a; Kneller et al.,

2018), but the superiority of the soil blend over 100 % topsoil is contrary to other studies. This result

could be explained in part by improved soil hydrology, with the incorporation of waste material

increasing infiltration within the pellet; on a larger scale, rock fragments have found to create

microsites and favourable soil water conditions (Golos et al., 2019).

23

Figure 13. Photographs of some pellet responses to the wetting and drying cycle with deleterious effects:

entire pellets lifted (left) and pellets cracking and rupturing (right) by the germination of Acacia seedlings

beneath.

4.2. Rainfall and pellet type effects on seedling emergence.

Species differed significantly in response to experimental factors. Triodia emergence was equivalent

under either rainfall regime but showed susceptibility to water stress. Indigofera emergence was

favoured by the higher rainfall regime (120 mm), however Indigofera survival was limited and Acacia

failed to survive under the fortnightly 10 mm simulated follow-up rainfalls. Low establishment levels

are consistent with comparable studies highlighting that early seedling life stages are vulnerable to

soil water deficits (Barnes, 2014; Larson et al., 2015; Lewandrowski et al., 2017). Studies continue to

show that there are difficulties with the emergence and establishment stages as young seedlings do

not develop drought tolerance until they have reached a certain stage of growth (Bateman et al.,

2018; Barnes, 2014). This transition from germination to emergence has been identified as the major

bottleneck in recruitment (James et al., 2011; Lewandrowski et al., 2017). The 2018-19 Pilbara

summer season included regular and extended heatwaves with very little precipitation, so ambient

air temperatures were elevated above what would usually be associated with rainfall, and may have

24

therefore increased sensitivity and evaporation despite seedlings receiving simulated rainfall. As

Triodia pungens is cued to recruit following heightened rainfall events (Lewandrowski et al., 2017b),

which may only occur infrequently, the importance of follow up rains after the initial event

associated with germination is highlighted.

Triodia emergence was inhibited in the pellets with the addition of the Triodia biomass amendment,

an unexpected effect. As this response was specific to Triodia, the inhibition is possibly due to the

presence of resins generating an allelopathic effect. Triodia pungens resin contains compounds that

allow polymerisation, which underlies its traditional use as an adhesive (de Silva et al., 2009). The

resin has also been found to have bioactive ingredients which can protect timber from termite

attack (Amiralian et al., 2014) and it was once surmised that the distinctive ring shape formed as

adult Triodia plants age may be due to inhibitors formed from the decomposing roots of the older

plants (Willis, 2007). That said, allelopathic effects of adult Triodia pungens plants on its own

seedlings has not been previously recorded. Kneller et al., (2018) had the opportunity but did not

observe an allelopathic effect in an experiment with Triodia wiseana. As Triodia emergence

increased in amended pellet types after the second rainfall event, it is likely that any allelopathic

effect would reduce over time. More research is needed to confirm allelopathic effects of Triodia

plant residues and their implications for recruitment.

4.3. Rainfall and pellet type effects on seedling survival.

Survival of the first round seedlings was related to the numbers that emerged, which is consistent

with studies that found emergence probability to be the strongest predictor of cumulative survival

(e.g., Larson et al., 2015). Of the first round Triodia seedlings, 40 % became vegetatively dormant at

the 6-leaf stage probably due to water stress, whereas only 23 % continued growing past the 20-leaf

stage. Barnes (2014) found a change in Triodia leaf anatomical features at the 12- to 20-leaf stage

which provided greater physiological capacity to withstand drought by conserving water and slowing

growth. Bateman et al. (2018) also showed that grasses species, when compared to other growth

forms, were more drought tolerant at the early seedling stage. Total survival did not show a clear

pattern with regard to experimental factors, possibly due to the shorter experimental period for the

second round emergents, less interference from pellet response effects, and the implications of

dependent probability. At the end of the experiment, three seedlings that were notably larger than

other survivors were growing in three different pellet types. Two of the plants were in the plot with

a higher clay content (Plot 41) so substrate may have been a dominant factor as Triodia roots

developed. However, it is not possible to generalise given so few survivors.

25

4.4 Microbial activity and community composition

Proteobacteria and Actinobacteria were the dominant bacterial communities in all soil types. There

was a higher percentage of Proteobacteria and Actinobacteria communities in the pellets with

amendment addition, however differences were not significant (Figure 11). Conversely there was a

slightly higher percentage of Cyanobacteria in the pellets without amendment. Proteobacteria and

Actinobacteria prefer topsoil and show positive correlation to C amendment (Bastida et al., 2013) so

this could explain the increased microbial activity in pellets with Triodia amendment. The addition of

carbon substrates in the form of organic amendment has been found to strongly increase

opportunistic microbial activity but the impacts on community composition vary over longer

timeframes (Bastida et al., 2013; Bastida et al., 2015; Hueso-González et al., 2018). Plant emergence

and survival were not correlated with microbial activity, a result similar to other studies (Kneller et

al., 2018; Gebhardt et al., 2017). Perhaps a microbial benefit will become apparent as seedlings

mature, as soil properties have been shown to significantly influence the development of plant

communities (for instance Wubs et al., 2016; You et al., 2016). Pellets contained the full set of

microbial communities that were also present in the topsoil from which pellets were constructed,

indicating that soil functionality was maintained (Wubs et al., 2016). This is a promising result

especially as the age and quality of stockpiled topsoil used may not represent what could be

achieved with fresh topsoil, given that the quality of microbial inoculum and generation of microbial

soil functions (analogous to reference sites) will depend on use of topsoil that has not been stored

for long periods (Kumaresan et al., 2017).

4.5. Implications for large-scale mine rehabilitation.

Removing the Triodia floret to overcome dormancy rapidly decreases seed persistence in the soil

(Lewandrowski et al., 2017; T. Erickson unpublished data). My results established that pre-treated

seeds could remain viable in the field within the seed pellets over the course of a season between

large rain events. The extended viability of ungerminated seeds within pellets will therefore benefit

seed-based rehabilitation programs as it demonstrates potential for multiple recruitment windows,

which increases the likelihood that seeds treated for dormancy and then pelleted will germinate and

emerge under varying conditions (Erickson et al., 2016a; Lewandrowski et al. 2017).

My results suggest that blending topsoil with waste is a useful strategy to maximise the use of this

limited resource, with the potential to dramatically reduce topsoil requirements using this method.

For example, standard practice is to spread 10 20 cm of topsoil over the rehabilitation site (Erickson

et al., 2017) (equating to 0.1 0.2 m3/m2 of topsoil). Even if one Triodia plant survives per pellet, at

5 pellets per square metre total Triodia density is consistent with required plants numbers (BGPA,

26

2017). However, the pellet method uses only 0.002 m3/m2 of topsoil; a reduction of 100 200 fold.

In addition, it was found that the relatively small quantities of topsoil blended into the pellets

maintained soil functionality, including autotrophic Cyanobacteria, which can assist in stabilising and

recovering soil processes in rehabilitation sites (Muñoz-Rojas et al., 2018b). Pelleting that

incorporates organic amendment can increase microbial activity, although the stability and maturity

of the organic material needs to be considered (Hueso-González et al., 2018), particularly given the

possible allelopathic effect on Triodia emergence. Despite the short length of the experiment, these

results serve as possible indicators that modified extruded seed pellets are a viable method to

transfer soil microbial communities to other substrates.

Overall Triodia recruitment numbers were low, with 18 % total emergence and only 9 % of first

round and 45 % of second round seedlings survived to the end of the experiment. The highest

emergence achieved in glasshouse conditions simulating the Pilbara summer season to date, by

applying a combination of treatments for Triodia, was 17 % (Kildisheva, 2019). Under field conditions

Triodia seedling emergence has been difficult, typically resulting in < 10% emergence, but it has

been as high as 40 % (Erickson et al 2017; Lewandrowski et al 2017). In this study, cumulative Triodia

seedling emergence was up to 18 %, which suggests that using irrigation to maintain soil moisture

could potentially increase establishment rates. Even so, 74 % of pellets had one or more surviving

Triodia seedlings at the end of the experiment. Targeted irrigation to supplement soil water

potentials has been recommended as a strategy to improve seedling recruitment, particularly with

the increasing rainfall variation projected under climate change (Muñoz-Rojas et al., 2016b;

Lewandrowski et al, 2017a; Loechel et al., 2011), but as substantial investment is required to

implement irrigation at the field scale, experiments are yet to be conducted.

Further research is required to develop better seed use efficiency as well as to determine the

optimal spatial density of pellets to obtain plant densities that match rehabilitation requirements.

Competition among seedlings on individual pellets was not apparent suggesting that seed densities

were appropriate for further testing, unless conditions favour increased emergence. I also

recommend that seeds selected for mixes would need to have compatible emergence requirements.

It has been noted that it is difficult to maximise establishment for all species in seed mixtures

(Madsen et al., 2016a), and this finding is confirmed by my results. Madsen (2012) found that

agglomerating seeds into a pellet improved emergence and survival in the early seedling stage by

generating enough collective force to penetrate the crust. However, unlike my experiment, he

planted only one species, so did not have deleterious interacting effects between different species.

The logistical and cost implications of optimising pellet types for species or groups of species may be

prohibitive especially given the scale of rehabilitation efforts.

27

Additional factors to be considered in translating the modified extruded seed pellet method to large

scale restoration are the erosional impacts of authentic raindrops, which can be torrential in a

tropical downpour, and the slope angle of the site (e.g., waste dump). The delivery of sprinkler water

in this experiment was gentle and the plots were levelled so field results may differ, however the

blended pellet types may also impart stability against erosion. An additional consideration is the

common practice of covering waste dumps with rock armouring to minimise erosion (Golos et al.,

2019) and it is unknown how the pellets would perform on this surface.

Conclusion

The main aim of this experiment was to assess modified extruded seed pellets as a method to

improve the soil physicochemical and microbiological environment for seedling emergence to

enhance the efficiency of seed-based rehabilitation in an arid system. This experiment demonstrated

that modified extruded seed pellets show promise as a viable alternative to spreading topsoil and

broadcasting seeds to recruit Triodia pungens. Furthermore, Triodia can establish from seed pellets

at a density that would match rehabilitation requirements but the method needs to be refined to

increase seed use efficiency. Seed viability was maintained within the pellet during the growing

season. It was demonstrated that blending waste material with topsoil improved the conditions for

seedling emergence which is a beneficial outcome for the effective use of a limited topsoil resource.

Adding Triodia biomass amendment inhibited Triodia seedling emergence but it did increase soil

microbial activity which could stimulate recovery of soil functional processes. The other species

tested need further research to overcome likely moisture limitations. I have suggested further

modifications to reduce hardsetting and to develop an extended moisture regime. My research

builds on that of others (e.g., Erickson et al., 2017) and in turn, provides a solid foundation for

additional research.

28

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Appendix

Table 4. Physicochemical properties of soils prior to the experiment (mean ± SE, *n=6, **n=3). EC:

electrical conductivity, TOC: total organic carbon.

Soil Plot soils (top

5cm)* Pellet soils **

pH 1:5 8.9 ± 0.0 8.2 ± 0.2

EC 1:5 (mS/m) 27.8 ± 1.7 149.3 ± 70.9

Total N (%) 0.013 ± 0.0 0.016 ± 0.0

TOC (%) 0.23 ± 0.02 0.27 ± 0.02

K (mg/kg) 70 ± 6 47 ± 10

Na (mg/kg) 150 ± 7 36 ± 8

P (mg/kg) 2.5 ± 0.2 1.6 ± 0.3

Sand (%) 62.9 ± 2.6 61.2 ± 7.7

Silt (%) 31.8 ± 2.0 33.7 ± 8.7

Clay (%) 5.2 ± 0.6 3.4 ± 0.2

Microbial activity (mg/kg CO2-C) 3.23 ± 0.41 2.59 ± 0.20