USE OF CROP RESIDUES AS SUBSTRATES FOR THE CULTIVATION OF KING STROPHARIA (STROPHARIA RUGOSOANNULATA) MUSHROOMS by Keaton Freel B.Sc., University of Northern British Columbia, 2020 THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN NATURAL RESOURCES AND ENVIRONMENTAL STUDIES UNIVERSITY OF NORTHERN BRITISH COLUMBIA March 2025 © Keaton Freel, 2025 ABSTRACT King stropharia (Stropharia rugosoannulata) is a white rot fungus that produces nutritious edible mushrooms. The species is prized among backyard mushroom cultivators due to its ability to grow on various lignocellulosic substrates in a range of environmental conditions and compete with contaminant microorganisms. These characteristics make king stropharia a great potential tool for enhancing crop residue decomposition in northern environments while producing a valuable crop of mushrooms. However, the species is understudied and underutilized since its production parameters have never been optimized. This study sought to determine 1) which readily available substrate (of alder chips, barley straw and hemp straw) produces the best yield of king stropharia mushrooms, 2) whether substrate impacts the nutritional content of king stropharia mushrooms, 3) how king stropharia chemically alters substrates, 4) how king stropharia alters substrate microbial communities, and 5) which spent substrate makes the best soil amendment for crop production. A cultivation trial was conducted at a farm in Prince George, British Columbia, Canada, from June to October 2022. Eight 1 m by 1 m wooden frames of each substrate were prepared. Five frames each of alder chips and barley straw were inoculated with king stropharia spawn and the three remaining frames served as uninoculated controls. Six frames of hemp straw were inoculated, leaving two uninoculated controls. Substrate samples were collected prior to inoculation and again after the cultivation period. The date, mass and count of mushrooms produced from each frame was recorded. Mushrooms samples were also collected for analysis. Mushroom and substrate samples were ii analysed for content of carbon, nitrogen and a suite of other elements. Mushrooms were analysed for protein and lipid content. Substrate sample lignocellulosic biomass fractions (lignin, cellulose and hemicellulose) were quantified, and substrate pH and electrical conductivity were tested. Substrate fungal and bacterial DNA were extracted, amplified, sequenced and analyzed. Hemp straw tended to be the fastest and highest yielding substrate in the cultivation trial. Hemp straw appears to have been the best performing substrate due to its water content, nutrient profile, lignin content and surface area to volume ratio compared to the other substrates. There also seemed to be a distinctive bacterial consortium associated with the successful cultivation of king stropharia in barley straw and hemp straw, with high relative abundance of the genera Bacillus and Paenibacillus in these samples. Further cultivation experiments using fresh spawn are necessary to properly assess king stropharia’s yield and effect on substrates since the results of this study were impacted by spawn contamination. All post-cultivation mushroom substrate types had beneficial properties for agricultural soil amendment, even though the substrates were not completely spent at the end of the cultivation trial. King stropharia presents a potential win-win scenario whereby farmers can produce nutritious mushrooms with their crop residues while enhancing crop residue decomposition and nutrient cycling with minimal technology and labour. Although further research is required to fully realize the potential of this species, this study demonstrates how king stropharia can contribute to sustainable agricultural practices. iii TABLE OF CONTENTS ABSTRACT ............................................................................................................................. ii TABLE OF CONTENTS .........................................................................................................iv LIST OF TABLES ....................................................................................................................ix LIST OF FIGURES ................................................................................................................... x GLOSSARY .......................................................................................................................... xiii Acknowledgements.................................................................................................................xiv 1 Introduction ........................................................................................................................ 1 1.1 2 Literature review ................................................................................................................ 5 2.1 Cultivation methods ................................................................................................... 5 2.1.1 Substrate selection ................................................................................................. 5 2.1.2 Substrate preparation ............................................................................................. 8 2.1.3 Bed preparation, inoculation, and myceliation ...................................................... 8 2.1.4 Tending to beds and triggering fruiting ............................................................... 10 2.1.5 Harvesting ............................................................................................................ 11 2.2 3 About King Stropharia............................................................................................... 2 Spent mushroom substrate applications .................................................................. 12 2.2.1 SMS as a soil amendment for crop production .................................................... 12 2.2.2 SMS as a peat substitute in horticulture .............................................................. 15 2.3 Knowledge gaps....................................................................................................... 17 2.4 Research objectives ................................................................................................. 18 Methods ............................................................................................................................ 19 3.1 Experimental design ................................................................................................ 19 3.2 Cultivation trial ........................................................................................................ 20 3.2.1 Frame construction .............................................................................................. 20 3.2.2 Study location ...................................................................................................... 21 3.2.3 Substrate sourcing and storage ............................................................................ 21 3.2.4 Substrate pasteurization ....................................................................................... 21 3.2.5 Substrate sampling ............................................................................................... 22 3.2.6 Inoculation and tending ....................................................................................... 22 3.2.7 Casing layer preparation and application ............................................................ 23 iv 3.2.8 3.3 Mushroom harvesting and sampling.................................................................... 24 Laboratory analysis .................................................................................................. 25 3.3.1 Sample processing ............................................................................................... 25 3.3.2 Mushroom protein and lipid analysis .................................................................. 26 3.3.3 Total carbon and nitrogen content by combustion elemental analysis (mushrooms and substrates) ............................................................................................. 27 3.3.4 Elemental analysis with Inductively coupled plasma – optical emission spectroscopy (ICP-OES) (mushrooms and substrates) .................................................... 28 3.3.5 Substrate Van Soest Fiber Analysis ..................................................................... 28 3.3.6 Substrate pH and Electrical Conductivity............................................................ 29 3.3.7 Genomics ............................................................................................................. 30 3.3.8 Microbial biomass ............................................................................................... 32 3.4 4 Statistical analysis.................................................................................................... 34 Results .............................................................................................................................. 36 4.1 Mushroom harvest timeline ..................................................................................... 36 4.1.1 Days to first yield................................................................................................. 36 4.1.2 Harvest period ...................................................................................................... 36 4.2 Mushroom yield and count ...................................................................................... 37 4.3 Mushroom composition ........................................................................................... 39 4.3.1 Mushroom moisture content ................................................................................ 39 4.3.2 Mushroom carbon and nitrogen content .............................................................. 40 4.3.3 Mushroom elemental analysis ............................................................................. 40 4.3.4 Mushroom protein and fat content ....................................................................... 41 4.4 Substrate composition.............................................................................................. 42 4.4.1 Substrate moisture content................................................................................... 42 4.4.2 Substrate carbon and nitrogen content ................................................................. 44 4.4.3 Substrate elemental analysis ................................................................................ 48 4.4.4 Substrate Van Soest fiber analysis ....................................................................... 69 4.5 Substrate pH and Electrical Conductivity................................................................ 78 4.5.1 pH ........................................................................................................................ 78 4.5.2 EC ........................................................................................................................ 80 4.6 4.6.1 Genomics ................................................................................................................. 81 Fungal diversity ................................................................................................... 81 v 4.6.2 4.7 5 Bacterial diversity ................................................................................................ 87 Chloroform Fumigation Extraction for the determination of microbial biomass .... 94 Discussion ........................................................................................................................ 95 5.1 Mushroom harvest timeline ..................................................................................... 95 5.1.1 Days to first yield................................................................................................. 95 5.1.2 Fruiting period ..................................................................................................... 97 5.2 Mushroom yield ....................................................................................................... 98 5.3 Mushroom composition ......................................................................................... 100 5.3.1 Mushroom moisture content .............................................................................. 100 5.3.2 Mushroom carbon and nitrogen content ............................................................ 100 5.3.3 Mushroom elemental analysis ........................................................................... 101 5.3.4 Mushroom protein and fat analysis.................................................................... 103 5.4 Substrate composition............................................................................................ 104 5.4.1 Substrate moisture content................................................................................. 104 5.4.2 Substrate carbon and nitrogen content ............................................................... 105 5.4.3 Substrate elemental analysis .............................................................................. 106 5.4.4 Substrate Van Soest fiber analysis ..................................................................... 114 5.5 Substrate pH and Electrical Conductivity.............................................................. 116 5.6 Genomics ............................................................................................................... 117 5.6.1 Fungal diversity ................................................................................................. 117 5.6.2 Bacterial diversity .............................................................................................. 120 5.6.3 Limitations of compositionality......................................................................... 121 5.6.4 Limitations of timeline ...................................................................................... 122 5.7 King stropharia post-cultivation substrate suitability as a soil amendment .......... 122 5.8 Areas for future research ....................................................................................... 127 5.8.1 More cultivation trials to improve yield data .................................................... 127 5.8.2 Biological efficiency .......................................................................................... 127 5.8.3 Litterbag study ................................................................................................... 128 5.8.4 Cultivation structure comparison experiments .................................................. 128 5.8.5 Nutrition experiments ........................................................................................ 129 5.8.6 Further analysis of genomics results.................................................................. 129 5.8.7 Post-cultivation substrate maturation and application experiments .................. 129 vi 5.8.8 Economic feasibility studies .............................................................................. 130 6 Conclusion ...................................................................................................................... 130 7 References ...................................................................................................................... 133 Appendices ............................................................................................................................ 148 Appendix A: Mushroom composition data tables.............................................................. 148 Appendix B: Substrate moisture content data tables ......................................................... 150 Substrate comparisons .................................................................................................... 150 Treatment comparisons .................................................................................................. 151 Appendix C: Substrate carbon and nitrogen data tables .................................................... 152 Substrate comparisons .................................................................................................... 152 Treatment comparisons .................................................................................................. 154 Pre- and post-cultivation comparisons ........................................................................... 155 Appendix D: Substrate macronutrient ICP-OES analysis data tables................................ 157 Substrate comparisons .................................................................................................... 157 Treatment comparisons .................................................................................................. 161 Pre- and post-cultivation comparisons ........................................................................... 164 Appendix E: Substrate micronutrient ICP-OES analysis data tables ................................. 166 Substrate comparisons .................................................................................................... 166 Treatment comparisons .................................................................................................. 169 Pre- and post-cultivation comparisons ........................................................................... 172 Appendix F: Substrate Al and Na ICP-OES analysis data tables ...................................... 174 Substrate comparisons .................................................................................................... 174 Treatment comparisons .................................................................................................. 175 Pre- and post-cultivation comparisons ........................................................................... 177 Appendix G: Van Soest fiber analysis data tables ............................................................. 178 Substrate comparisons .................................................................................................... 178 Treatment comparisons .................................................................................................. 181 Pre- and post-cultivation comparisons ........................................................................... 183 Appendix H: pH and EC data tables .................................................................................. 185 Substrate comparisons .................................................................................................... 185 Treatment comparisons .................................................................................................. 186 Pre- and post-cultivation comparisons ........................................................................... 188 vii viii LIST OF TABLES Table Title Page 1 Yield (g) and count of king stropharia mushrooms on 15 different substrates 6 2 Yield of king stropharia mushrooms (g/kg substrate) and rate of contamination (%) of trials with respect to substrate and soaking method 8 3 Optimal compost physical, chemical, and biological properties for use in vegetable production and other production systems 14 4 Summary of experimental design 19 5 Timeline of inoculation and pasteurization 23 6 Median mushroom elemental contents for elements for which significant differences were not detected between mushrooms grown in different substrates 40 7 Summary of king stropharia mushroom protein and fat content analysis results 41 8 Comparison of the lignocellulosic content of substrates (% dry matter) from literature data 77 9 Chloroform Fumigation Extraction data 94 10 Compost quality assessment of inoculated postcultivation alder chips 122 11 Compost quality assessment of inoculated postcultivation barley straw 123 12 Compost quality assessment of inoculated postcultivation hemp straw 124 ix LIST OF FIGURES Figure Title Page 1 Photos of the garden frames used for the cultivation of king stropharia in this study 20 2 Boxplot of days to first mushroom yield by king stropharia inoculation date 36 3 Boxplot of harvest period lengths grouped by substrate and inoculation date 37 4 Boxplot of fresh mushroom yield (g) per m2 by substrate 38 5 Boxplot of the number of mushrooms harvested per frame by substrate 39 6 Boxplot of substrate sample moisture content 43 7 Boxplots of total N content of substrate samples 45 8 Boxplots of total C content of substrate samples 46 9 Boxplots of C:N ratio of substrate samples 47 10 Boxplots of alder chip dry matter content of a) Ca, b) K, c) Mg, d) P, and e) S 48-50 11 Boxplots of barley straw dry matter content of a) Ca, b) K, c) Mg, d) P, and e) S 51-53 12 Boxplots of hemp straw dry matter content of a) Ca, b) K, c) Mg, d) P, and e) S 54-56 13 Boxplots of alder chip dry matter content of a) B, b) Cu, c) Fe, d) Mn and e) Zn 57-59 14 Boxplots of barley straw dry matter content of a) B, b) Cu, c) Fe, d) Mn and e) Zn 60-62 15 Boxplots of hemp straw dry matter content of a) B, b) Cu, c) Fe, d) Mn and e) Zn 63-65 16 Boxplots of alder chip dry matter content of a) Al and b) Na 66 17 Boxplots of barley straw dry matter content of a) Al and b) Na 67 18 Boxplots of hemp straw dry matter content of a) Al and b) Na 68 x 19 Boxplots of pre-cultivation substrate content of a) lignin, b) cellulose, c) hemicellulose and d) total lignocellulosic biomass (all expressed a percentage of dry matter) 69-71 20 Boxplots of post-cultivation inoculated substrate content of a) lignin, b) cellulose, c) hemicellulose and d) total lignocellulosic biomass (all expressed a percentage of dry matter) 73-74 21 Boxplots of post-cultivation control substrate content of a) lignin, b) cellulose, c) hemicellulose and d) total lignocellulosic biomass (all expressed a percentage of dry matter) 75-76 22 Boxplot of substrate pH a) pre-cultivation and b) postcultivation 79 23 Boxplot of substrate electrical conductivity a) pre-cultivation and b) post-cultivation 80-81 24 Relative abundance of fungal ASVs in alder chips by taxonomic order 83 25 Relative abundance of fungal ASVs in barley straw by taxonomic order 83 26 Relative abundance of fungal ASVs in hemp straw by taxonomic order. 84 27 Relative abundance of fungal ASVs in alder chip samples, grouped by primary lifestyle at the genus level based on the FungalTraits database. 86 28 Relative abundance of fungal ASVs in barley straw samples, grouped by primary lifestyle at the genus level based on the FungalTraits database. 86 29 Relative abundance of fungal ASVs in hemp straw samples, grouped by primary lifestyle at the genus level based on the FungalTraits database. 87 30 Relative abundance of bacterial ASVs in alder chip samples by phylum 89 31 Relative abundance of bacterial ASVs in barley straw samples by phylum 89 32 Relative abundance of bacterial ASVs in hemp straw samples by phylum 90 33 Relative abundance of bacterial ASVs in inoculated alder chip samples by family within the phylum Firmicutes 91 xi 34 Relative abundance of bacterial ASVs in inoculated alder chip samples by genus within the phylum Firmicutes 91 35 Relative abundance of bacterial ASVs in inoculated barley straw samples by family within the phylum Firmicutes 92 36 Relative abundance of bacterial ASVs in inoculated barley straw samples by genus within the phylum Firmicutes 92 37 Relative abundance of bacterial ASVs in inoculated hemp straw samples by family within the phylum Firmicutes 93 38 Relative abundance of bacterial ASVs in inoculated hemp straw samples by genus within the phylum Firmicutes 93 xii GLOSSARY Term Meaning AD Air Dry ASV Amplicon Sequence Variant BE Biological Efficiency CFE Chloroform Fumigation Extraction DM Dry Matter EC Electrical Conductivity FTIR Fourier Transformed Infrared Radiation FW Fresh weight GFIC Guelph Food Innovation Centre ICP-OES Inductively Couple Plasma - Optical Emission Spectrometry MPN Most Probable Number NALS Northern Analytical Laboratory Service NRAL Natural Resources Analytical Laboratory OD Oven Dry OTU Operational Taxonomic Unit PCR Polymerase Chain Reaction SMS Spent Mushroom Substrate WRF White Rot Fungi/Fungus xiii Acknowledgements I acknowledge with gratitude that I completed this thesis on the unceded traditional territory of the Lheidli T’enneh First Nation, part of the Dakelh (Carrier) peoples’ territory. Many thankyous are owed in the completion of this thesis, starting with my outstanding supervisor Dr. Lisa Wood. Lisa has been a wise and kind support through my master’s studies from start to finish. Thank you to my committee members Drs. Michael Preston and Guillermo Hernandez-Ramirez for your scientific guidance. Thank you to John Orlowsky and Doug Thompson for helping me collect alder branches and allowing me to chip and store them in the Enhanced Forestry Lab compound. Thank you to Jay Bang of Halltray Farm in Vanderhoof for donating barley straw and hemp straw for use in my experiment. Thank you to Deniz Divanlí and Angus Ball. Deniz provided much appreciated help during the construction of the garden frames and walked me through the DNA extraction process with guidance from Angus Ball. Angus also provided resources and support for the analysis of the bioinformatics data resulting from the DNA samples submitted to Genome Quebec. Thank you to Stephanie Hurst, with whom I shared a plate to submit my DNA samples to Genome Quebec, and who handled correspondence with Genome Quebec along with Lisa Wood. Thank you to Karen Dayton, who generously allowed me to conduct my field trial on her farmland, and to Roanne Whitticase and Jane Markin, who helped tend to the mushrooms. Thank you to Dr. Kaila Fadock for assistance with the CFE procedure. Thank you to Charles Bradshaw and Northern Analytical Laboratory Services for technical services and guidance with regards to my sample analyses. Thank you to Dr. Kelvin Lien at the University of Alberta for detailed guidance through the fiber analysis procedure. And, of course, thank you to my partner Bo White and to my friends and family for supporting me throughout this academic journey. I would also like to acknowledge the financial support that made this research possible. I received a British Columbia Graduate Scholarship from the provincial government. The University of Northern British Columbia granted me a Research Project Award and a UNBC Graduate Scholarship. My stipend was provided through Dr. Wood’s Ecosystem Science and Management departmental funding. xiv xv 1 Introduction Worldwide consumption of mushrooms has been rising steeply since the 1990s, both in overall and per capita consumption (De Cianni et al., 2023; Royse, 2014). This rise has been driven in part by increased consumer awareness of the health benefits of mushrooms as a low-calorie source of protein, nutrients and medicinal compounds. To keep pace with demand, mushroom production must be increased and diversified. There is increasing interest in the sustainable use of agricultural waste streams, such as crop residues, in the face of mounting pressure on global food systems from climate change, population growth and land degradation (Grimm & Wösten, 2018; Selvaraju et al., 2011). The use of crop residues as a substrate for mushroom production presents a potential win-win situation in which the decomposition and nutrient cycling of crop residues is enhanced while producing mushrooms as an additional food source (Grimm & Wösten, 2018; McKoy, 2016; Sheldrake, 2021; Stamets, 2000). Saprophytic (decomposer) mushroom cultivation can convert low-quality lignocellulosic crop residues into high quality food products (Grimm & Wösten, 2018). The cultivation of white rot fungi (WRF) reduces the lignin content of crop residues, potentially making the spent mushroom substrate suitable for many applications such as a soil amendment, a peat substitute in horticulture, a substrate for further mushroom cultivation, a component of feed for ruminants, or as a bioenergy feedstock (Madadi & Abbas, 2017; Paula et al., 2017; Zied et al., 2020). Roughly eighty-five percent of the world’s mushroom production consists of just five genera of mushrooms: Lentinula (L. edodes, shiitake), Pleurotus (P. ostreatus, oyster mushroom and 1 P. eryngii, king oyster), Agaricus (A. bisporus, button mushrooms), Auricularia (a genus of jelly fungi) and Flammulina (F. velutipes, enoki) (Royse, 2014; Singh et al., 2020). Higher crop species diversity can result in greater stability and resilience in food systems (Merlos & Hijmans, 2020). In consideration of climate change, population growth and supply chain challenges, creating more resilient food systems is an urgent priority. Therefore, it is important to explore the potential of underutilized mushroom species. One promising species and the focus of this thesis is king stropharia (Stropharia rugosoannulata Farl. ex. Murrill). 1.1 About King Stropharia King stropharia, also known as wine cap mushroom or garden giant, is a nutritious, gourmet mushroom. It is reported to contain 22% protein on a dry matter basis, though the mushrooms are only 8% dry matter (Szudyga, 1978). King stropharia mushrooms have also been found to contain antioxidant polysaccharides and to be a source of niacin (vitamin B3) (Liu et al., 2020; Szudyga, 1978). The mushrooms have a mild, umami flavour and dense, white flesh (Stamets, 2000). The species has a cosmopolitan distribution, having been reported growing wild in Europe, North and South America, Japan and Oceania (Gibson, 2020). It was first cultivated in the 1960s in Eastern Europe but remains underutilized (Bonenfant-Magné, 2000; Szudyga, 1978). The species has had some limited industrial applications as a biological agent for delignification to make cereal straws more digestible for animals, and to make paper pulp (Bonenfant-Magné, 2000). King stropharia possesses characteristics that make it popular among amateur and hobby mushroom growers (Mercy, 2021; Szudyga, 1978). King stropharia is a white rot fungus (WRF) (Buta et al., 1989). WRF, along with a limited number of bacterial species, are the 2 only aerobic organisms able to degrade lignin; they are also able to degrade cellulose and hemicellulose (de Gonzalo et al., 2016; Rodríguez-Couto, 2017). Lignin is a bulky, complex aromatic heteropolymer that provides structure and protection in woody plant cell walls, and is resistant to decay by most microorganisms (Bugg et al., 2011; Zabel & Morrell, 2020). Lignin decomposition occurs through oxidative reactions that break C-C bonds or ether linkages and separate functional groups, aromatic rings and side chains from lignin macromolecules (Zabel & Morrell, 2020). WRF produce various extracellular enzymes involved in the degradation of lignin, including cellulases, laccases, and peroxidases (Bonenfant-Magné, 2000; Bugg et al., 2011). King stropharia is considered to have high resistance to diseases, pests, and adverse environmental conditions (Szudyga, 1978). This fungus produces fruiting bodies at temperatures as low as 4.5°C and as high as 30°C (Sharma et al., 2007). Mycologist Paul Stamets calls king stropharia “the premier mushroom for outdoor bed culture by mycophiles in temperate climates” (Stamets, 2000). The ruggedness of this species allows for it to thrive in relatively inexpensive, low-tech cultivation systems. King stropharia can be grown indoors or outdoors on hardwood chips, straw and other crop residues (Bonenfant-Magné, 2000; Sharma et al., 2007; Szudyga, 1978). While its hardiness lends it well to outdoor cultivation, there are practical limitations to the indoor production of king stropharia. Indoor commercial cultivation of this species is considered uneconomical due to the long period between inoculation and fruiting (6 to10 weeks for king stropharia, compared to 2 to 4 weeks for some Pleurotus ostreatus strains, for example) and inconsistent yields (Bonenfant-Magné, 2000; Bruhn et al., 2010; Stamets, 2000). Indoor mushroom cultivation is resource- and energy-intensive. Temperature, humidity, light levels, CO2 levels 3 and contaminants are carefully monitored and controlled. King stropharia does not produce a consistent return on investment that would justify this level of intensive management. However, given the time and space, it has the potential to be fruitful outdoors with minimal effort, making it a good candidate for on-site inoculation of crop residues. 4 2 Literature review 2.1 Cultivation methods Literature on the cultivation of king stropharia is sparse and scattered, and several key publications are now out of print (Domondon & Poppe, 2000; Szudyga, 1978). Below is a summary of information largely drawn from four papers: Szudyga (1978), Bonenfant-Magné (2000), Domondon and Poppe (2000) and Bruhn et al (2010), with supplemental information from Stamets (2000) and web sources. 2.1.1 Substrate selection King stropharia can been grown on many different lignocellulosic materials, including various crop residues and hardwood chips. Table 1 summarizes Domondon and Poppe (2000)’s findings on yield from different substrates, resulting from a decade of experimentation. The researchers note that there was a correlation between mycelial growth and mushroom yield, i.e., if king stropharia mycelium grew vigorously in a substrate, a higher yield of mushrooms was likely to follow. 5 Table 1. Yield (g) and count of king stropharia mushrooms on 15 different substrates. Numbers are averaged from four replicates. All replicates contained 10 kg moistened substrates. Biological efficiency = (mass of fresh mushrooms produced)/ (mass of dry substrate) x 100%. Modified from Domondon and Poppe (2000). Substrates % of mycelium growth Winter pruning wood Sawdust Sunflower peels + sawdust Hammermilled wheat straw Winter pruning + sunflower peels Winter pruning + grass chaff Summer pruning w/ dry leaves Grass chaff + sawdust Grass chaff + black peat Summer pruning w/ green leaves Winter pruning + Agaricus compost Sunflower peels Grass chaff Coconut fibers Corn cobs + black peat 100% 90% 65% 90% 55% 45% 65% 70% 55% 45% 30% 50% 35% 30% 5% Total on 10 kg substrate No Wt. . (g) 71 1608 37 818 36 668 30 705 28 746 25 520 22 378 21 492 21 279 18 340 17 474 16 441 13 296 10 134 0 0 Mean weight per mushroom (g) Biological efficiency (%) 22.6 22.1 18.5 23.4 26.6 20.8 17.8 23.4 13.2 18.8 27.8 27.5 22.7 13.4 0 48.2 24.5 20 21.1 22.3 15.6 11.3 14.7 8.3 10.2 14.2 13.2 8.8 4 0 The wood chips used in Domondon and Poppe (2000)’s experiments came from Tilia and Populus trees, but Stamets has reported good results with Alnus as well, albeit without accompanying yield data (Stamets, 2000). Pea plant tops have also been successfully used as a fruiting substrate (Bonenfant-Magné, 2000). Substrates should be fresh and uncontaminated with other fungi (Szudyga, 1978). Substrates should not be supplemented with inorganic fertilizers, as this results in poor mycelium development. If substrates must be stored prior to inoculation, they should be stored in cool, dry conditions to prevent contamination. 6 2.1.1.1 Hemp: a new substrate for king stropharia mushrooms Hemp straw has not previously been studied as a substrate for king stropharia in the academic literature. The area of hemp cultivated in Canada increased from 2,400 hectares when industrial hemp production was legalized in 1998 to a peak of 37,400 hectares in 2019 following the legalization of recreational cannabis in October 2018 (Health Canada, 2023). Some hemp producers believed that the legalization of cannabis would bring about a massive expansion in the market for products containing cannabidiol (CBD), a non-psychoactive cannabinoid in hemp that research suggests may help to treat epileptic seizures, anxiety, insomnia, and chronic pain (Arnason, 2024; Grinspoon, 2021). Unfortunately, the growth of the CBD market did not live up to expectations. The area of hemp cultivated in Canada declined to 13,700 hectares in 2022 (Arnason, 2024). While hemp production in Canada varies with market and other conditions, the crop is unlikely to return to its pre-legalization obscurity. Worldwide sales of hemp products continue to grow rapidly (Kaur & Kander, 2023). Business and policy changes, infrastructure investments, and improved cultivation methods help to support the growth and sustainability of the hemp industry. Some hemp is cultivated to use its fiber for textiles, paper products and even building materials. Other varieties of hemp are produced for flower, grain, or seed, which results in hemp straw as a by-product (Health Canada, 2023). Inoculation with king stropharia is an opportunity to generate another revenue stream from the cultivation of hemp for flower, grain or seed while accelerating the decomposition and nutrient cycling of the crop residue. 7 2.1.2 Substrate preparation Before inoculation, substrates should be moistened to 65-75% water content (wet weight basis) via soaking or showering (Domondon & Poppe, 2000; Szudyga, 1978). Adequate moistening of the substrate is one of the most important steps for successful cultivation. This is because, while king stropharia mycelium needs moisture for its growth, once the mycelium has established too much free water is detrimental to it. Soaking for 2 h at 65°C has been found to enhance the leaching of soluble sugars and amino acids from substrates, resulting in lower rates of contamination (Table 2) (Bonenfant-Magné, 2000). Table 2. Yield of king stropharia mushrooms (g/kg substrate) and rate of contamination (%) of trials with respect to substrate and soaking method. Adapted from Bonenfant-Magné (2000). Translated from French by the author. The results are the mean of several replicates. Aged straw 17 h, 20°C Yield (g/kg) 77 Contamination (%) 80 Fresh straw 2 h, 65°C 2 h, 65°C Pea plant tops 2 h, 65°C 135 75 25-160 18 220 0 Corn cobs 150 75 17 h, 20°C 2.1.3 Bed preparation, inoculation, and myceliation The best sites for cultivation are warm and sheltered from the wind (Szudyga, 1978). Total shade will greatly decrease fruiting body development, but partial shade (60-80%) is ideal. A suitable microclimate for king stropharia can be created by preparing beds inside of wooden garden frames covered with an opaque material (Szudyga, 1978). Partial shade can be achieved by propping the frames open. The frames’ lids should be sloping to allow rain to 8 run off. Moist substrate is placed in frames and thoroughly compacted by treading. The beds are filled to a depth of 20 to 30 cm (Szudyga, 1978). King stropharia can also be grown in rounded heaps under 70% shade from forest cover or another source (Bruhn et al., 2010; Domondon & Poppe, 2000). Eliminating the use of garden frames reduces the time and capital expenses required to start production but also results in inconsistent microclimate conditions compared to when garden frames are used. In their forest edge cultivation experiment, Bruhn, Albright, and Mihail (2010) reported significant differences in mushroom yield depending on plot location. Substrates can be inoculated with 3-4 cm diameter pieces of spawn buried at even spacing to a depth of 5-8 cm (Szudyga, 1978). Alternatively, spawn can be crumbled and distributed uniformly over the surface of the bed and then covered with the last 5-8 cm of humid substrate. The substrate should then be covered with moist burlap or cardboard (Domondon & Poppe, 2000; Szudyga, 1978). This covering material should be kept humid, but care should be taken so that free water doesn’t drip down into the substrate (Szudyga, 1978). Myceliation of the substrate, also known as spawn run, requires 3-5 weeks outdoors, depending on conditions. It should be noted that spawn run and the time to first yield are not equivalent due the additional time elapsed between the application of the casing layer and the growth of the first fruiting bodies. Spawn run occurs at temperatures between 20-29°C, with an optimum range of 25-28°C (Domondon & Poppe, 2000; Szudyga, 1978). Below 20°C, myceliation will be slow, while prolonged periods above 30°C will damage the mycelium. If the top layer of substrate becomes desiccated, it should be removed to a depth where mycelium appears (Szudyga, 1978). 9 2.1.4 Tending to beds and triggering fruiting Once the mycelium has grown through the substrate and begun to penetrate the covering material, this material should be removed (Szudyga, 1978). The substrate is then covered in a casing layer of 50/50 (v/v) peat and humus-rich soil (plain mineral soil is not suitable) with a pH of 5.5-6.5 (Domondon & Poppe, 2000; Szudyga, 1978). The requirement for a casing layer is not unusual. Several commonly cultivated mushrooms also benefit from a casing layer, including Agaricus bisporus (button mushrooms) and Pleurotus spp. (oyster mushrooms) (Shields, 2018). The casing layer provides the conditions for the fusion of hyphae into knots from which the fruiting bodies are formed, and therefore greatly enhances the yield of mushrooms (Bruhn et al., 2010; Szudyga, 1978). However, unlike many other cultivated mushroom species, king stropharia will yield little to no fruiting bodies if the casing layer is sterilized. It is believed that soil microbes are necessary to initiate fruiting, but the relationship between king stropharia and its microbial associates is not understood (Shields, 2018; Stamets, 2000). About 50 L of casing material is needed per 1 m2 bed (Szudyga, 1978). Stamets (2000) recommended light pasteurization of the casing layer at 55-60°C for 30 minutes. The intent of light pasteurization is to eliminate potential pests and pathogens, without killing beneficial soil bacteria that enhances fruiting. The casing layer must be kept moist, but care must be taken not to excessively wet it. The beds should be aerated and exposed to partial light 10-14 days after casing by propping open the garden frame lids (Szudyga, 1978). Domondon and Poppe (2000) also experimented with adding fruit peels (apple, banana, and citrus) to the casing layer to test whether hormones from the decomposing fruit would affect mushroom fruiting. They only made one replicate with each type of fruit peel. They found 10 that the beds with fruit peels fruited an average of four days earlier than the control, but total yield from the fruit peel beds was not significantly different. Domondon and Poppe also remarked that 10-20% of fruiting bodies grew between grasses and herbs at the edge of beds. Stropharia mycelia were observed growing around fine plant roots. The authors suggest that this could be peritrophic mycorrhization and that king stropharia could be using sugars and amino acids from the plant roots. This is a potential area for further study. 2.1.5 Harvesting Fruiting has been reported to begin 44 to 58 days after inoculation in central Missouri and 56 to 70 days after inoculation in the pacific northwest (Bruhn et al., 2010; Stamets, 2000). Mushrooms reach full maturity 10-12 days after fruit body setting, also known as pin set. Successive crops will continue to occur at 10-12 days intervals, with the first and second crops producing the highest yields (Stamets, 2000; Szudyga, 1978). Under natural conditions in Eastern Europe, fruiting begins in early August and lasts until frost reaches the beds (Szudyga, 1978). The optimum temperature range for fruiting is 17-26°C. However, day temperatures of up to 32°C can be tolerated for fruiting if the nights are approximately 10°C cooler, and fruiting can also occur at temperatures below 15°C (Bruhn et al., 2010; Domondon & Poppe, 2000). The optimal growth stage to harvest king stropharia mushrooms is immediately before the veil breaks from the cap (Stamets, 2000). Harvesting at this stage allows the mushrooms to reach a medium size, with firm flesh and a longer shelf life. If allowed to develop longer, the mushrooms can attain a larger size, but this is achieved at the cost of a shorter shelf life. To harvest, the mushrooms are twisted (rather than cut) out of the casing layer. The ends of the stipes are cleaned or cut off. The mushrooms can be stored at 2°C to 5°C for two to three 11 days. According to Szudyga (1978), yields can range from 2-33 kg/m2. The unpredictability of yields and the lack of understanding of the factors that control fruiting are among the reasons that large-scale cultivation of this species has not yet taken off (Bonenfant-Magné, 2000; Szudyga, 1978). 2.2 Spent mushroom substrate applications Spent mushroom substrate (SMS), also referred to as mushroom compost, is the leftover biomass remaining at the end of mushroom cultivation, i.e., post-cultivation substrates. Although SMS can be considered a “waste product,” its nutrient content, microbial activity, and chemical and physical properties give it many potential uses. The documented applications of SMS include feed for livestock, bioenergy feedstock, fertilizer, peat substitute in horticulture, a material in wastewater treatment, a source of degradative enzymes and biopesticides and more (Grimm & Wösten, 2018; Mohd Hanafi et al., 2018; Paula et al., 2017; Stamets, 2000). SMS can also be treated and amended for further mushroom cultivation (Grimm & Wösten, 2018; Stamets, 2000; Zied et al., 2020). 2.2.1 SMS as a soil amendment for crop production SMS can improve soil structure by increasing organic matter content, nutrient retention, water holding capacity and microbial activity, and by decreasing compaction (Grimm & Wösten, 2018). SMS can also provide nutrients for crop production, which can help offset the use of financially and environmentally costly synthetic fertilizers. There is a large body of literature on the use of SMS as a soil amendment to improve crop production. The bulk of this literature concerns itself with the most widely commercially cultivated mushrooms (Agaricus bisporus, Pleurotus spp. and Lentinula edodes) (Rinker, 2017). Since king stropharia is a white rot fungus like Pleurotus spp. and L. edodes, there will likely be some 12 similarities in the characteristics of the compost produced, but this remains to be confirmed. The quality of the compost produced also depends in large part on the mushroom cultivation substrate. Composts are chemically complex, and they vary in their quality as soil amendments (Bernal et al., 2009; Ozores-Hampton, 2017). Table 3 provides a list of parameters compiled by Ozores-Hampton (2017) for assessing compost quality. 13 Table 3. Optimal compost physical, chemical, and biological properties for use in vegetable production and other production systems. Compiled information is from various sources, listed below. The composts studied to determine these parameters were made from a variety of feedstocks, including crop residues, manures, paper products and vegetable scraps. This table is from “Guidelines for Assessing Compost Quality for Safe and Effective Utilization in Vegetable Production” (https://journals.ashs.org/horttech/view/journals/horttech/27/2/article-p162.xml) by Monica OzoresHampton. This article is currently licensed under CC BY-NC 4.0 (https://creativecommons.org/licenses/by-nc/4.0/). © 2017 Monica Ozores-Hampton. The terms maturity and stability are sometimes used interchangeably, but they have different, albeit overlapping, meanings. Stability refers to an advanced degree of organic matter decomposition, with resistance to further decomposition. A mature compost is one that does 14 not cause adverse effects to crop plants when applied (i.e., phytotoxins, pathogens and weed seeds have been broken down by the heat generated from the composting process) (OzoresHampton, 2017; Rynk et al., 2021; Wichuk & McCartney, 2010). Complete decomposition is important to produce good quality compost. If decomposition is incomplete, the high degree of microbial activity can cause dangerous levels of self-heating if the compost is stored in large heaps or windrows (Rynk et al., 2021; Wichuk & McCartney, 2010). Continued decomposition can also cause odours and disease vector attraction. Immature composts can be phytotoxic due to high levels of intermediate decomposition byproducts such as ammonia and short-chain organic acids. SMS is not guaranteed to be stable or mature after mushroom cultivation and may require further decomposition before optimal use as a soil amendment (Paula et al., 2017). Fortunately, SMS is considered low risk for pathogens compared to some other compost feedstocks (e.g., manures, infected plant materials, biosolids). One potential issue with SMS compost is high electrical conductivity (EC), which indicates high salt concentrations. Excessive salinity can negatively affect plant growth and development (Paula et al., 2017). In cases where EC is high, irrigation can help leach the excess salts from the SMS. 2.2.2 SMS as a peat substitute in horticulture Peat is a spongy material formed by the partial decomposition of organic matter, often sphagnum moss, in wetlands (Kopp, 2024). It is widely used in horticulture due to its favourable physical characteristics, including high water availability, water buffering capacity and wettability. These properties of peat support the germination of seedlings and the growth of plants in containers and soilless mediums (Eudoxie & Alexander, 2011; 15 Michel, 2010). However, peat bogs are ecologically important for the unique plant and wildlife habitat they provide and the vast amounts of carbon they store (Alexander et al., 2008). Sphagnum moss is slow-growing, and the regeneration of peat falls woefully short of the pace of peat harvesting, leading to destructive environmental consequences (Keddy, 2010). This has created an imperative to find effective substitutes for peat in horticulture. Several experiments have been conducted on the use of commercial SMS as a peat substitute (da Silva Alves et al., 2024; Eudoxie & Alexander, 2011; Gao et al., 2015; Paula et al., 2017; Prasad et al., 2021). The results of these experiments support the idea that SMS can at least partially, and sometimes completely, substitute peat in horticultural applications without sacrificing plant growth, yield, or quality. The limiting factors of using SMS as a peat substitute include high EC and particle size. High EC results from high available K (da Silva Alves et al., 2024; Eudoxie & Alexander, 2011; Prasad et al., 2021). It is possible to use untreated post-mushroom crop SMS as a component up to 25% (v/v) of soilless growing media and seed starting mixes without negative effects on horticultural crops, depending on the properties of the SMS. Particle size is another limiting factor (Abad et al., 2001; Eudoxie & Alexander, 2011). Ideal seed germination substrates have a particle size range of 0.25-2.0 mm to provide even water holding capacity throughout the substrate, but most SMS contains a significant portion of larger particles. Fortunately, both high EC and large particle size can be overcome through simple treatments. Composting and washing/leaching can be used to reduce the EC of SMS to appropriate levels such that it can completely replace peat as a growing medium while maintaining or even increasing horticultural outcomes (da Silva Alves et al., 2024; Eudoxie & Alexander, 2011; 16 Paula et al., 2017). Further composting has the added benefit of simultaneously reducing particle size. Eudoxie & Alexander (2011) found that sieving sugarcane bagasse-based SMS through a 2 mm mesh significantly improved its performance as a medium for producing tomato seedlings compared to both un-sieved replicates of the same SMS and to peat-based Pro-Mix. SMS also provides significantly more nutrients to plants than peat (da Silva Alves et al., 2024; Eudoxie & Alexander, 2011). Therefore, if the appropriate treatments are used to reduce EC and particle size, SMS can outperform peat while reducing fertilizer requirements in horticulture. 2.3 Knowledge gaps Literature on the cultivation of king stropharia is limited. Some documents are out of print or difficult to locate. Most of the material published on king stropharia from the 1980s onward cites Szudyga’s chapter in Biology and Cultivation of Edible Mushrooms (1978). Despite these frequent references, I was unable to find a more recent study that has replicated Szudyga’s garden frame cultivation method. Szudyga reports king stropharia mushroom yields ranging from 2-33 kg/m2 but provides no explanation of how these numbers were obtained. Outdoor cultivators report growing king stropharia in partially shaded patches or beds, but they either have not achieved the yields Szudyga reports (Bruhn et al., 2010), reported yields in a different format to which a direct comparison cannot be made (Bonenfant-Magné, 2000) or do not address yield quantitatively because the publications are non-commercial and nonacademic (Stamets, 2000, and many resources for hobby growers e.g. Mercy, 2021b). There is no consensus on best practices for cultivating this mushroom outdoors. Although king 17 stropharia has a history of being cultivated at temperate latitudes, there is no academic literature on its cultivation in subboreal zones like that of the Prince George area. We know it is possible to grow the mushroom in this region since there is a producer (Michael Doyle from Ancient Forest Mushroom Farm) who grows king stropharia outside in Dome Creek, approximately 125 km east of Prince George. There is scant data on how substrates are chemically altered by inoculation with king stropharia. There is one published paper on the topic (Buta et al., 1989), but the experiment only examined lignin content and was designed to simulate the conditions in a solid-state fermenter, not outdoor cultivation conditions. There is virtually no literature on the specific nutritional needs of king stropharia to optimize yield. 2.4 Research objectives The objectives of this research were to determine: 1. Which readily available substrate (alder chips, barley straw or hemp straw) produces the best yield of king stropharia, 2. If substrate impacts the nutritional content of king stropharia, 3. How king stropharia chemically alters substrates, 4. How king stropharia alters microbial communities in substrates, and 5. Which spent substrate makes the best soil amendment for crop production. 18 3 Methods 3.1 Experimental design Three lignocellulosic substrates, alder chips, barley straw and hemp straw were chosen for the cultivation of king stropharia mushrooms in 1 m x 1 m outdoor garden frames. Eight frames of each substrate were prepared, for a total of 24 frames. Five frames each of alder chips and barley straw were inoculated with king stropharia spawn and three frames were kept as uninoculated controls. It was decided that there should be more inoculated replicates than uninoculated replicates to generate more robust data with regards to king stropharia mushroom yield and nutrition. While a larger number of replicates would have desirable to increase statistical power and therefore be able to draw stronger conclusions, funding and time constraints limited the number of garden frames I could build and tend to. Six frames of hemp straw were inoculated (one extra frame was inoculated due to a labeling error), leaving two frames as uninoculated controls. Table 4 summarizes the experimental design. Table 4: Summary of experimental design. The independent variable was the mushroom growing substrate, of which there were three types (alder chips, barley straw and hemp straw). There were 5 to 6 inoculated (treated) replicates of each substrate and 2 to 3 uninoculated (untreated/control) replicates of each substrate for a total of 8 each. The Dependent variables field lists the primary types of data collected from the experiment. The Constants field shows factors which were kept consistent regardless of substrate type or treatment. Independent variable: substrate Inoculated (treated) replicates Uninoculated (untreated/control) replicates Dependent variables Alder chips Barley straw Hemp straw 5 5 6 3 3 2 Number and mass of fruiting bodies (yield) Micro and macronutrient Fungal and bacterial communities in substrates Micro and macronutrient content of substrates Substrate chemical composition preand post-cultivation Quality of spent substrate as a soil amendment 19 Constants content of fruiting bodies Garden frame Preparation of design and materials substrates (relative age; pasteurization procedure) Casing layer composition (NorGrow compost and peat) 3.2 Cultivation trial 3.2.1 Frame construction Mushrooms were grown in 1m x 1m frames made with 2 x 4 SPF dimensional lumber and foundation wrap (a type of waterproof, corrugated plastic) as siding (Figure 1). The frames had inclined plywood roofs on hinges to allow for easy access to the frame contents and to shed precipitation. The bottoms of the frames were left open to allow for substrate-soil contact. Figure 1. Photos of the garden frames used for the cultivation of king stropharia in this study. The first photo shows the 2x4 wooden structure used. The second photo shows a complete frame with foundation wrap siding and a hinged plywood roof. The frame in the second photo is filled with pasteurized hemp straw. 20 3.2.2 Study location The mushroom cultivation trial was conducted at Three Seeds Farm, located at 1679 Foreman Road, Prince George, BC, from June to October 2022. The frames were laid out along a fence line running northeast to southwest in a hay and vegetable field that slopes gently (<5%) northeast towards the Fraser River. 3.2.3 Substrate sourcing and storage All substrates were sourced in October 2021. The hemp (Cannabis sativa var. Finola®) straw and barley (Hordeum vulgare) straw were baled at Halltray Farm in Vanderhoof, BC. Finola® is a hemp variety developed for oil seed (Smeriglio et al., 2015). The hemp and barley were conventionally grown but were not sprayed with herbicides in the weeks prior to harvest to minimize herbicide residues on the crops. The straw bales were stored in a barn at Halltray Farm from October 2021 to May 2022. The wood chips were obtained from Sitka alder (Alnus viridis ssp. sinuata) branches harvested within 2 km of the Enhanced Forestry Lab at the University of Northern British Columbia and chipped using a 6” (15 cm) auto-feed Vermeer BC600X chipper. The chips were stored in tarped bins in the Enhanced Forestry Lab compound over the same period. 3.2.4 Substrate pasteurization Substrates were pasteurized at ≥ 60 °C, with fluctuations up to 90 °C, for 2 h to reduce the content of soluble sugars and amino acids, based on methods described by Bonenfant-Magné (2000). This was achieved by heating the substrates in well water sourced from the study location using a 30-gallon food-grade metal drum over a propane burner while monitoring temperature using a probe thermometer. Two 30-gallon drums’ worth of substrate were prepared for each garden frame. The contents of the drum took about one hour to reach 60°C, 21 resulting in a 3h total substrate soak time. After two hours at temperatures at or above 60°C, the drum was tipped over into a strainer box made of wooden pallets lined with hardware mesh to drain the water. The frames were then filled with substrate to a depth of 20–30 cm as described by Szudyga (1978). Substrates were then thoroughly compacted by walking on them. 3.2.5 Substrate sampling Before fungal inoculation, ten subsamples of substrate were taken from each frame for a total fresh weight of approximately 500 g. I sampled in a three-dimensional spiral pattern through the frames so that material was taken from different areas and depths of the frames. The samples were packed into large Ziplock bags and stored at -18˚C until analysis. The same sampling procedure was used for the second round of substrate sampling, which was done after the mushrooms ceased to fruit for the season. 3.2.6 Inoculation and tending King stropharia spawn was purchased from Mr. Mercy’s Mushrooms, based in Nelson, BC. Spawn was kept in sealed bags in cold storage (4°C) for approximately eight months before inoculation. Over the course of the storage period, visible contamination of the spawn occurred, but was not discovered until inoculation time, at which point it was too late to order fresh spawn. I visually assessed the spawn for the growth of contaminants (e.g., Trichoderma spp. green mold). Only the cleanest spawn was used, and the rest was discarded. However, it is unlikely that sorting by eye resulted in the exclusion of contaminants from the inoculant used in the experiment. Also, even among the uncontaminated inoculant, mycelial vigour may have been reduced due to the prolonged storage period, although it was suggested that 22 spawn could be stored indefinitely in the bags when refrigerated (R. Mercy, personal communication, July 6, 2021). Spawn was inoculated into substrates in 3 to 4 cm diameter chunks at even spacing to a depth of 5–8 cm at a rate of 250 g/m2 (Mercy 2021b, 2021c, Szudyga 1978). Due to the timeconsuming nature of pasteurizing the substrates, inoculation was done in three rounds, as outlined in Table 5 below. Table 5: Timeline of inoculation and pasteurization. The garden frames were sequentially divided into three groups of eight. The pasteurization date range shows the timeframe during which the substrates for each group of eight frames was pasteurized. Once pasteurization was finished for a group, all the frames assigned a treatment within that group were inoculated on the same day. Date of inoculation 2022-06-15 2022-06-24 2022-07-02 Inoculation group number 1 2 3 Frames in group 1 to 8 9 to 16 17 to 24 Pasteurization date range 2022-05-30 to 2022-06-15 2022-06-15 to 2022-06-23 2022-06-24 to 2022-07-02 Following inoculation, the contents of the frames were covered with a layer of moistened, unbleached cardboard to improve moisture retention. During spawn run (the period during which the mycelium colonizes the substrate), the lids of the frames were kept shut except to tend to and monitor the contents. Moisture content was assessed qualitatively by feel, and the substrates were moistened to approximately field capacity every 1-3 days. 3.2.7 Casing layer preparation and application According to general mushroom cultivation principles, the casing layer is applied to a substrate once spawn run is complete (Stamets, 2000). In this experiment, to maintain consistency between treatments, when a frame full of inoculated substrate was ready to be 23 cased, I also applied the casing layer to the uninoculated control frames containing the same substrate which were pasteurized in the same batch. The casing layer was made by thoroughly mixing a 27-gallon (100 L) tote of Norgrow compost sourced from the Foothills Boulevard Regional Landfill with one 3 cu.Ft. (85 L) bag of peat moss and gradually hydrating the mixture to field capacity. One batch of this recipe covered four frames. The packaged peat moss was assumed to have consistent composition between bags. The compost was thoroughly mixed before dividing it among batches. The mixture was pasteurized at ≥ 60˚C for 30 minutes using a propane burner and a stainless-steel canning pot bathed in hot water within the 30-gallon drum based on Stamets (2000). The casing mixture was set aside to cool to ambient temperature before spreading it on the substrate surface in a 5 cm-deep layer. The resulting pH of the mixture was approximately 6.5, measured with a garden pH probe. 3.2.8 Mushroom harvesting and sampling During the fruiting period, I recorded the date, frame number, substrate type and mass to the nearest gram of each mushroom harvested. I harvested the mushrooms as near as possible to the developmental stage when the veil on the mushroom breaks from the cap. This is considered the best time to harvest to balance between optimizing the yield and the shelf life of the mushrooms (Stamets, 2000). Mushrooms were twisted out of the substrate at the stipe base and gently brushed with a soft-bristle brush to remove attached substrate and casing soil before weighing. The first 200 to 220 g of fresh mushrooms harvested from each frame were set aside for sample processing and analysis. I kept the first 200 to 220 g of mushrooms instead of sampling throughout the fruiting period due to uncertainty about what the total yield would 24 be and how long the fruiting period would last, as well as based on the principle that the first flush of mushrooms from a substrate is usually the largest (McKoy, 2016; Stamets, 2000). 3.3 Laboratory analysis 3.3.1 Sample processing 3.3.1.1 Mushroom sample processing Mushrooms collected for analysis were gently brushed to remove dirt, rinsed with tap water, and patted dry with clean towels. The mushrooms were cut into thin (~0.5 cm) slices and dehydrated at 71˚C for 48 h in a Hamilton Beach food dehydrator (model 32100C) based on the drying methods described in Kumar et al. (2013). After dehydration, the mushrooms were weighed again, and moisture content was calculated on a fresh weight basis, i.e.: Moisture content = (fresh weight – air-dry weight)/fresh weight x 100%. The dried mushrooms were later ground into a fine powder using an A11 basic Analytical mill from IKA mills with a single beater. Dried and ground mushroom samples were used for the following analyses: carbon and nitrogen content analysis, ICP-OES elemental analysis, and lipid and protein analysis. 3.3.1.2 Substrate sample processing After field collection, substrate samples were stored in large freezer bags at -18˚C until later use. A fresh weight of approximately 80 g was subsampled from each substrate sample bag. Material was taken from several different parts of the bag to create a more representative subsample. The subsamples were weighed into paper bags and dried in a kiln oven for 72 hours at 55˚C. Dry weights were recorded and used to calculate sample moisture content on a fresh weight basis, i.e.: 25 Moisture content = (fresh weight – air dry weight)/fresh weight x 100%. The subsamples were then ground into a fine powder using a Wiley mill fitted with a #20 mesh screen, which corresponds to a particle size of 850 μm. After drying and grinding, the samples were stored at room temperature in plastic containers until further use. Dried and ground substrate samples were used for the following analyses: carbon and nitrogen content analysis, ICP-OES elemental analysis, Van Soest fibre analysis, and pH and EC measurements. 3.3.1.2.1 Liquid nitrogen grinding Substrate subsamples from which DNA was extracted were ground with liquid nitrogen to prevent heat-related DNA degradation. I wanted to have approximately 10 g dry mass equivalent to subsample from for the DNA extraction, so I calculated the fresh sample weight required for 10 g dry matter based on the moisture contents previously calculated. Samples were ground using an A11 basic Analytical mill from IKA mills with a single beater. Based on the mill’s user manual guidelines, I filled the grinding chamber with substrate, then poured in a sufficient volume of liquid nitrogen to submerge the substrate. Liquid nitrogen was allowed to boil off and then the samples were ground. After processing, the samples were stored in Falcon tubes in a freezer at -18˚C until ready for use. 3.3.2 Mushroom protein and lipid analysis Two samples of 15 to 20 g of dried, ground mushrooms from each substrate were submitted to the Guelph Food Innovation Centre (GFIC) at the University of Guelph for protein and lipid analysis. Only two samples per substrate were submitted due to the mass of sample available and high laboratory fees. Barley straw and hemp straw-grown mushroom samples 26 were composited. Since only two alder chip frames produced mushrooms, the mushrooms from each of these frames were submitted as individual samples. 3.3.3 Total carbon and nitrogen content by combustion elemental analysis (mushrooms and substrates) Total carbon and nitrogen in dried, ground fruiting body samples were measured on a Costech 4010 elemental combustion system by Northern Analytical Laboratory Services (NALS) at UNBC. Total carbon and nitrogen in dried, ground substrate samples were measured on a Thermo FLASH 2000 Organic Elemental Analyzer (Thermo Fisher Scientific Inc., Bremen, Germany 2016) by the Natural Resources Analytical Laboratory (NRAL) at the University of Alberta. The dry combustion method begins by dropping a known mass of sample in a tin or silver capsule into a combustion tube containing chromium (III) oxide and silvered cobaltous oxide catalysts. An aliquot of purified oxygen is added to the quartz tube to generate a flash combustion reaction. The carbon in the sample is converted to CO2, and the nitrogen is converted to N2 and NOx. The combustion gases are carried through a reduction furnace, reducing NOx species to N2, then through sorbent traps to remove water. The resulting N2 and CO2 gases are separated on a 2m x 6mm OD stainless steel Porapak QS 80/100 mesh packed chromatographic column and detected quantitatively by a Thermal Conductivity Detector (TCD). The integrated TCD peak signal in the resulting chromatogram is directly proportional to the amount of C and N present in the sample which, along with the sample weight, is used to calculate %C and %N (w/w). 27 3.3.4 Elemental analysis with Inductively coupled plasma – optical emission spectroscopy (ICP-OES) (mushrooms and substrates) ICP-OES was performed on dried, ground fruiting body samples at NALS (UNBC) and on dried, ground substrate samples at NRAL (University of Alberta). The fruiting bodies and substrates were tested for different standard suites of elements offered at the respective labs. The fruiting bodies were tested for Al, As, B, Ba, Ca, Cd, Co, Cr, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, Pb, S, Sb, Se, Sn, U, V and Zn. The substrates were tested for Al, B, Ca, Cu, Fe, K, Mg, Mn, Mo, Na, Ni, P, S and Zn. For ease of statistical analysis and reporting results for the substrate samples, I divided the elements into three categories: macronutrients (Ca, K, Mg, P, S), micronutrients (B, Cu, Fe, Mn, Zn) and non-plant nutrient elements (Al and Na). Mo and Ni, which were below detectable limits in most or all samples, were omitted from analysis. 3.3.5 Substrate Van Soest Fiber Analysis The Van Soest method of fiber analysis was selected to quantify lignin, cellulose and hemicellulose content in substrate samples (Van Soest et al., 1991; Van Soest & McQueen, 1973; Van Soest & Robertson, 1980). The method was originally developed to study the nutritional quality of animal feeds but is also useful in understanding fungal decomposition. I also considered Fourier-Transformed Infrared Spectroscopy (FTIR) as a method to analyze substrate lignin, cellulose and hemicellulose content, since FTIR is faster and more modern (Maceda et al., 2020). However, while the absorbance spectra curves generated by FTIR are effective in demonstrating the presence of organic compounds, it is difficult to convert this information into quantitative data. Conversely, the Van Soest method generates quantitative results which can be analysed with simple statistical methods. 28 Air-dry (A.D.), ground substrate samples were analyzed using an ANKOM fiber analyzer at the University of Alberta. A mass of 0.45 - 0.50 g of each air-dry substrate was weighed into an ANKOM bag. Initial sample weights were corrected to oven dry (O.D.) weights using the formula: O.D. weight = A.D. weight * (100 – % A.D. moisture content)/100. The samples then underwent a series of chemical digestions. After each digestion, the samples were rinsed in deionized water, oven-dried and reweighed. The samples were first digested in a neutral detergent, then in an acid detergent and finally in sulfuric acid. The lignin content of the samples was taken to be the sample mass remaining in the bag after sulfuric acid digestion. Cellulose content was calculated as acid detergent fiber minus lignin, and hemicellulose was calculated as neutral detergent residue minus acid detergent fiber. These values were then converted to percentages of the original O.D. sample weights. 3.3.6 Substrate pH and Electrical Conductivity A mass of 5.0 g of each A.D. ground substrate was mixed with 50.0 mL deionized water in an Erlenmeyer flask on a VWR DS-500E orbital shaker at 150 rpm for twenty minutes. Mineral soil pH and EC are typically measured using a ratio of 1.0 g soil: 2.0 mL water (Weil & Brady, 2017). Due to the high water-holding capacity of the substrates compared to mineral soil, a ratio of 1.0 g substrate: 10 mL water was required to be able to produce sufficient filtrate to measure with the probe meter. After shaking, the flask contents were left to settle for one hour, then poured through Whatman No. 1 filter papers into falcon tubes. The pH and EC of the filtrates were measured using a freshly calibrated Hanna HI9813-61 portable pH/EC/TDS/temperature meter. 29 3.3.7 Genomics 3.3.7.1 Substrate fungal and bacterial DNA extraction DNA in substrate samples was extracted using the DNEasy PowerSoil Pro Kit (Qiagen, USA). Based on the substrate moisture content data, the equivalent of approximately 0.125 g A.D. weight per sample was used to extract DNA following the steps outlined in the kit. Samples were then screened for a minimum DNA concentration of 10 ng DNA/ μL solution using the Nanodrop test. The quality of DNA was assessed via spectrophotometer using the A260/A280 ratio and A260/A230 ratio (Francioli et al., 2021). The viability of replication of the DNA extracts was verified using polymerase chain reaction (PCR) and gel electrophoresis. Fungal DNA was amplified with primers ITS1-F-KYO1 5' CTTGGTCATTTAGAGGAAGTAA-3' and ITS2-KYO1 5'-CTRYGTTCTTCATCGDT-3', which target the internal transcribed spacer region of the nuclear ribosomal repeat (Toju et al., 2012). This pair of primers has been used to examine soil fungal community composition (Bui et al., 2020). Prokaryotic DNA was amplified with primers 515F 5’-GTGCCAGCMGCCGCGGTAA-3’ and 806R 5’-GGACTACHVGGGTWTCTAAT-3’ targeting the V4 regions of the 16S rRNA gene (Walters et al., 2011). This primer pair is recommended by the international scientific consortium Earth Microbiome Project (EMP) for the identification of bacteria and archaea from soils (Caporaso et al., 2023). DNA extract samples were stored at -18˚C from the time of their extraction in April 2023 until shipment in January 2024. At this time, samples were thawed briefly at room temperature, vortexed and centrifuged to redistribute the extracted DNA, and then loaded 30 onto an Eppendorf full skirt 96-well plate. The plate was sealed, bagged, and packed in dry ice for shipment to Genome Quebec. 3.3.7.2 DNA Sequencing and Bioinformatics At Genome Quebec, the extracted DNA was amplified, then sequenced with the NextSeq 2000 system (Illumina, San Diego, USA). Samples 4Ht-P, 20Bt-P and 14Bu-C failed to amplify with the ITS primers and therefore were not included in the fungal genomics analysis. Samples 9Ht-P, 10At-P, 11Bt-P and 12Ht-P failed to amplify with the 16S primers and therefore were not included in the bacterial genomics analysis. A Genome Quebec technician then cleaned and processed the reads through the following bioinformatics pipeline to prepare tables of counts of operational taxonomic units (OTU’s). 3.3.7.3 Bioinformatics Analysis Pipeline Raw reads were trimmed and clipped to remove technical sequences and low-quality regions using cutadapt v2.10. Low-quality regions and adapter clipping was performed with trimmomatic v0.36. Cleaned reads from the same R1/R2 pair were overlapped and merged with FLASH v1.2.11 to obtain the complete sequence of the targeted amplicon. Potentially chimeric amplicons were detected using usearch61 (via vsearch 1.11.1) with the ChimeraSlayer’s “gold” database from Broad Microbiome Utilities. Chimeric amplicons were excluded from the analysis. The non-chimeric amplicons for every sample were compared to a reference database (greengenes v138 for the 16S pipeline and UNITE v1211 for the custom ITS pipeline). Amplicons presenting sequence homology higher than 97% with a reference sequence were considered to belong to the same taxon and were combined to form an OTU with qiime 1.9.1 pick_otus (usearch61 via vsearch 1.11.1). Each OTU’s most 31 abundant sequence was selected as its representative sequence. This sequence was then compared to those of the reference databases. The top three hits were used to assign a taxonomic rank to the OTU. To be considered valid, a hit had to have a minimum of 90% sequence homology and cover 51% of the OTU’s sequence. 3.3.7.4 Genomics Data Analysis and Visualization The count data were cleaned in Microsoft Excel (e.g., removing OTUs that were not assigned a kingdom and removing kingdom Archaea entries from the bacterial data). The XLOOKUP function was used to map primary lifestyle data from the FungalTraits database onto the fungal genera identified in the count data (Põlme et al., 2020). Relative abundance graphs were generated to represent fungal and bacterial diversity in R Studio (R version 4.3.3) using the ggplot2 package (Wickham, 2016). 3.3.8 Microbial biomass Substrate sample microbial biomass carbon was estimated based on a method developed for soil microbial carbon with several modifications (Vance et al., 1987). Because the method was developed for soils and not for lignocellulosic biomass substrates, an initial run of nine samples was conducted to verify that the method would work and that the extracted organic carbon would be in the detectable range. Samples were chosen to represent the different substrate, treatment, and cultivation stage combinations. A pre-cultivation sample, a treated post-cultivation sample and an untreated post-cultivation sample were run for each substrate type. For each sample, two portions of approximately 40 mL of frozen substrate were measured into 100 mL beakers. The substrates were weighed, and an oven dry weight was calculated 32 based on prior moisture data. 1.0 mL of deionized water was added to each beaker. The samples were covered in aluminum foil to block light and left to thaw for 24 hours at room temperature. After thawing, one of each pair of samples was placed in a vacuum desiccator with a beaker containing approximately 40 mL of chloroform (CHCl3) to be fumigated. The desiccator was connected to a vacuum pump and a Schlenk line and was evacuated until the chloroform had boiled for over two minutes. At this time, the unfumigated samples were extracted following the same protocol as the fumigated samples described below. Meanwhile, the vacuum desiccator was sealed and left to incubate in the dark under a plastic tote for 24 hours at room temperature. Following the incubation period, the desiccator was evacuated six times to remove most of the remaining chloroform gas. For the extraction, 0.5 M K2SO4 was added to the sample beakers at a rate of 4.0 mL solution per 1.0 g of oven dried substrate. The beakers were covered in parafilm and shaken on a VWR DS-500E orbital shaker at a speed of 150 rpm for 30 minutes. A shaking speed was not specified in the original method, so this speed was selected because it was the highest setting at which the beakers would not slide around on the shaker platform. The suspensions were filtered through Whatman no. 42 filter papers into Falcon tubes. The resulting filtrate was submitted to NALS for total organic carbon (TOC) analysis. Due to an issue with the TOC analyser at NALS, the filtrate samples had to be forwarded to AGAT Laboratories in Calgary. Before sending, the samples were diluted by a factor of 30 by a NALS technician to reduce the K2SO4 concentration to appropriate levels for the TOC 33 analyser. The TOC values resulting from AGAT Laboratories’ analysis were corrected for the dilution. The data were received in mg C/L 0.5 M K2SO4 solution and were converted to μg C/g substrate dry matter by multiplying by 4.0 mL solution/1.0 g substrate dry matter and performing the appropriate unit conversions. The carbon extractable by fumigation, Ec, was calculated as Ec = (organic C extracted by 0.5 M K2SO4 from a fumigated soil) – (organic C extracted by 0.5 M K2SO4 from a nonfumigated soil) (Powlson & Jenkinson, 1976). Biomass C was calculated as (2.64 ± 0.060)*Ec (Vance et al., 1987). 3.4 Statistical analysis Substrate samples were grouped into four categories: pre-cultivation substrate samples designated for inoculation, pre-cultivation samples designated control, post-cultivation inoculated samples and post-cultivation control samples. Unless otherwise noted, there were no significant differences between the pre-cultivation samples that were and were not designated for inoculation. However, for statistical thoroughness, I still compared data between these two pre-cultivation groups for each analysis. The results can be found in the “Treatment comparisons” section of each Appendix. Data were analysed statistically in Minitab 21 (Minitab LLC, USA) statistical software. Datasets were tested for normality quantitively using the Ryan-Joiner test (similar to ShapiroWilk) and visually by examining histograms. Many data sets were not normally distributed. The Kruskal-Wallace test was used for unpaired data (i.e., comparing among substrates, and treatments) and the Wilcoxon signed rank confidence interval for paired data (i.e., comparing 34 before and after cultivation). For unpaired data where there were more than two groups to be compared (i.e., comparing between substrates), the Kruskal-Wallace multiple comparison test was run using the %KRUSMC macros in Minitab. The Kruskal-Wallace multiple comparison test performs Dunn’s post-hoc test and uses a family alpha value of 0.2, a Bonferroni individual alpha of 0.067 and a Bonferroni 2-sided Z-value of 1.834. Differences between unpaired data were considered significant if p < 0.05 (denoted *** in data tables) and of borderline significance of 0.05 P > S > Mg > Ca > Na > Fe > Zn > Cu, Mn > Al, B > Cd, Ba) also does not match the order of abundance of elements in the pre-cultivation substrates (Ca > K > Mg > S > P > Na > Fe > Al > Mn > Zn > Cu, B). These results provide clues about king stropharia mushrooms’ use of nutrients. When researchers compared the elemental content of the fruiting bodies of Pleurotus eryngii, Flammulina velutipes, and Hypsizigus marmoreus with that of their lignocellulosic growing substrates, they found a similar pattern: high Ca content in the substrates compared to the fruiting bodies, and high K content in the fruiting bodies compared to the substrates (Lee et al., 2009). Lee et al. (2009) suggest that Ca in the substrates may not be bioavailable to the fungi or that the mushrooms lack efficient Ca uptake channels, compared to their ability to take up K. High levels of K have also been reported in Agaricus bisporus, Lentinus edodes and Pleurotus ostreatus (Mattila et al., 2001). This suggests commonalities between king stropharia’s nutrient uptake and that of other commonly cultivated mushrooms. Fruiting body formation causes the selective removal of nutrients from the substrate to meet the needs of the developing mushrooms (Zadražil, 1978). Therefore, K likely plays an important role in fruiting body formation in many fungal species. 101 It should be noted however, that mycelia and mushrooms differ in their nutritional composition, and mycelia make up the majority of fungal biomass (Ulziijargal & Mau, 2011). Therefore, elemental analyses of the isolated mycelia would be required for a deeper understanding of the nutrient uptake of this species. 5.3.3.2 Significant differences in elemental content by substrate Mushrooms grown in hemp straw contained significantly more B and Fe than those grown in other substrates, and significantly more Mn than those grown in alder chips. With regards to human nutrition, B and Mn are extremely minor trace elements in the human body, but Fe is more abundant and plays an essential role in human health as a component of hemoglobin in red blood cells (Davey, 2021). These differences in mushroom micronutrient content correspond with the differences in the initial concentration of these nutrients in the substrates. Pre-cultivation hemp straw contained the highest median levels of B, Fe and Mn. These results mirror the findings of a study that measured mineral content in Pleurotus ssp. mushrooms and the various substrates they were grown in (Hoa et al., 2015). According to Hoa et al. (2015), differences in oyster mushroom mineral content depend on the species of mushroom, the mineral concentration of the substrate and on the EC of the substrate. As it does in plants, high EC can inhibit the uptake of nutrients in fungi by increasing osmotic pressure outside mycelia (Hoa et al., 2015). The threshold at which this occurs in king stropharia is unknown. However, EC’s effect on nutrient uptake could help explain why some but not all the substrate nutrient trends are reflected in the mushroom nutrient trends. 102 5.3.3.3 Cadmium concerns Mushrooms grown in alder chips contained significantly more Cd than those grown in barley straw. Since substrate Cd content was not analysed, we do not know whether the mushrooms’ elevated Cd content correlated with alder chip Cd content. The question that follows is, are these Cd levels a concern for human health? The toxicological reference value for cadmium is 0.21-0.36 μg/kg bodyweight/day (Schaefer et al., 2023). Therefore, a 70-kg person could safely consume 92.5-158.6 g of alder chip-grown king stropharia mushrooms per day. Assuming king stropharia mushrooms have a similar density to common button mushrooms (Agaricus bisporus, approximately 70 g chopped raw mushrooms/cup), the safe consumption limit is well over what most people would consume in a typical serving (Cervonie, 2022). Therefore, moderation is advised, but toxicity is not of grave concern. 5.3.4 Mushroom protein and fat analysis The differences in protein and fat content between mushrooms grown in different substrates are of unknown significance due to the small number of samples submitted. The rankings of the results differ based on whether they are considered on a dry or fresh weight basis due to significant differences in moisture content. Therefore, the relative nutritional merit of mushrooms grown in different substrates depends on whether they are consumed fresh or dried. However, the results for protein and fat content between mushrooms grown in different substrates fell within a narrow enough range that it may not affect consumer choice. Consumers show a general preference for simplified nutrition information, and numbers on Nutrition Facts labels are often rounded to the nearest gram (Kiesel et al., 2011). Regardless 103 of substrate, fresh king stropharia mushrooms contained negligible fat (<1%) and modest protein content (2-3%). This, in addition to their elemental nutrient content, will place them into the “healthy food” category for most consumers. The average %DM protein values for the substrates tested in this experiment, which ranged from 27.41-33.20%, were higher than the 22.0 %DM protein reported by Szudyga (1978) for king stropharia mushrooms grown on cereal straw or flax straw. Szudyga did not explain how the reported values were obtained. Reasons for the discrepancy may include a) the substrates tested in this experiment produce more protein-rich mushrooms than those Szudyga used, b) different growing conditions affected the protein content of the mushrooms, and/or c) different laboratory procedures were used to determine protein content. 5.4 Substrate composition 5.4.1 Substrate moisture content The literature suggests that before inoculation, substrates should be moistened to 65-75% humidity (Domondon & Poppe, 2000; Szudyga, 1978). In this experiment, the median preinoculation moisture content of alder chips fell slightly below this range and that of barley straw and hemp straw fell slightly above it. Post-cultivation inoculated alder chip and hemp straw samples contained less water than the uninoculated controls. Imaging technology could be used to compare the particle size distribution and pore sizes in the inoculated substrates and the controls (Lu et al., 2017). I hypothesise that as king stropharia mycelium consumes and replaces lignocellulosic biomass, there is a decrease in the substrates’ proportion of water-holding pores. 104 5.4.2 Substrate carbon and nitrogen content Alder chips had the highest pre-cultivation C:N ratio. The C:N ratio of a soil or substrate affects how much N is available to plants and microorganisms (Weil & Brady, 2017). Generally, the lower the C:N ratio, the greater the availability of N to plants and microorganisms such as fungi and bacteria. Materials with a high C:N ratio will tend to have a lower rate of decomposition due to the limited N available to decomposers (Gilmour et al., 1998; Pérez Harguindeguy et al., 2008). In a cultivation study of WRF Pleurotus ostreatus and P. cystidiosus on a variety of substrates, Hoa et al. (2015) found a negative correlation between substrate C:N ratio and myceliation period, mushroom weight and yield, biological efficiency, and protein content of the mushrooms. This suggests that N availability can be a limiting factor in mushroom production from WRF. Lignocellulosic peroxidase production in Bjerkandera sp., a genus of WRF, was also found to be limited by substrate N availability (Kaal et al., 1993). The ideal nutritional profile of king stropharia is not known, but there could be similarities between its nutritional requirements and those of other WRF. A controlled cultivation experiment using a lignocellulosic substrate amended with known proportions of N could help to understand the relationship between C:N ratio and king stropharia mushroom yield. Inoculated barley straw and hemp straw’s N content increased, and C content decreased over the cultivation period, resulting in lower post-cultivation C:N ratios in these substrates. Inoculated barley straw and hemp straw also contained significantly less C than the uninoculated controls. This is likely because C was lost in the form of CO2 due to cellular respiration, which occurred at a higher rate in the inoculated samples, resulting in a relative increase in the proportion of N (Sales-Campos et al., 2009). 105 Domondon and Poppe (2000) also suggested the possibility of periotrophic mycorrhizal associations between king stropharia and plants. Other WRF species such as Ceriporia lacerata have been found to increase biological N fixation of crop plants (Yin et al., 2022). The wooden frames used in this experiment were open bottomed to allow for soil contact, and king stropharia mycelium could have expanded into the soil to associate with plants in the surrounding field. King stropharia can obtain N by trapping and killing nematodes using cells with finger-like projections called acanthocytes (Luo et al., 2006). This may have contributed to higher N levels in the inoculated substrate samples, but the magnitude of the effect is unknown. 5.4.3 Substrate elemental analysis 5.4.3.1 Macronutrients (Ca, K, Mg, P, S) 5.4.3.1.1 Substrate comparisons Pre-cultivation hemp straw had the highest Ca and Mg content, and barley straw had the highest K, P and S content. The alder chips were comparatively low in all nutrients. These initial differences in macronutrient content reflect the different management histories, nutrient uptake characteristics and growth habits of the plants the substrates are made of (Pourazari, 2016). The hemp straw and barley straw were both obtained from Halltray farm in Vanderhoof and were grown with conventional fertilizers, which increased the nutrient content of the plant tissues (Iványi & Izsáki, 2009; McKenzie et al., 2004). The alder chips came from wild-grown alder branches along a dirt road behind UNBC’s Prince George campus and were not fertilized. 106 Alder is a perennial woody shrub, whereas barley is an annual grass, and hemp is an annual herb (Jacobs, 2016; MacKinnon et al., 1999; Pancaldi et al., 2025). These functional groups of plants have different life histories and strategies, which are connected to different biomass and nutrient allocation patterns (Pourazari, 2016). Annual grass and herb crops have been artificially selected for high resource allocation (including nutrient allocation) to reproductive parts and associated aboveground structures, likely at the expense of allocation to belowground parts (Van Tassel et al., 2010). In contrast, perennial plants tend to allocate more resources below ground. Metabolically active (e.g., photosynthesizing) tissues tend to contain more nutrients than woody structural tissues, which are usually carbon-rich and contain a lower relative proportion of other nutrients (Orji & Wali, 2021; Zhao et al., 2020). Although the optimal nutrient profile for cultivating king stropharia mushrooms is not yet known, king stropharia growers should bear in mind the possible impact of substrate nutrient content on the yield of this mushroom species. It may be that annual crop plants are better able to meet king stropharia’s nutritional needs than woody annual plants. 5.4.3.1.2 Treatment and pre- and post-cultivation comparisons 5.4.3.1.2.1 Alder chips There were no significant differences in macronutrient content between inoculated and control alder chip samples. The low rate of myceliation of the alder chips may have resulted in a weak treatment effect. Regardless of treatment, Ca, Mg and S content in alder chips increased over the cultivation period, while P and K did not. Since there was no treatment effect, the difference in the behaviour of Ca, Mg and S versus P and K must have resulted from the differing mobility of these nutrients in alder chips. 107 A limitation of ICP-OES is that it does not detect specific nutrient-containing compounds and instead only measures elements. The ability of a nutrient to be leached from a substrate depends on its molecular species and its mobility within the substrate, which is in turn affected by pH (Lehmann & Schroth, 2003; Weil & Brady, 2017). The inoculated alder chips were more acidic than the controls, but they were both still acidic. When pH is close to or below 5.0, P, K, S, Ca and Mg are all less mobile and less bioavailable (Hopkins & Hüner, 2009). Ca and S are immobile in living plant tissues, whereas P, K and Mg are mobile. However, as plant matter decomposes, cell walls break down and the patterns of nutrient mobility in living plant tissues no longer apply (Hopkins & Hüner, 2009). Therefore, one possible explanation for the relative increase in Ca and S content over the cultivation period is that these nutrients were less mobile in the relatively undecomposed alder chip tissues. However, this still does not explain the relative increase in Mg content. 5.4.3.1.2.2 Barley straw Ca, Mg and S content in barley straw increased over the cultivation period regardless of treatment. K and P increased in control samples but did not change significantly in inoculated samples. This difference could be partially explained by the acidification of the inoculated substrates by king stropharia mycelium’s extracellular enzymes, which resulted in a median pH of 4.0 in the inoculated samples compared to 7.0 in the control samples. Although acidification tends to increase K+ leaching, phosphate (PO43-) leaching does not correlate linearly with decreasing pH (Deveau et al., 2018; Haynes & Swift, 1986). Instead, P behaviour depends partly on pH and partly on the presence of cations with which it can precipitate and substances that it can adsorb onto. In most mineral soils, P is most mobile at 108 pH 6.5 (Weil & Brady, 2017). At lower pH, it precipitates as Al/Fe-P minerals and/or absorbs to clays and Al/Fe oxides. At higher pH, P precipitates as Ca-P minerals and/or adsorbs to clays and CaCO3. One might expect the relative concentration of P to be lower in the control samples, where the median pH of 7.0 was closer to P’s peak mobility at pH 6.5, but this is not what happened. The treatment differences may result partly from king stropharia mycelium’s manipulation of nutrients through factors other than pH. At least one other WRF, Ceriporia lacerata GH2011, has been shown to mobilize P through several different biochemical mechanisms (Sui et al., 2022). This could also be the case for king stropharia. However, research also indicates that the effect of inoculation of biomass with WRF on nutrient concentration and cycling varies by species (Ostrofsky et al., 1997). 5.4.3.1.2.3 Hemp straw There were unintended pre-cultivation treatment differences in the hemp straw samples. These differences may have been due to inadequate sample homogenization. Hemp straw samples designated for inoculation contained significantly more Ca, K and Mg than their counterparts designated as controls. This makes it more difficult to analyse the treatment differences in post-cultivation Ca and Mg content (the inoculated samples had higher levels of these nutrients). The Wilcoxon signed rank interval test indicates increases over the cultivation period in Ca, Mg, K and S content in the treated hemp samples, with no trend in P content. Based on the boxplots, it appears that among the control samples, Ca and P content did not change meaningfully, while K and Mg content increased. It is unclear whether S increased in the 109 control samples. The difference in trends between the inoculated and control samples over the cultivation period indicates that there were treatment differences in Ca’s behaviour. Of the substrates tested, hemp straw had the highest initial Ca content. It is possible that less Ca was leached from the inoculated samples due to Ca’s decreased mobility at a lower pH in the inoculated samples compared to the controls (Hopkins & Hüner, 2009). However, if pH were the explanation, we would expect a similar trend in barley straw, for which the inoculated samples were also more acidic than the controls. Yet we do not see the same trend for Ca in barley straw. More research is required to determine if the apparent behaviour of Ca in this study is a fluke of the data or if it is related to actual phenomena in the king stropharia cultivation process. Hemp straw S content was higher in the inoculated samples than in the controls. Why S content would be higher in the inoculated hemp samples but not the inoculated barley samples is puzzling. The same can be said of P content, which displayed in different trends in hemp straw versus barley straw. Treated hemp straw and barley straw were both strongly myceliated and acidified by king stropharia, and S and P contents did not vary significantly between hemp straw and barley straw in the pre-cultivation samples. For reasons yet unknown, the nutrients did not behave the same way in these substrates. 110 5.4.3.2 Micronutrients (B, Cu, Fe, Mn, Zn) 5.4.3.2.1 Substrate comparisons As discussed in Section 5.4.3.1, the initial differences in nutrient content between substrates reflect the different management histories, nutrient uptake characteristics and growth habits of the substrate plants. 5.4.3.2.2 Treatment and pre- and post-cultivation comparisons The general trend in all substrates towards an increase in micronutrient content over the cultivation period likely results from the relative decrease in substrate carbon content due to microbial cellular respiration as the substrates decomposed. The treatment differences in the post cultivation samples varied puzzlingly between substrates and could neither be explained by the initial nutrient content differences or by changes in pH. The micronutrient needs of king stropharia cannot be deduced from this small data set. 5.4.3.2.2.1 Alder chips The post-cultivation alder chip samples contained more Mn and Zn that their untreated counterparts. This difference cannot be explained by the difference in pH between the inoculated and control samples (pH 3.5 and pH 5.7 respectively). The availability of Mn and Zn does not change greatly in this pH range (Hopkins & Hüner, 2009). Instead, this difference may result from microbial activity (M. Preston, personal communication, March 7, 2025). 111 5.4.3.2.2.2 Barley straw The post-cultivation barley straw samples contained less Cu than their untreated counterparts. The inoculated samples were more acidic (pH 4.0) than the control samples (pH 7.0), but Cu is less mobile at lower pH values (Hopkins & Hüner, 2009). Therefore, we would expect the inoculated samples to contain more Cu due to reduced leaching. One could speculate that the explanation lies in the nutrient cycling effects of the other microorganisms present in the barley straw frames. 5.4.3.2.2.3 Hemp straw Post-cultivation inoculated hemp samples contained significantly more B, Cu and Mn than their corresponding controls, whereas Fe and Zn did not vary significantly between treatments. The inoculated samples had a median pH of 4.2 and the control samples had a median pH of 7.4. B and Cu are less mobile at lower pH, so this could potentially explain why the content of these micronutrients was higher in the treated samples. However, Mn is more mobile at a neutral pH than an acidic one. The concentration of Mn was also higher in the inoculated alder chip samples than the controls. Mn’s increase in the inoculated samples of both substrates suggests that king stropharia uses Mn in its tissues and causes it to accumulate. However, the same trend was not observed in barley straw. 5.4.3.3 Aluminum and sodium There were no significant treatment differences in Al and Na content in any substrate. Concentrations of these elements tended to increase over the cultivation period. This is in keeping with the idea that substrate C content decreased over the cultivation period because 112 of the cellular respiration of decomposer microbes, resulting in a relative increase in the concentration of other elements. 113 5.4.4 Substrate Van Soest fiber analysis The proportion of lignocellulosic biomass in all inoculated substrates decreased over the cultivation period as substrate tissues were consumed and replaced with mycelium. This occurred because WRF like king stropharia degrade lignocellulosic compounds through the secretion of extracellular ligninolytic enzymes (Rodríguez-Couto, 2017). The decrease in lignocellulosic biomass was strongest for hemp straw and weakest for alder chips. This mirrors the trend in king stropharia mushroom yield, implying that the more effectively the mycelium can consume the substrate, the greater the mushroom yield will be. This is supported previously observed correlation between mycelial growth and mushroom yield in this species (Domondon & Poppe, 2000). The proportion of lignocellulosic biomass decreased over time to a lesser extent in the control samples. This is due to the presence of other decomposer microorganisms naturally present in the cultivation environment, including soil bacteria and fungi. WRF have three main enzymatic systems: cellulases, polysaccharidases other than cellulases, and ligninases (Bonenfant-Magné, 2000). There are two modes of lignin degradation in WRF: selective and non-selective decay (Isroi et al., 2011). Selective decay targets lignin and hemicellulose while leaving the cellulose fraction relatively intact, whereas in the case of non-selective decay, all lignocellulose fractions are decayed. Only certain white rot fungal species are capable of selective decay, and this ability is affected by the lignocellulosic species, the cultivation time, and other factors. In all substrates tested in this study, the proportion of cellulose in the inoculated and control samples did not differ significantly. This suggests that king stropharia is capable of selective decay of lignin and hemicellulose, and that other microorganisms are responsible for the decrease in cellulose content. 114 The proportion of lignin increased over time relative to the other lignocellulosic fractions in the control samples due to lignin’s natural resistance to decay. WRF are one of the only known groups of organisms that can decompose lignin, which is a recalcitrant and bulky heteropolymer (Rodríguez-Couto, 2017). There are anaerobic fungi capable of breaking down lignin, such as those present in the gut flora of ruminants (Lankiewicz et al., 2023). Some aerobic actinomycetes (filamentous soil bacteria) can also degrade lignin (Kirby, 2005; Wei et al., 2019). However, under aerobic conditions, lignin breakdown is primarily associated with WRF. There were volunteer fungi growing in the mushroom frames during the cultivation period. The most common of these was a Coprinopsis species which produced mushrooms in several barley straw and hemp straw frames. Coprinopsis ssp. are saprotrophic, but without a definitive identification of the species we cannot know which compounds it was consuming (MacKinnon & Luther, 2021). However, the amount of mycelium the volunteer fungi produced and the amount of substrate they consumed was likely inconsequential compared to king stropharia, which was inoculated at a rate of 250 g spawn/m2 and is a strong competitor with other fungi (Stamets, 2000; Szudyga, 1978). The lignin content in inoculated hemp straw samples decreased significantly over the cultivation period. By contrast, lignin content in inoculated alder chips was relatively unchanged, and there was a slight increase in the lignin content of inoculated barley straw. Hemp straw and alder chips had similar starting proportions of lignin, cellulose, and hemicellulose, yet alder chips produced the lowest mushroom yield and hemp straw the highest yield. The greater surface area to volume ratio in straw versus chips may have resulted in higher decay rates (Fukasawa & Kaga, 2022; Stamets, 2000). It would have 115 improved the rigour of comparison between substrates in this study if the alder had been processed into smaller pieces. However, the alder chips were prepared using the most readily available equipment (a woodchipper), which makes the experimental results more applicable to real-world cultivation scenarios. Barley straw had a more similar surface area to volume ratio to hemp straw and similar precultivation water content and total lignocellulosic biomass content. However, barley straw did not contain as much lignin as hemp straw and produced a median mushroom yield less than half that of hemp straw (although the difference in yields between the substrates was not found to be significant due to high variability). This suggests a link between lignin content and king stropharia mushroom yield; however, more data is needed. Post-cultivation, among the inoculated samples, hemp straw had the lowest lignocellulosic biomass content, while in the control samples, barley straw had the lowest content. This contrast illustrates the difference between the ease of decomposition in the presence of the study site’s native decomposer microbes, which were more effective at decomposing barley straw, versus the ease of decomposition by king stropharia, a WRF. Overall, alder chips were the most decomposition resistant. 5.5 Substrate pH and Electrical Conductivity The main trend in the pH data was that all inoculated post-cultivation substrate samples were found to be significantly more acidic than the corresponding controls. Many decomposer fungi can convert products of lignocellulosic biomass degradation, such as glucose, into organic acids, resulting in an increase in substrate acidity over the cultivation period (Liaud et al., 2014; Philippoussis et al., 2003). The results of the present study support the idea that king stropharia secretes enzymes that acidify substrates as it consumes them. 116 Likewise, all inoculated post-cultivation substrate samples had significantly higher EC than their corresponding controls. An increase in EC is typical in protected mushroom cultivation (Philippoussis et al., 2003; Zied et al., 2020). In the absence of precipitation to translocate salts deeper into the substrate, evaporation can cause salts accumulate in the surface layers, leading to an increase in EC (H. Sun et al., 2019). The increase in EC could be lessened if the mushrooms were cultivated in the shade of a forest edge rather than under plywood lids, as this would enhance the leaching of salts deeper into the soil profile. High EC can be an issue for several applications of spent mushroom substrate (SMS), including its use as a soil amendment for crops, as a peat substitute in horticulture and as casing layer material for further mushroom cultivation (Ozores-Hampton, 2017; Paula et al., 2017; Zied et al., 2020). Fortunately, EC can be reduced through SMS washing, although there may be an accompanying loss of beneficial water-soluble nutrients in this process. 5.6 Genomics 5.6.1 Fungal diversity 5.6.1.1 Relative abundance by taxonomic order The results demonstrate that there were qualitative differences in the fungal communities associated with each substrate in the pre-cultivation samples. Alder chips likely contained a higher initial diversity of fungal orders because of the plants’ life history and substrate storage. As perennials growing on a forest edge, they would likely have been exposed to more fungi than conventionally grown annual crops in a farm field (Balami et al., 2020). As previously discussed, the alder chips’ storage conditions were moister than those for barley straw and hemp straw, which led to higher levels of contaminants in the alder chips. 117 The post-cultivation control samples suggest that each substrate supports different assemblages of fungal volunteers from the study site environment. The differing physical and chemical characteristics of the substrates (e.g. particle size distribution, lignocellulosic biomass content, elemental composition etc.) provide slightly different niches to different fungi. However, the similarities in the change in fungal orders from pre-cultivation to postcultivation among the control samples (namely the decrease in the abundance of Filobasidiales) is evidence of fungal succession from opportunistic species consuming easily degraded compounds towards wood-rotting fungi consuming more resistant compounds (Tian et al., 2014). In the post-cultivation inoculated samples, the high abundance of the order Agaricales, which includes the genus Stropharia indicates successful colonization by the desired species. That this trend appeared to be weaker in alder chips correlates with the lower yield of mushrooms from this substrate. 5.6.1.2 Relative abundance by primary lifestyle 5.6.1.2.1 Trichoderma in pre-cultivation alder chip samples Trichoderma spp. green mould, a common contaminant in mushroom production, was abundant in the pre-cultivation alder chips (Colavolpe et al., 2014). This contaminant had more favourable conditions to spread through the alder chips due to its moist storage conditions. Since the pre-cultivation substrate samples were collected after pasteurization, the presence of Trichoderma spp. also indicates that soaking for 2 hours at ≥60˚C was not effective in destroying this contaminant. However, the low abundance of Trichoderma spp. 118 and other mycoparasites in the inoculated post-cultivation alder samples suggests that king stropharia was able to outcompete it or create unfavourable conditions for it. 5.6.1.2.2 Cercophora, Schizothecium and Coprinopsis in post-cultivation barley straw control samples FungalTraits classifies the primary lifestyle of the genera Cercophora and Schizothecium as dung saprotrophs. However, primary lifestyle is a broad designation that may not apply to every species within a genus (MacKinnon & Luther, 2021; Rodriguez & Redman, 1997). Individual species may also be capable of different lifestyles depending on environmental conditions (Sheldrake, 2021). Cercophora spp. are described as lignicolous (growing on wood) as well as coprohilous (growing on animal dung) (Bundhun et al., 2020). Schizothecium ASVs have been found to be enriched in wheat and maize straw (Zhang et al., 2023). Similarly, although FungalTraits lists the primary lifestyle of Coprinopsis as a soil saprotroph, species within the genus have also been documented living on lignocellulosic biomass and dung (Kombrink et al., 2019; Ragasa et al., 2016). The question that remains is why these genera only successfully colonized uninoculated barley straw and not hemp straw and alder chips. This is difficult to ascertain without species-level fungal identification and a detailed knowledge of their needs. The physical and chemical conditions provided by barley straw (nutrient content, pH, EC, fibre content etc.) could have been better suited to these fungi than the other substrates. 5.6.1.2.3 Increase in the abundance of litter saprotrophs in treated post-cultivation samples The sizeable increase in the abundance of litter saprotrophs in inoculated post-cultivation samples of all substrate types indicates the dominance of king stropharia in those samples since FungalTraits categorizes the genus Stropharia as a litter saprotroph. 119 5.6.2 Bacterial diversity 5.6.2.1 Relative abundance by bacterial phylum At the phylum level, the pre-cultivation substrate samples appeared to have similar bacterial communities. However, at the phylum level, this apparent similarity may not be meaningful. Greater diversity and differences in abundance of phyla between substrates in the postcultivation control samples result from bacteria in the environment colonizing the different substrates after pasteurization. The very high abundance of the phylum Firmicutes among post-cultivation inoculated barley straw and hemp straw, which were the substrates most effectively colonized by king stropharia, suggests possible symbiotic relationships between bacteria in this phylum and king stropharia. Similar associations have been noted elsewhere. For example, Firmicutes enrichment has been observed in paper birch (Betulina papyrifera) rotted by WRF Fomes fomentarius, suggesting that wood decomposer fungi may exert selection effects on bacteria or vice versa (Haq et al., 2022). 5.6.2.2 Family and genus-level relative abundance within the phylum Firmicutes among post-cultivation inoculated samples Two families (Bacillaceae and Paenibacilaceae) and two genera within these families (Bacillus and Paenibacillus, respectively) made up the majority of Firmicutes reads in all post-cultivation inoculated substrate samples. The two genera share several similarities. They have both been found to degrade lignin (Chandra et al., 2008). This could partially explain why they would occur in the same environment as WRF (they can use the same food source), 120 but it does not explain why they had higher relative abundance in the substrates that were successfully colonized by WRF, where lignin content was lower. Bacillus and Paenibacillus are Gram-positive aerobic endospore-forming bacteria that are ubiquitous in agricultural soils (Govindasamy et al., 2011; McSpadden Gardener, 2004). They have been studied as potential plant-growth promoting rhizobacteria with sustainable agriculture applications thanks to a suite of benefits observed from these bacteria in crop plants, including atmospheric nitrogen fixation, solubilization of soil phosphorous, micronutrient uptake, and production of phytohormones and antimicrobial metabolites (Grady et al., 2016). There is emerging research on how these genera interact with WRF, though not king stropharia specifically. Bacillus and Paenibacillus have been found to promote mycelial growth in Pleurotus ostreatus (Shamugam & Kertesz, 2023). Cultivation of P. ostreatus in the presence of these bacterial genera has been found to result in an increase in mycelial laccase activity, possibly as a defence response to the bacteria (Shamugam & Kertesz, 2023). Bacillus and Paenibacillus have also been found to inhibit the growth of competitor Trichoderma spp. fungi (Velázquez-Cedeño et al., 2008). It has also been suggested that, through their N-fixing ability, these bacterial genera could increase N availability to WRF in exchange for greater access to embedded carbohydrates (Haq et al., 2022). 5.6.3 Limitations of compositionality Interpretation of the results of genetic sequencing are limited by their inherent compositionality. Compositional data is constrained to an arbitrary constant sum, which is this case is the total number of reads (Douglas & Langille, 2021). Because of the compositionality of the data, it is possible to compare relative proportions of different 121 taxonomic constituents, but the total population of these constituents cannot be known. This constrains our understanding of fungal-bacterial interactions because we do not know if bacterial biomass increased or decreased over the cultivation period, depending on substrate type and treatment. Knowing the change in bacterial biomass could be important because bacterial biomass can be a major nutrient source for WRF such as P. ostreatus. The growth of P. ostreatus corresponds with a 5 to 10-fold decrease in bacterial 16S rRNA levels, and this species has been shown to actively penetrate and lyse bacterial colonies, followed by profuse P. ostreatus hyphal growth (Bánfi et al., 2021). King stropharia may also use bacteria growing on the substrates and its own decomposition byproducts as a food source. 5.6.4 Limitations of timeline The microbial community in the inoculated post-cultivation substrates at the time of analysis is unlikely to resemble the community that will be present later because of succession processes that occur throughout the decomposition process. Paula et al. (2017) found bacterial community structure to be a potential predictor of the stability of mushroom compost. 5.7 King stropharia post-cultivation substrate suitability as a soil amendment I assessed the quality of king stropharia post-cultivation substrates by comparing their properties to the optimal ranges of the compost quality parameters compiled by OzoresHampton (2017) (Table 3). Since all inoculated substrate types continued to produce mushrooms for at least two years after the experimental cultivation period, they cannot be considered true “spent” mushroom substrates (SMS). Each post-cultivation substrate type was assessed separately (Tables 10, 11 and 12). 122 Not all the parameters listed in Table 3 were measured in this study, but there are a few unmeasured parameters for which I can provide context. Based on handling the post-cultivation substrates, the alder chips would not meet the particle size criteria (98% of the material being able to pass through a ¾-inch screen). The straws contained narrower particles to begin with and were more substantially decomposed over the cultivation period. Most of the postcultivation straw could be forced through a ¾-inch screen, but it is uncertain whether the 98% threshold would be met. Because the substrates were each prepared from one kind of single-origin plant biomass, they would have been virtually free of weed seeds. The post-cultivation substrates met many parameters for optimal compost quality, but each substrate also failed to meet some parameters. All substrate types had pH values that were below the optimal range, C:N ratios above the optimal range and insufficient P content. Fortunately, values for these parameters can be improved through further maturation of the mushroom compost under aerobic conditions (Paula et al., 2017; Sundberg, 2005). Sufficient aeration is one of the most important factors determining the creation of high-quality compost (Guo et al., 2012). While these studies used ventilated in-vessel composters, compost aeration can be achieved simply by turning the piles of decomposing material (Rhoades, 2012). Further decomposition would increase C loss, which results in an increase in the relative concentration of nutrients such as N and P. Over time, the biochemistry of compost also tends to shift towards a decrease in the buildup of organic acids (Rynk et al., 2021; Sundberg, 2005). Inoculated post-cultivation alder chip values were the furthest from meeting OzoresHampton’s criteria. Alder chips had the largest particle size, lowest pH, and lowest nutrient content (Table 10). Hemp straw was the closest to meeting the criteria overall since it had the 123 highest pH, highest N content and its C:N ratio was almost in the optimal range (Table 12). However, barley straw had a higher mean P content than hemp straw (Table 11). All three substrate types could be made into suitable soil amendments to improve the physical, chemical, and biological properties of soil for crop production. However, the alder chips would require the longest period of maturation before achieving optimal quality. Table 10: Compost quality assessment of inoculated post-cultivation alder chips. Parameter (units) Moisture (%) Optimal range Median value in alder chips Within optimal range? 30 (dry) - 60 (wet) 54 Yes Organic matter (%) 40-60 >72 No Physical contaminants (%) <2 <2 Yes Comments Based on total lignocellulosic biomass pH could be increased through further maturation of compost with aeration pH 5.0-8.0 3.5 No EC (mmho/cm) <6 0.69 Yes Carbon:nitrogen ratio 10-25 55 No Nitrogen (%) 0.5-6.0 0.86 Yes Phosphorous (%) 0.2-3.0 0.04 No Potassium (%) 0.10-3.5 0.14 Yes Copper (ppm) <450 5 Yes Molybdenum (ppm) <75 <3 Yes Most values were below ICP-OES quantification limits Most values were below ICP-OES quantification limits Nickel (ppm) <50 <3 Yes Zinc (ppm) <900 32 Yes Fecal coliform (MPN/g total solids) <1000 NA Genera of fecal coliforms detected in 16S bacterial genome sequencing Very likely counts at extremely low levels 124 Salmonella (MPN/4 g) <3 0 Yes No reads of Salmonella detected in 16S bacterial genome sequencing Table 11: Compost quality assessment of inoculated post-cultivation barley straw. Optimal range Median value in barley straw Within optimal range? Comments Moisture (%) 30 (dry) 60 (wet) 80 No Easy to fix by allowing it to dry out Organic matter (%) 40-60 >56 Likely Based on total lignocellulosic biomass Physical contaminants (%) <2 <2 Yes Parameter (units) pH 5.0-8.0 4.0 No EC (mmho/cm) <6 2.0 Yes Carbon:nitrogen ratio 10-25 31 No Nitrogen (%) 0.5-6.0 1.4 Yes Phosphorous (%) 0.2-3.0 0.16 Almost Potassium (%) 0.10-3.5 0.41 Yes Copper (ppm) <450 8.4 Yes Molybdenum (ppm) <75 <3 Most values were below ICP-OES quantification limits <3 Yes Zinc (ppm) <900 44.9 Yes Salmonella (MPN/4 g) <3 NA 0 P content could be increased through further maturation of compost Yes <50 <1000 Ratio could be decreased through further maturation of compost Most values were below ICP-OES quantification limits Nickel (ppm) Fecal coliform (MPN/g total solids) pH could be increased through further maturation of compost with aeration Very likely Genera of fecal coliforms detected in 16S bacterial genome sequencing counts at extremely low levels Yes No reads of Salmonella detected in 16S bacterial genome sequencing Table 12: Compost quality assessment of inoculated post-cultivation hemp straw. 125 Parameter (units) Optimal Median value in range hemp straw Within optimal range? Comments Moisture (%) 30 (dry) - 60 (wet) 69 No Easy to fix by allowing it to dry out Organic matter (%) 40-60 >44 Likely Based on total lignocellulosic biomass Physical contaminants (%) <2 <2 Yes pH 5.0-8.0 4.2 No EC (mmho/cm) <6 2.5 Yes Carbon:nitrogen ratio 10-25 26 Almost Nitrogen (%) 0.5-6.0 1.5 Yes Phosphorous (%) 0.2-3.0 0.1 No Potassium (%) 0.10-3.5 0.29 Yes Copper (ppm) <450 13 Yes Molybdenum (ppm) <75 <3 pH could be increased through further maturation of compost with aeration Ratio could be decreased through further maturation of compost P content could be increased through further maturation of compost Yes Most values were below ICP-OES quantification limits Most values were below ICP-OES quantification limits Nickel (ppm) <50 <3 Yes Zinc (ppm) <900 34 Yes NA Very likely Genera of fecal coliforms detected in 16S bacterial genome sequencing counts at extremely low levels Yes No reads of Salmonella detected in 16S bacterial genome sequencing Fecal coliform (MPN/g total solids) Salmonella (MPN/4 g) <1000 <3 0 126 5.8 Areas for future research 5.8.1 More cultivation trials to improve yield data The use of contaminated spawn in this experiment likely impacted mushroom yield. This was also a small-scale cultivation trial. More and larger trials with fresh, high-quality spawn are needed to assess the true yield potential of king stropharia. 5.8.2 Biological efficiency Biological efficiency (BE) is a measure of how efficiently a fungus converts substrate biomass into mushroom biomass. It is calculated as: BE % = total fresh weight of mushrooms (kg)/dry weight of substrate (kg) x 100 (Biswas & Layak, 2014). BE is a common metric for assessing the suitability of different substrates and techniques for mushroom production (Cueva et al., 2017; Hoa et al., 2015; Onyeka et al., 2018). In future king stropharia cultivation experiments, the initial mass of the substrates should be recorded so that BE can be calculated. This data would help cultivators predict yields based on the type, preparation, and amount of substrate used. It will take longer to collect BE data for king stropharia than it does for many other cultivated mushroom species due to king stropharia’s slow growth rate. None of the substrates in the present study were completely spent at the end of one cultivation season, as evidenced by the presence of mushrooms in the garden frames the following two seasons. To know the total yield from a given substrate would require multiple seasons of data collection until mushroom production in the substrates tapered off. Long term yield data collection would also be helpful to cultivators wanting to know the longevity of their mushroom substrates. 127 5.8.3 Litterbag study Analysis of pre- and post-cultivation substrate data (i.e., fiber analysis and nutrient content) in this study was limited by compositionality. Mass loss data would enhance our understanding substrate decomposition by king stropharia. The litterbag method is a commonly used technique for studying decomposition rates under field conditions (Bärlocher, 2020; Kurz-Besson et al., 2005; Xie, 2020). In this method, a known mass of substrate is enclosed in a mesh litterbag and laid on the substrate surface or buried in the substrate. Litterbags are then periodically collected and weighed, and decomposition rates are determined by mass loss modeling (Xie, 2020). It would also be possible to approximate mass loss based on the ratio of non-volatile solids (NVS) or ash before and after cultivation, as is sometimes done for compost (Breitenbeck & Schellinger, 2004). 5.8.4 Cultivation structure comparison experiments There is no published literature comparing the yield of king stropharia from different cultivation structures. This hardy, versatile mushroom has been intercropped with strawmulched vegetables (Stamets, 2000), grown in wooden boxes (Szudyga, 1978) and produced in piles or beds of substrates in partially shaded areas (e.g., along a forest edge) (Bruhn et al., 2010; Stamets, 2000). I think king stropharia could also be grown in inexpensive tent structures to provide shade, and some ability to moderate temperature and humidity, as has been done successfully for the cultivation of mushrooms in developing countries (Oei & van Nieuwenhuijzen, 2005). Research is needed to compare different growing structures. 128 5.8.5 Nutrition experiments More controlled experiments are needed to determine the ideal substrate nutrient profile for growing king stropharia mushrooms. The nutrient data in the present study are difficult to interpret because there are so many variables and confounding factors. King stropharia could also be investigated for vitamin and medicinal compound content. Szudyga (1978) mentions that the niacin (vitamin B-3) content of king stropharia mushrooms is 3.41 mg. He does not write what mass of mushrooms this measurement corresponds to (Is it 100 g? or 1 kg?), nor whether king stropharia is rich in any other vitamins. Many mushrooms are rich in healthful compounds, but king stropharia has yet to be thoroughly studied in this regard (Stamets, 2000). 5.8.6 Further analysis of genomics results Our understanding of the genomics results could be deepened through further analysis, such as compositional data analysis and calculation of alpha and beta diversity (Gloor & Reid, 2016; Martino et al., 2022; Willis, 2019). This would allow for a more detailed and quantitative understanding of the differences in microbial communities between cultivation stages and treatment types. 5.8.7 Post-cultivation substrate maturation and application experiments The post-cultivation substrate data collected in the present study provide a basis for evaluating their use in applications such as a soil amendment. However, as previously noted, the substrates were not completely spent at the end of the experimental cultivation period. Re-evaluation after a second season of cultivation or longer would provide a better sense of the potential applications of the materials. 129 Experiments using the SMS in various potential applications would bring this area from theory into practice. Such experiments could include potted plant studies comparing the effects of king stropharia SMS to other growing mixes. Another area for experimentation is the use of king stropharia SMS for the cultivation of other mushroom species (Stamets, 2000; Zied et al., 2020). 5.8.8 Economic feasibility studies For the cultivation of king stropharia mushrooms to become widespread, the economic feasibility of commercial production must be improved. This would involve reducing production costs, increasing yields through improved cultivation techniques, and generating additional value through SMS applications. For example, pasteurization must be achieved using more efficient methods than the small batch heating done in this study. The pursuit of the research areas identified above would result in a better understanding of how to achieve the greatest yields and improve the value of the SMS. Because king stropharia mushrooms are relatively unknown to consumers, improving the economic potential of cultivating them would also involve market research about how to increase consumer awareness and appreciation of the species. 6 Conclusion This study provides insights into the potential for cultivating king stropharia mushrooms in northern environments. Of alder chips, barley straw and hemp straw, hemp straw tended to be the fastest and highest yielding substrate in the cultivation trial but was not significantly different than barley straw in terms of yield. Hemp straw’s good quality as a substrate for cultivating king stropharia mushrooms is likely due to its nutrient profile and lignin content 130 compared to the other substrates. There also appeared to be a distinctive bacterial consortium associated with the successful cultivation of king stropharia in barley straw and hemp straw, with high relative abundance of the genera Bacillus and Paenibacillus in these samples. Additional cultivation experiments using fresh spawn are necessary to properly assess king stropharia’s yield potential and effects on substrates since the results of this study were impacted by spawn contamination. Furthermore, a longer-term trial is needed to assess the performance of alder chips as a substrate. This experiment showed a lower yield from the alder chip substrate, but compounding factors such as spawn run time and lack of casing layers applied due to a lack of full myceliation, likely affected the yield results. It is recommended that, for outdoor experimentation, the trial period begins as soon as possible in the growing season, and continues for 2 or 3 years to get a full picture of absolute yield differences between these three substrates. All post-cultivation mushroom substrate types, and especially hemp straw, had beneficial properties for agricultural soil amendment, even though the substrates were not completely spent at the end of the cultivation trial. Further decomposition of the substrates from another season of mushroom cultivation likely would have further improved their soil amendment properties. These findings highlight king stropharia’s potential for enhancing northern agricultural security and sustainability. King stropharia presents a win-win scenario whereby farmers can produce nutritious mushrooms on their crop residues while enhancing the crop residue decomposition and nutrient cycling with minimal technology and labour. In conclusion, the cultivation of king stropharia mushrooms in northern environments, specifically using hemp straw, is a promising endeavor with several environmental and agricultural benefits. Although a lot more research is required to fully realize the potential of 131 this species, this study demonstrates how king stropharia can contribute to both local agriculture and sustainable waste management practices. 132 7 References Abad, M., Noguera, P., & Burés, S. (2001). National inventory of organic wastes for use as growing media for ornamental potted plant production: Case study in Spain. Bioresource Technology, 77(2), 197–200. https://doi.org/10.1016/S09608524(00)00152-8 Abdul Khalil, H. P. S., Hossain, Md. S., Rosamah, E., Azli, N. A., Saddon, N., Davoudpoura, Y., Islam, Md. N., & Dungani, R. (2015). The role of soil properties and it’s interaction towards quality plant fiber: A review. Renewable and Sustainable Energy Reviews, 43, 1006–1015. https://doi.org/10.1016/j.rser.2014.11.099 Alexander, P. D., Bragg, N. C., Meade, R., Padelopoulos, G., & Watts, O. (2008). Peat in horticulture and conservation: The UK response to a changing world. Mires and Peat, 3(8). Arnason, R. (2024, January 25). Hemp proponents optimistic crop will rebound. The Western Producer. https://www.producer.com/markets/hemp-proponents-optimistic-crop-willrebound/ Balami, S., Vašutová, M., Godbold, D., Kotas, P., & Cudlín, P. (2020). Soil fungal communities across land use types. iForest - Biogeosciences and Forestry, 13(6), 548–558. https://doi.org/10.3832/ifor3231-013 Bánfi, R., Pohner, Z., Szabó, A., Herczeg, G., Kovács, G. M., Nagy, A., Márialigeti, K., & Vajna, B. (2021). Succession and potential role of bacterial communities during Pleurotus ostreatus production. FEMS Microbiology Ecology, 97(10), 1–12. https://doi.org/10.1093/femsec/fiab125 Bärlocher, F. (2020). Leaf Mass Loss Estimated by the Litter Bag Technique. In F. Bärlocher, M. O. Gessner, & M. A. S. Graça (Eds.), Methods to Study Litter Decomposition: A Practical Guide (pp. 43–51). Springer International Publishing. https://doi.org/10.1007/978-3-030-30515-4_6 Bernal, M. P., Alburquerque, J. A., & Moral, R. (2009). Composting of animal manures and chemical criteria for compost maturity assessment. A review. Bioresource Technology, 100(22), 5444–5453. https://doi.org/10.1016/j.biortech.2008.11.027 Biswas, M. K., & Layak, M. (2014). Techniques for Increasing the Biological Efficiency of Paddy Straw Mushroom (Volvariella Volvacea) in Eastern India. Food Science and Technology, 2(4), 2–57. https://doi.org/10.13189/fst.2014.020402 133 Bonenfant-Magné, M. (2000). Préparation d’un substrat de culture pour le strophaire (Stropharia rugoso-annulata) par trempage de résidus ligno-cellulosiques agricoles. Canadian Journal of Botany. 78, 6. Borah, T. R., Singh, A. R., Paul, P., Talang, H., Kumar, B., & Hazarika, S. (2019). Spawn production and mushroom cultivation technology. 46. Breitenbeck, G. A., & Schellinger, D. (2004). Calculating the Reduction in Material Mass and Volume during Composting. Compost Science & Utilization, 12(4), 365–371. https://doi.org/10.1080/1065657X.2004.10702206 Bruhn, J. N., Abright, N., & Mihail, J. D. (2010). Forest farming of wine-cap Stropharia mushrooms. Agroforestry Systems, 79(2), 267–275. https://doi.org/10.1007/s10457009-9257-3 Bugg, T. D. H., Ahmad, M., Hardiman, E. M., & Rahmanpour, R. (2011). Pathways for degradation of lignin in bacteria and fungi. Natural Product Reports, 28(12), 1883– 1896. https://doi.org/10.1039/C1NP00042J Bui, A., Orr, D., Lepori-Bui, M., Konicek, K., Young, H. S., & Moeller, H. V. (2020). Soil fungal community composition and functional similarity shift across distinct climatic conditions. FEMS Microbiology Ecology, 96(12), fiaa193. https://doi.org/10.1093/femsec/fiaa193 Bundhun, D., Maharachchikumbura, S., Jeewon, R., Senanayake, I., Jayawardena, R., Hongsanan, S., Samarakoon, M., Dayarathne, M., Huang, S., Perera, R., & Hyde, K. (2020). Https://sordariomycetes.org/, a platform for the identification, ranking and classification of taxa within Sordariomycetes. Asian Journal of Mycology, 3(1), 13– 21. https://doi.org/10.5943/ajom/3/1/2 Buta, J. G., Zadrazil, F., & Galletti, G. C. (1989). FT-IR determination of lignin degradation in wheat straw by white rot fungus Stropharia rugosoannulata with different oxygen concentrations. Journal of Agricultural and Food Chemistry, 37(5), 1382–1384. https://doi.org/10.1021/jf00089a038 Caporaso, J. G., Ackermann, G., Apprill, A., Bauer, M., Berg-Lyons, D., Betley, J., Fierer, N., Fraser, L., Fuhrman, J. A., Gilbert, J. A., Gormley, N., Humphrey, G., Huntley, J., Jansson, J. K., Knight, R., Lauber, C. L., Lozupone, C. A., McNally, S., Needham, D. M., … Weber, L. (2023). EMP 16S Illumina Amplicon Protocol. PLOS One. https://doi.org/dx.doi.org/10.17504/protocols.io.kqdg3dzzl25z/v2 134 Cervonie, B. (2022, September 22). Mushroom Nutrition Facts and Health Benefits. Verywell Fit. https://www.verywellfit.com/mushroom-nutrition-facts-calories-andhealth-benefits-4117115 Chandra, R., Singh, S., Reddy, M. M. K., Patel, D. K., Purohit, H. J., & Kapley, A. (2008). Isolation and characterization of bacterial strains Paenibacillus sp. And Bacillus sp. For kraft lignin decolorization from pulp paper mill waste. The Journal of General and Applied Microbiology, 54(6), 399–407. https://doi.org/10.2323/jgam.54.399 Climate-Data.org. (2019). Climate data for cities worldwide. https://en.climate-data.org/ Colavolpe, M. B., Mejía, S. J., & Albertó, E. (2014). Efficiency of treatments for controlling Trichoderma spp. during spawning in cultivation of lignicolous mushrooms. Brazilian Journal of Microbiology, 45, 1263–1270. https://doi.org/10.1590/S151783822014000400017 Cueva, M. B. R., Hernández, A., & Niño-Ruiz, Z. (2017). Influence of C/N ratio on productivity and the protein contents of Pleurotus ostreatus grown in different residue mixtures. Revista de La Facultad de Ciencias Agrarias, 49(2), 331–344. da Silva Alves, L., Tolardo, G., Caitano, C. E. C., Vieira Júnior, W. G., Gomes Freitas, P. N., & Cunha Zied, D. (2024). Use of spent mushroom substrate for cherry tomato seedlings; a potential alternative to peat in horticulture. Biological Agriculture & Horticulture, 1–14. https://doi.org/10.1080/01448765.2024.2308301 Davey, R. (2021, May 19). What Chemical Elements are Found in the Human Body? Medical News. https://www.news-medical.net/life-sciences/What-ChemicalElements-are-Found-in-the-Human-Body.aspx De Cianni, R., Pippinato, L., & Mancuso, T. (2023). A systematic review on drivers influencing consumption of edible mushrooms and innovative mushroom-containing products. Appetite, 182, 106454. https://doi.org/10.1016/j.appet.2023.106454 de Gonzalo, G., Colpa, D. I., Habib, M. H. M., & Fraaije, M. W. (2016). Bacterial enzymes involved in lignin degradation. Journal of Biotechnology, 236, 110–119. https://doi.org/10.1016/j.jbiotec.2016.08.011 Deveau, A., Bonito, G., Uehling, J., Paoletti, M., Becker, M., Bindschedler, S., Hacquard, S., Hervé, V., Labbé, J., Lastovetsky, O. A., Mieszkin, S., Millet, L. J., Vajna, B., Junier, P., Bonfante, P., Krom, B. P., Olsson, S., van Elsas, J. D., & Wick, L. Y. (2018). Bacterial–fungal interactions: Ecology, mechanisms and challenges. FEMS Microbiology Reviews, 42(3), 335–352. https://doi.org/10.1093/femsre/fuy008 135 Dias, E. S., Zied, D. C., & Pardo-Gimenez, A. (2021). Revisiting the casing layer: Casing materials and management in Agaricus mushroom cultivation. Ciência e Agrotecnologia, 45, e0001R21. https://doi.org/10.1590/1413-70542021450001R21 Domondon, D., & Poppe, J. (2000). Fruit optimization with wastes used for outdoor cultivation of King Stropharia. In L. Van Griensven (Ed.), Science and Cultivation of Edible Fungi (Vol. 2, pp. 909–918). Balkema. Douglas, G. M., & Langille, M. G. I. (2021). A primer and discussion on DNA-based microbiome data and related bioinformatics analyses. OSF Preprints, ver. 4 peerreviewed and recommended by Peer Community in Genomics. https://doi.org/10.31219/osf.io/3dybg Eudoxie, G. D., & Alexander, I. A. (2011). Spent Mushroom Substrate as a Transplant Media Replacement for Commercial Peat in Tomato Seedling Production. Journal of Agricultural Science, 3(4). Fengel, D., & Wegener, G. (1984). Wood—Chemistry, ultrastructure, reactions. De Gruyter. Francioli, D., Lentendu, G., Lewin, S., & Kolb, S. (2021). DNA Metabarcoding for the Characterization of Terrestrial Microbiota—Pitfalls and Solutions. Microorganisms, 9(2), Article 2. https://doi.org/10.3390/microorganisms9020361 Fukasawa, Y., & Kaga, K. (2022). Surface Area of Wood Influences the Effects of Fungal Interspecific Interaction on Wood Decomposition—A Case Study Based on Pinus densiflora and Selected White Rot Fungi. Journal of Fungi, 8(5), Article 5. https://doi.org/10.3390/jof8050517 Gao, W., Liang, J., Pizzul, L., Feng, X. M., Zhang, K., & Castillo, M. del P. (2015). Evaluation of spent mushroom substrate as substitute of peat in Chinese biobeds. International Biodeterioration & Biodegradation, 98, 107–112. https://doi.org/10.1016/j.ibiod.2014.12.008 Ghimire, A., Pandey, K. R., Joshi, Y. R., & Subedi, S. (2021). Major Fungal Contaminants of Mushrooms and Their Management. International Journal of Applied Sciences and Biotechnology, 9(2), Article 2. https://doi.org/10.3126/ijasbt.v9i2.37513 Gibson, I. (2020). Stropharia rugosoannulata. E-Flora BC Distribution Map. https://linnet.geog.ubc.ca/eflora_SMaps/indexStatic.html?sciname=Stropharia%20rug osoannulata&synonyms=(%27none%27)&mapservice=fungi Gilmour, J. T., Norman, R. J., Mauromoustakos, A., & Gale, P. M. (1998). Kinetics of Crop Residue Decomposition: Variability among Crops and Years. Soil Science Society of 136 America Journal, 62(3), 750–755. https://doi.org/10.2136/sssaj1998.03615995006200030030x Gloor, G. B., & Reid, G. (2016). Compositional analysis: A valid approach to analyze microbiome high-throughput sequencing data. Canadian Journal of Microbiology, 62(8), 692–703. https://doi.org/10.1139/cjm-2015-0821 Govindasamy, V., Senthilkumar, M., Magheshwaran, V., Kumar, U., Bose, P., Sharma, V., & Annapurna, K. (2011). Bacillus and Paenibacillus spp.: Potential PGPR for Sustainable Agriculture. In D. K. Maheshwari (Ed.), Plant Growth and Health Promoting Bacteria (pp. 333–364). Springer. https://doi.org/10.1007/978-3-64213612-2_15 Grady, E. N., MacDonald, J., Liu, L., Richman, A., & Yuan, Z.-C. (2016). Current knowledge and perspectives of Paenibacillus: A review. Microbial Cell Factories, 15(1), 203. https://doi.org/10.1186/s12934-016-0603-7 Grimm, D., & Wösten, H. A. B. (2018). Mushroom cultivation in the circular economy. Applied Microbiology and Biotechnology, 102(18), 7795–7803. https://doi.org/10.1007/s00253-018-9226-8 Grinspoon, P. (2021, September 24). Cannabidiol (CBD): What we know and what we don’t. Harvard Health. https://www.health.harvard.edu/blog/cannabidiol-cbd-what-weknow-and-what-we-dont-2018082414476 Guo, R., Li, G., Jiang, T., Schuchardt, F., Chen, T., Zhao, Y., & Shen, Y. (2012). Effect of aeration rate, C/N ratio and moisture content on the stability and maturity of compost. Bioresource Technology, 112, 171–178. https://doi.org/10.1016/j.biortech.2012.02.099 Haq, I. U., Hillmann, B., Moran, M., Willard, S., Knights, D., Fixen, K. R., & Schilling, J. S. (2022). Bacterial communities associated with wood rot fungi that use distinct decomposition mechanisms. ISME Communications, 2(1), 1–9. https://doi.org/10.1038/s43705-022-00108-5 Haynes, R. J., & Swift, R. S. (1986). Effects of soil acidification and subsequent leaching on levels of extractable nutrients in a soil. Plant and Soil, 95(3), 327–336. https://doi.org/10.1007/BF02374613 Health Canada. (2023, June 12). Industrial hemp licensing statistics [Datasets; statistics]. Government of Canada. https://www.canada.ca/en/health-canada/services/drugsmedication/cannabis/producing-selling-hemp/about-hemp-canada-hempindustry/statistics-reports-fact-sheets-hemp.html 137 Hoa, H. T., Wang, C.-L., & Wang, C.-H. (2015). The Effects of Different Substrates on the Growth, Yield, and Nutritional Composition of Two Oyster Mushrooms (Pleurotus ostreatus and Pleurotus cystidiosus). Mycobiology, 43(4), 423–434. https://doi.org/10.5941/MYCO.2015.43.4.423 Hopkins, W. G., & Hüner, N. P. A. (2009). Introduction to Plant Physiology (Fourth). John Wiley & Sons, Inc. Isroi, Millati, R., Syamsiah, S., Niklasson, C., Cahyanto, M. N., Ludquist, K., & Taherzadeh, M. J. (2011). Biological pretreatment of lignocelluloses with white-rot fungi and its applications: A review. BioResources, 6(4), Article 4. Iványi, I., & Izsáki, Z. (2009). Effect of Nitrogen, Phosphorus, and Potassium Fertilization on Nutritional Status of Fiber Hemp. Communications in Soil Science and Plant Analysis, 40(1–6), 974–986. https://doi.org/10.1080/00103620802693466 Jacobs, A. A. (2016, November). Plant guide for common barley (Hordeum vulgare L.). USDA - Natural Resources Conservation Service, Jamie L. Whitten Plant Materials Center. Coffeeville, Mississippi. Kaal, E. E. J., De Jong, E., & Field, J. A. (1993). Stimulation of Ligninolytic Peroxidase Activity by Nitrogen Nutrients in the White Rot Fungus Bjerkandera sp. Strain BOS55. Applied and Environmental Microbiology, 59(12), 4031–4036. https://doi.org/10.1128/aem.59.12.4031-4036.1993 Kaur, G., & Kander, R. (2023). The Sustainability of Industrial Hemp: A Literature Review of Its Economic, Environmental, and Social Sustainability. Sustainability, 15(8), Article 8. https://doi.org/10.3390/su15086457 Keddy, P. A. (2010). Chapter 7. In Wetland ecology: Principles and conservation (2nd ed., p. 497). Cambridge University Press. Khan, M. W., Ali, M. A., Khan, N. A., Khan, M. A., Rehman, A., & Javed, N. (2013). Effect of different levels of lime and pH on mycelial growth and production efficiency of oyster mushroom (Pleurotus spp.). Pakistan Journal of Botany, 45(1), 297–302. Kiesel, K., McCluskey, J. J., & Villas-Boas, S. B. (2011). Nutritional Labeling and Consumer Choices. Annual Review of Resource Economics, 3, 141–158. Kirby, R. (2005). Actinomycetes and Lignin Degradation. In A. I. Laskin, J. W. Bennett, G. M. Gadd, & S. Sariaslani (Eds.), Advances in Applied Microbiology (Vol. 58, pp. 125–168). Academic Press. https://doi.org/10.1016/S0065-2164(05)58004-3 138 Kombrink, A., Tayyrov, A., Essig, A., Stöckli, M., Micheller, S., Hintze, J., van Heuvel, Y., Dürig, N., Lin, C., Kallio, P. T., Aebi, M., & Künzler, M. (2019). Induction of antibacterial proteins and peptides in the coprophilous mushroom Coprinopsis cinerea in response to bacteria. The ISME Journal, 13(3), 588–602. https://doi.org/10.1038/s41396-018-0293-8 Kopp, O. C. (2024, February 18). Peat. Britannica. https://britannica.com/technology/peat Kumar, R., Tapwal, A., Pandey, S., Borah, R. K., Borah, D., & Borgohain, J. (2013). Macrofungal diversity and nutrient content of some edible mushrooms of Nagaland, India. Nusantara Bioscience, 5(1), 1–5. https://doi.org/10.13057/nusbiosci/n050101 Kurz-Besson, C., Coûteaux, M.-M., Thiéry, J. M., Berg, B., & Remacle, J. (2005). A comparison of litterbag and direct observation methods of Scots pine needle decomposition measurement. Soil Biology and Biochemistry, 37(12), 2315–2318. https://doi.org/10.1016/j.soilbio.2005.03.022 Lankiewicz, T. S., Choudhary, H., Gao, Y., Amer, B., Lillington, S. P., Leggieri, P. A., Brown, J. L., Swift, C. L., Lipzen, A., Na, H., Amirebrahimi, M., Theodorou, M. K., Baidoo, E. E. K., Barry, K., Grigoriev, I. V., Timokhin, V. I., Gladden, J., Singh, S., Mortimer, J. C., … O’Malley, M. A. (2023). Lignin deconstruction by anaerobic fungi. Nature Microbiology, 8(4), Article 4. https://doi.org/10.1038/s41564-02301336-8 Lee, C.-Y., Park, J.-E., Kim, B.-B., Kim, S.-M., & Ro, H.-S. (2009). Determination of Mineral Components in the Cultivation Substrates of Edible Mushrooms and Their Uptake into Fruiting Bodies. Mycobiology, 37(2), 109–113. https://doi.org/10.4489/MYCO.2009.37.2.109 Lehmann, J., & Schroth, G. (2003). Chapter 7—Nutrient Leaching. In Trees, Crops and Soil Fertility—Concepts and Research Methods (pp. 151–166). CABI Publishing. Liaud, N., Giniés, C., Navarro, D., Fabre, N., Crapart, S., Gimbert, I. H.-, Levasseur, A., Raouche, S., & Sigoillot, J.-C. (2014). Exploring fungal biodiversity: Organic acid production by 66 strains of filamentous fungi. Fungal Biology and Biotechnology, 1(1), 1. https://doi.org/10.1186/s40694-014-0001-z Liu, Y., Hu, C.-F., Feng, X., Cheng, L., Ibrahim, S. A., Wang, C.-T., & Huang, W. (2020). Isolation, characterization and antioxidant of polysaccharides from Stropharia rugosoannulata. International Journal of Biological Macromolecules, 155, 883–889. https://doi.org/10.1016/j.ijbiomac.2019.11.045 139 Lu, Z., Hu, X., & Lu, Y. (2017). Particle Morphology Analysis of Biomass Material Based on Improved Image Processing Method. International Journal of Analytical Chemistry, 2017(1), 5840690. https://doi.org/10.1155/2017/5840690 Luo, H., Li, X., Li, G., Pan, Y., & Zhang, K. (2006). Acanthocytes of Stropharia rugosoannulata Function as a Nematode-Attacking Device. Applied and Environmental Microbiology, 72(4), 2982–2987. https://doi.org/10.1128/AEM.72.4.2982-2987.2006 Maceda, A., Soto-Hernández, M., Peña-Valdivia, C. B., Trejo, C., & Terrazas, T. (2020). Characterization of lignocellulose of Opuntia (Cactaceae) species using FTIR spectroscopy: Possible candidates for renewable raw material. Biomass Conversion and Biorefinery. https://doi.org/10.1007/s13399-020-00948-y MacKinnon, A., & Luther, K. (2021). Mushrooms of British Columbia. Royal BC Museum. MacKinnon, A., Pojar, J., & Coupe, R. (Eds.). (1999). Sitka alder. In Plants of Northern British Columbia (Second Edition, p. 37). Lone Pine Publishing. Madadi, M., & Abbas, A. (2017). Lignin Degradation by Fungal Pretreatment: A Review. Journal of Plant Pathology & Microbiology, 8, 1–6. https://doi.org/10.4172/21577471.1000398 Mahesh, M. S., & Mohini, M. (2013). Biological treatment of crop residues for ruminant feeding: A review. African Journal of Biotechnology, 12(27), Article 27. https://doi.org/10.5897/AJB2012.2940 Martino, C., McDonald, D., Cantrell, K., Dilmore, A. H., Vázquez-Baeza, Y., Shenhav, L., Shaffer, J. P., Rahman, G., Armstrong, G., Allaband, C., Song, S. J., & Knight, R. (2022). Compositionally Aware Phylogenetic Beta-Diversity Measures Better Resolve Microbiomes Associated with Phenotype. mSystems, 7(3), e00050-22. https://doi.org/10.1128/msystems.00050-22 Mattila, P., Könkö, K., Eurola, M., Pihlava, J.-M., Astola, J., Vahteristo, L., Hietaniemi, V., Kumpulainen, J., Valtonen, M., & Piironen, V. (2001). Contents of Vitamins, Mineral Elements, and Some Phenolic Compounds in Cultivated Mushrooms. Journal of Agricultural and Food Chemistry, 49(5), 2343–2348. https://doi.org/10.1021/jf001525d McKenzie, R. H., Middleton, A. B., Hall, L., DeMulder, J., & Bremer, E. (2004). Fertilizer response of barley grain in south and central Alberta. Canadian Journal of Soil Science, 84(4), 513–523. https://doi.org/10.4141/S04-013 140 McKoy, P. (2016). Radical Mycology. Chthaeus Press. McSpadden Gardener, B. B. (2004). Ecology of Bacillus and Paenibacillus spp. In Agricultural Systems. Phytopathology, 94(11), 1252–1258. Mercy, R. (2021, January 12). Grow Your Own—Mr. Mercy’s Mushrooms. https://mrmercysmushrooms.com/grow-your-own/ Merlos, F. A., & Hijmans, R. J. (2020). The scale dependency of spatial crop species diversity and its relation to temporal diversity. Proceedings of the National Academy of Sciences, 117(42), 26176–26182. https://doi.org/10.1073/pnas.2011702117 Michel, J.-C. (2010). The physical properties of peat: A key factor for modern growing media. Mires and Peat, 6(2). https://institut-agro-rennes-angers.hal.science/hal00729716 Mohd Hanafi, F. H., Rezania, S., Mat Taib, S., Md Din, M. F., Yamauchi, M., Sakamoto, M., Hara, H., Park, J., & Ebrahimi, S. S. (2018). Environmentally sustainable applications of agro-based spent mushroom substrate (SMS): An overview. Journal of Material Cycles and Waste Management, 20(3), 1383–1396. https://doi.org/10.1007/s10163018-0739-0 Odero, G. M. O. (2009). Substrates Evaluation and Effects of pH and Nutritional Supplementation on Production of Oyster Mushroom (Pleurotus Ostreatus) [Master’s]. University of Nairobi. Oei, P., & van Nieuwenhuijzen, B. (2005). Small-scale mushroom cultivation (J. de Feijter, Ed.; 1st ed.). Onyeka, E. U., Udeogu, E., Umelo, C., & Okehie, M. A. (2018). Effect of substrate media on growth, yield and nutritional composition of domestically grown oyster mushroom (Pleurotus ostreatus). African Journal of Plant Science, 12(7), 141–147. https://doi.org/10.5897/AJPS2016.1445 Orji, O. A., & Wali, C. (2021). Effect of different sources of “browns” and “greens” on the quality of compost. IOSR Journal of Agriculture and Veterinary Science, 14(6), 8–12. https://doi.org/10.9790/2380-1406010812 Ostrofsky, A., Jellison, J., Smith, K. T., & Shortle, W. C. (1997). Changes in cation concentrations in red spruce wood decayed by brown rot and white rot fungi. Canadian Journal of Forest Research, 27(4), 567–571. https://doi.org/10.1139/x96188 141 Ozores-Hampton, M. (2017). Guidelines for Assessing Compost Quality for Safe and Effective Utilization in Vegetable Production. HortTechnology, 27(2), 162–165. https://doi.org/10.21273/HORTTECH03349-16 Pancaldi, F., Salentijn, E. M. J., & Trindade, L. M. (2025). From fibers to flowering to metabolites: Unlocking hemp (Cannabis sativa) potential with the guidance of novel discoveries and tools. Journal of Experimental Botany, 76(1), 109–123. https://doi.org/10.1093/jxb/erae405 Paula, F. S., Tatti, E., Abram, F., Wilson, J., & O’Flaherty, V. (2017). Stabilisation of spent mushroom substrate for application as a plant growth-promoting organic amendment. Journal of Environmental Management, 196, 476–486. https://doi.org/10.1016/j.jenvman.2017.03.038 Pérez Harguindeguy, N., Blundo, C. M., Gurvich, D. E., Díaz, S., & Cuevas, E. (2008). More than the sum of its parts? Assessing litter heterogeneity effects on the decomposition of litter mixtures through leaf chemistry. Plant and Soil, 303(1), 151–159. https://doi.org/10.1007/s11104-007-9495-y Philippoussis, A. N., Diamantopoulou, P. A., & Zervakis, G. I. (2003). Correlation of the properties of several lignocellulosic substrates to the crop performance of the shiitake mushroom Lentinula edodes. World Journal of Microbiology and Biotechnology, 19(6), 551–557. https://doi.org/10.1023/A:1025100731410 Põlme, S., Abarenkov, K., Henrik Nilsson, R., Lindahl, B. D., Clemmensen, K. E., Kauserud, H., Nguyen, N., Kjøller, R., Bates, S. T., Baldrian, P., Frøslev, T. G., Adojaan, K., Vizzini, A., Suija, A., Pfister, D., Baral, H.-O., Järv, H., Madrid, H., Nordén, J., … Tedersoo, L. (2020). FungalTraits: A user-friendly traits database of fungi and fungus-like stramenopiles. Fungal Diversity, 105(1), 1–16. https://doi.org/10.1007/s13225-020-00466-2 Pourazari, F. (2016). Nutrient Economy in Annual and Perennial Crops [Doctoral dissertation]. Swedish University of Agricultural Sciences. Powlson, D. S., & Jenkinson, D. S. (1976). The effects of biocidal treatments on metabolism in soil—II. Gamma irradiation, autoclaving, air-drying and fumigation. Soil Biology and Biochemistry, 8, 179–188. Prasad, R., Lisiecka, J., Antala, M., & Rastogi, A. (2021). Influence of Different Spent Mushroom Substrates on Yield, Morphological and Photosynthetic Parameters of Strawberry (Fragaria × ananassa Duch.). Agronomy, 11(10), Article 10. https://doi.org/10.3390/agronomy11102086 142 Ragasa, C. Y., Tan, M. C. S., & Shen, C.-C. (2016). Chemical Constituents of Coprinopsis lagopus. International Journal of Toxicological and Pharmacological Research, 8(6), 421–424. Raud, M., Tutt, M., Olt, J., & Kikas, T. (2015). Effect of lignin content of lignocellulosic material on hydrolysis efficiency. Agronomy Research, 13(2), 405–412. Rhoades, H. (2012, March 2). Turning Your Compost Heap—How to Aerate A Compost Pile. Gardening Knowhow. https://www.gardeningknowhow.com/composting/basics/turning-compost-pile.htm Rinker, D. (2017). Spent Mushroom Substrate Uses. In Edible and Medicinal Mushrooms: Technology and Applications (First, pp. 427–454). Wiley. https://doi.org/10.1002/9781119149446.ch20 Rodriguez, R. J., & Redman, R. S. (1997). Fungal Life-Styles and Ecosystem Dynamics: Biological Aspects of Plant Pathogens, Plant Endophytes and Saprophytes. In J. H. Andrews, I. C. Tommerup, & J. A. Callow (Eds.), Advances in Botanical Research (Vol. 24, pp. 169–193). Academic Press. https://doi.org/10.1016/S00652296(08)60073-7 Rodríguez-Couto, S. (2017). Industrial and environmental applications of white-rot fungi. Mycosphere, 8(3), 456–466. https://doi.org/10.5943/mycosphere/8/3/7 Royse, D. (2014). A Global Perspective on the High Five: Agaricus, Pleurotus, Lentinula, Auricularia & Flammulina. Proceedings of the 8th International Conference on Mushroom Biology and Mushroom Products (ICMBMP8). Rynk, R., Black, G., Gilbert, J., Biala, J., Bonhotal, J., Schwarz, M., & Cooperband, L. (Eds.). (2021). The Composting Handbook (1st ed.). Academic Press. Sakamoto, Y. (2018). Influences of environmental factors on fruiting body induction, development and maturation in mushroom-forming fungi. Fungal Biology Reviews, 32(4), 236–248. https://doi.org/10.1016/j.fbr.2018.02.003 Sales-Campos, C., Eira, A. F., Minhoni, M. T. A., & Andrade, M. C. N. (2009). Mineral composition of raw material, substrate and fruiting bodies of Pleurotus ostreatus in culture. Interciencia, 34(6), 432–436. Schaefer, H. R., Flannery, B. M., Crosby, L. M., Pouillot, R., Farakos, S. M. S., Van Doren, J. M., Dennis, S., Fitzpatrick, S., & Middleton, K. (2023). Reassessment of the cadmium toxicological reference value for use in human health assessments of foods. 143 Regulatory Toxicology and Pharmacology, 144, 105487. https://doi.org/10.1016/j.yrtph.2023.105487 Selvaraju, R., Gommes, R., & Bernardi, M. (2011). Climate science in support of sustainable agriculture and food security. Climate Research, 47(1), 95–110. https://doi.org/10.3354/cr00954 Shamugam, S., & Kertesz, M. A. (2023). Bacterial interactions with the mycelium of the cultivated edible mushrooms Agaricus bisporus and Pleurotus ostreatus. Journal of Applied Microbiology, 134(1), 1–10. https://doi.org/10.1093/jambio/lxac018 Sharma, V. P., Sharma, S. R., & Kumar, S. (2007). Cultivation of Least Exploited Commercial Mushrooms. In R. Rai, S. Singh, Dr. M. Yadav, & R. P. Tewari (Eds.), Mushroom Biology and Biotechnology (pp. 167–192). Mushroom Society of India. Sheldrake, M. (2021). Entangled Life. Random House. Shields, T. (2018, March 5). Growing Mushrooms Using a Casing Layer. FreshCap Mushrooms. https://learn.freshcap.com/growing/growing-mushrooms-using-a-casinglayer/ Singh, M., Kamal, S., & Sharma, V. P. (2020). Status and trends in world mushroom production-III -World Production of Different Mushroom Species in 21st Century. Mushroom Research, 29(2), Article 2. https://epubs.icar.org.in/index.php/MR/article/view/113703 Smeriglio, A., Galati, E. M., Monforte, M. T., Gaia, F., & Circosta, C. (2015). Antioxidant Properties of Finola Hemp (Cannabis sativa L.) Seed Oil. 51. https://iris.unime.it/handle/11570/3064662 Španić, N., Jambreković, V., & Klarić, M. (2018). Basic chemical composition of wood as a parameter in raw material selection for biocomposite production. Cellulose Chemistry and Technology, 52(3–4), 163–169. Stamets, P. (2000). Growing Gourmet and Medicinal Mushrooms (3rd ed.). Ten Speed Press. Sui, Z., Yin, J., Huang, J., & Yuan, L. (2022). Phosphorus mobilization and improvement of crop agronomic performances by a new white-rot fungus Ceriporia lacerata HG2011. Journal of the Science of Food and Agriculture, 102(4), 1640–1650. https://doi.org/10.1002/jsfa.11501 144 Sun, H., Wei, C., Xu, W., Yang, J., Wang, X., & Qiu, Y. (2019). Characteristics of salt contents in soils under greenhouse conditions in China. Environmental Science and Pollution Research, 26(4), 3882–3892. https://doi.org/10.1007/s11356-018-3865-2 Sun, R. C., & Tomkinson, T. (2000). Essential guides for isolation/purification of polysaccharides. Encyclopedia of Separation Science, 6, 4568–4574. https://doi.org/10.1016/B0-12-226770-2/07271-9 Sundberg, C. (2005). Improving Compost Process Efficiency by Controlling Aeration, Temperature and pH [Doctoral dissertation]. Swedish University of Agricultural Sciences. Szudyga, K. (1978). Stropharia rugoso-annulata. In S. Chang & W. Hayes (Eds.), Biology and Cultivation of Edible Mushrooms (pp. 559–571). Academic Press, Inc. Tian, B., Wang, C., Lv, R., Zhou, J., Li, X., Zheng, Y., Jin, X., Wang, M., Ye, Y., Huang, X., & Liu, P. (2014). Community Structure and Succession Regulation of Fungal Consortia in the Lignocellulose-Degrading Process on Natural Biomass. The Scientific World Journal, 2014(1), 845721. https://doi.org/10.1155/2014/845721 Toju, H., Tanabe, A. S., Yamamoto, S., & Sato, H. (2012). High-Coverage ITS Primers for the DNA-Based Identification of Ascomycetes and Basidiomycetes in Environmental Samples. PLOS ONE, 7(7), e40863. https://doi.org/10.1371/journal.pone.0040863 Tutt, M., Kikas, T., & Olt, J. (2013). Influence of harvesting time on biochemical composition and glucose yield from hemp. Agronomy Research, 11(1), 215–220. Ulziijargal, E., & Mau, J.-L. (2011). Nutrient Compositions of Culinary-Medicinal Mushroom Fruiting Bodies and Mycelia. International Journal of Medicinal Mushrooms, 13(4). https://doi.org/10.1615/IntJMedMushr.v13.i4.40 Van Soest, P. J., & McQueen, R. W. (1973). The chemistry and estimation of fibre. Proceedings of the Nutrition Society, 32(3), 123–130. https://doi.org/10.1079/PNS19730029 Van Soest, P. J., & Robertson, J. B. (1980). Standardization of analytical methodology for feeds: Proceedings of a workshop held in Ottawa, Canada, 12 - 14 March 1979 (W. J. Pigden, C. C. Balch, & M. Graham, Eds.). International Development Research Centre. Van Soest, P. J., Robertson, J. B., & Lewis, B. A. (1991). Methods for Dietary Fiber, Neutral Detergent Fiber, and Nonstarch Polysaccharides in Relation to Animal Nutrition. 145 Journal of Dairy Science, 74(10), 3583–3597. https://doi.org/10.3168/jds.S00220302(91)78551-2 Van Tassel, D. L., DeHaan, L. R., & Cox, T. S. (2010). Missing domesticated plant forms: Can artificial selection fill the gap? Evolutionary Applications, 3(5–6), 434–452. https://doi.org/10.1111/j.1752-4571.2010.00132.x Vance, E. D., Brookes, P. C., & Jenkinson, D. S. (1987). An extraction method for measuring soil microbial biomass C. Soil Biology and Biochemistry, 19(6), 703–707. https://doi.org/10.1016/0038-0717(87)90052-6 Velázquez-Cedeño, M., Farnet, A. M., Mata, G., & Savoie, J.-M. (2008). Role of Bacillus spp. in antagonism between Pleurotus ostreatus and Trichoderma harzianum in heattreated wheat-straw substrates. Bioresource Technology, 99(15), 6966–6973. https://doi.org/10.1016/j.biortech.2008.01.022 Walters, W. A., Caporaso, J. G., Lauber, C. L., Berg-Lyons, D., Fierer, N., & Knight, R. (2011). PrimerProspector: De novo design and taxonomic analysis of barcoded polymerase chain reaction primers. Bioinformatics, 27(8), 1159–1161. https://doi.org/10.1093/bioinformatics/btr087 Wei, Y., Wu, D., Wei, D., Zhao, Y., Wu, J., Xie, X., Zhang, R., & Wei, Z. (2019). Improved lignocellulose-degrading performance during straw composting from diverse sources with actinomycetes inoculation by regulating the key enzyme activities. Bioresource Technology, 271, 66–74. https://doi.org/10.1016/j.biortech.2018.09.081 Weil, R., & Brady, N. (2017). The Nature and Properties of Soils (15th ed.). Pearson. Wichuk, K. M., & McCartney, D. (2010). Compost stability and maturity evaluation—A literature review. Canadian Journal of Civil Engineering, 37(11), 1505–1523. https://doi.org/10.1139/L10-101 Wickham, H. (2016). Ggplot2. Springer International Publishing. https://doi.org/10.1007/978-3-319-24277-4 Willis, A. D. (2019). Rarefaction, Alpha Diversity, and Statistics. Frontiers in Microbiology, 10. https://doi.org/10.3389/fmicb.2019.02407 Xie, Y. (2020). A meta-analysis of critique of litterbag method used in examining decomposition of leaf litters. Journal of Soils and Sediments, 20(4), 1881–1886. https://doi.org/10.1007/s11368-020-02572-9 146 Yin, J., Sui, Z., Li, Y., Yang, H., Yuan, L., & Huang, J. (2022). A new function of white-rot fungi Ceriporia lacerata HG2011: Improvement of biological nitrogen fixation of broad bean (Vicia faba). Microbiological Research, 256, 126939. https://doi.org/10.1016/j.micres.2021.126939 Zabel, R. A., & Morrell, J. J. (2020). Chemical changes in wood caused by decay fungi. In Wood Microbiology (Second). Zadražil, F. (1978). Cultivation of Pleurotus. In S. Chang & W. Hayes (Eds.), The Biology and Cultivation of Edible Mushrooms (pp. 521–557). Academic Press. https://doi.org/10.1016/B978-0-12-168050-3.50031-1 Zhang, L., Tang, C., Yang, J., Yao, R., Wang, X., Xie, W., & Ge, A.-H. (2023). Salinitydependent potential soil fungal decomposers under straw amendment. Science of The Total Environment, 891, 164569. https://doi.org/10.1016/j.scitotenv.2023.164569 Zhao, N., Yu, G., Wang, Q., Wang, R., Zhang, J., Liu, C., & He, N. (2020). Conservative allocation strategy of multiple nutrients among major plant organs: From species to community. Journal of Ecology, 108(1), 267–278. https://doi.org/10.1111/13652745.13256 Zhao, X., Guo, P., Zhang, Z., Yang, Y., & Zhao, P. (2020). Wood Density, Anatomical Characteristics, and Chemical Components of Alnus sibirica Used for Industrial Applications. Forest Products Journal, 70(3), 356–363. https://doi.org/10.13073/FPJD-20-00006 Zied, D. C., Sánchez, J. E., Noble, R., & Pardo-Giménez, A. (2020). Use of Spent Mushroom Substrate in New Mushroom Crops to Promote the Transition towards A Circular Economy. Agronomy, 10(9), Article 9. https://doi.org/10.3390/agronomy10091239 147 Appendices Appendix A: Mushroom composition data tables Table A-1: Results of Kruskal-Wallace Multiple Comparison test on mushroom sample carbon and nitrogen content. Response variable Total carbon (% dry matter) Total nitrogen (% dry matter) Substrate N Median Alder chips 2 43.25 Barley straw 5 42.7 Hemp straw 6 43.3 Overall 13 43.2 Alder chips 2 4.55 Barley straw 5 5.6 Hemp straw 6 4.5 Overall 13 4.7 Mean rank 7.8 4.8 8.6 7 5.3 9 5.9 7 Z-value DF H-value 0.3 -1.61 1.36 2 2.71 -0.69 1.46 -0.93 2 2.26 P-value 0.258 0.323 Table A-2: Significant results of Kruskal-Wallace Multiple Comparisons test on mushroom ICP-OES elemental analysis data. P-values < 0.05 are indicated by *. All values are reported on a dry matter basis. Element B Cd Fe Substrate Alder chips Barley straw Hemp straw Overall Barley straw vs. Hemp straw Alder chips vs. Hemp straw Alder chips Barley straw Hemp straw Overall Alder chips vs. Barley straw Alder chips Barley straw Hemp straw Overall N Median Mean Rank Z-value 2 1 4 -1.18 5 1 4.9 -1.54 6 5.5 9.8 2.36 13 7 2 5 6 13 2 5 6 13 1.55 0.4 0.45 12.5 4.9 6.9 DF H value P value 2 0.035* 2.23946 0.0251* 1.96902 2.17 -1.54 -0.07 0.049* 2 86.5 109 145 3.5 4.8 10 7 6.68 5.78 2.40283 -1.38 -1.61 2.57 0.056 0.0163* 2 6.79 0.034* 148 Mn Barley straw vs. Hemp straw Alder chips vs. Hemp straw Alder chips Barley straw Hemp straw Overall Alder chips vs. Hemp straw 2 5 6 13 14 20 23.5 2.5 5.7 9.6 7 2.20811 0.0272* 2.04697 -1.78 -0.95 2.21 0.0407* 2 2.26526 6.07 0.048* 0.0235* 149 Appendix B: Substrate moisture content data tables Substrate comparisons Table B-1: Results of Kruskal-Wallace multiple comparison test on the relationship between substrate type and substrate moisture content (fresh weight basis). Substrate: A = alder chips, B = barley straw, H = hemps straw. Treatment: t = treated (inoculated), u = untreated (uninoculated). Cultivation stage: P = pre-cultivation, C = post-cultivation. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation Treatment stage t u P P Median % Substrate N moisture content Mean Rank ZHValue DF value P-value A 5 61.2 3 -3.12 B 5 78.1 10.2 0.96 H 6 78.9 11.7 2.06 Overall 16 8.5 u C C 9.98 0.007*** A vs. H 3.01 0.0026*** A vs. B 2.39 0.0167*** A 3 60.8 2 -2.24 B 3 78.8 5.7 1.04 H 2 80.15 6.5 1.33 Overall 8 4.5 A vs. H t 2 2 2.01 A 5 54 3 -3.12 B 5 80.1 14 3.12 H 6 69.4 8.5 0 Overall 16 8.5 5.14 0.077 0.0442*** 2 13.35 0.001*** A vs. B 3.65 0.0003*** A vs. H 1.91 0.0564* B vs. H 1.91 0.0564* A 3 61.7 2 -2.24 B 3 84.1 7 2.24 H 2 79.05 4.5 0 Overall 8 A vs. B 4.5 2 2.5 6.25 0.044*** 0.0124*** 150 Treatment comparisons Table B-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and substrate moisture content (wet weight basis). Substrate: A = alder chips, B = barley straw, H = hemp straw. Cultivation stage: P = pre-cultivation, C = post-cultivation. Treatment: t = treated (inoculated), u = untreated (uninoculated). Cultivation Substrate stage A B H A B H P P P C C C Median % moisture Mean Treatment N content Rank ZHValue DF value t 5 61.2 5.1 0.89 u 3 60.8 3.5 -0.89 Overall 8 t 5 78.1 4 -0.75 u 3 78.8 5.3 0.75 Overall 8 t 6 78.9 4.2 -0.67 u 2 80.15 5.5 0.67 Overall 8 t 5 54 3 -2.24 u 3 61.7 7 2.24 Overall 8 t 5 80.1 3.4 -1.64 u 3 84.1 6.3 1.64 Overall 8 t 6 69.4 3.5 -2 u 2 79.05 7.5 2 Overall 8 4.5 4.5 4.5 4.5 4.5 1 0.82 0.365 1 0.56 0.456 1 0.44 0.505 1 4.5 P-value 1 1 5 0.025*** 2.69 0.101 4 0.046*** 151 Appendix C: Substrate carbon and nitrogen data tables Substrate comparisons Table C-1 (a) – (d). Results of Kruskal-Wallace Multiple Comparisons test on the relationship between substrate type and carbon, nitrogen (expressed as a percentage of oven dry sample mass) and C:N ratio. Substrates: A = alder chips, B = barley straw, H = hemp straw. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. (a) Treated (inoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value P-value A 5 0.67 4.6 -2.21 B 5 0.89 11.4 1.64 Total nitrogen H 6 0.84 9.3 0.54 Overall 16 8.5 2 5.39 0.067* A vs. B 2.26 0.0239*** A 5 49.26 14 3.12 B 5 45.59 7.6 -0.51 H 6 45.25 4.7 -2.49 Total carbon Overall 16 8.5 2 10.74 0.005*** A vs. H 3.24 0.0012*** A vs. B 2.13 0.0335*** A 5 74.21 13.2 2.66 B 5 50.97 5.2 -1.87 H 6 54.015 7.3 -0.76 C:N Overall 16 8.5 2 7.64 0.022*** A vs. B 2.66 0.0079*** A vs. H 2.03 0.0419*** (b) Untreated (uninoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value P-value A 3 0.66 3 -1.34 B 3 0.8 6.7 1.94 Total nitrogen H 2 0.71 3.5 -0.67 Overall 8 4.5 2 3.81 0.149 A 3 48.79 7 2.24 B 3 45.33 3.3 -1.04 Total carbon H 2 44.71 2.5 -1.33 Overall 8 4.5 2 5.14 0.077 A vs. H 2.012 0.0442*** 152 C:N A B H Overall A vs. B 3 73.92 3 55.29 2 62.98 8 6.7 2 5 4.5 1.94 -2.24 0.33 2 2.33 5.56 0.062* 0.0196*** (c) Treated, post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value P-value A 5 0.86 5.8 -1.53 B 5 1.44 9.8 0.74 Total nitrogen H 6 1.525 9.7 0.76 Overall 16 8.5 2 2.34 0.31 A 5 49.1 14 3.12 B 5 43.97 9 0.28 H 6 39.845 3.5 -3.25 Total carbon Overall 16 8.5 2 13.35 0.001*** A vs. H 3.64 0.0003*** B vs. H 1.91 0.0564* A 5 55.36 11.2 1.53 B 5 30.53 8.4 -0.06 C:N H 6 26.24 6.3 -1.41 Overall 16 8.5 2 2.85 0.24 (d) Untreated, post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value P-value A 3 2.95 4 -0.45 B 3 3.52 6 1.34 Total nitrogen H 2 1.87 3 -1 Overall 8 4.5 2 2 0.368 A 3 48.81 7 2.24 B 3 45.43 2 -2.24 Total carbon H 2 46.15 4.5 0 Overall 8 4.5 2 6.25 0.044*** A vs. B 2.5 0.0124*** A 3 16.55 5 0.45 B 3 12.82 3 -1.34 C:N H 2 30.75 6 1 Overall 8 4.5 2 2 0.368 153 Treatment comparisons Table C-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and carbon, nitrogen (expressed as a percentage of oven dry sample mass) and C:N. Treatment: t = treated (inoculated), u = untreated (uninoculated). P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation stage Substrate Alder chips Precultivation Barley straw Hemp straw Postcultivation Alder chips Response variable Mean Treatment N Median Rank t 5 0.67 4.2 Total u 3 0.66 5 nitrogen Overall 8 4.5 t 5 49.26 5.4 Total carbon u 3 48.79 3 Overall 8 4.5 t 5 74.21 4.8 C:N u 3 74.27 4 Overall 8 4.5 t 5 0.89 4.8 Total u 3 0.8 4 nitrogen Overall 8 4.5 t 5 45.59 5.5 Total carbon u 3 45.33 2.8 Overall 8 4.5 t 5 50.97 4.2 C:N u 3 55.37 5 Overall 8 4.5 t 6 0.84 4.8 Total u 2 0.71 3.5 nitrogen Overall 8 4.5 t 6 45.25 5.3 Total carbon u 2 44.71 2 Overall 8 4.5 t 6 54.015 4.2 C:N u 2 63.32 5.5 Overall 8 4.5 t 5 0.86 4 Total u 3 2.95 5.3 nitrogen Overall 8 4.5 Total carbon t 5 49.1 4.4 ZHvalue DF value P-value -0.45 0.45 1 0.2 0.655 1.34 -1.34 1 1.8 0.18 0.45 -0.45 1 0.2 0.655 0.45 -0.45 1 0.2 0.655 1.49 -1.49 1 2.25 0.134 -0.45 0.45 1 0.2 0.655 0.67 -0.67 1 0.44 0.505 1.67 -1.67 1 2.78 0.096 -0.67 0.67 1 0.44 0.505 -0.75 0.75 1 0.56 0.456 -0.15 154 C:N Total nitrogen Barley straw Total carbon C:N Total nitrogen Hemp straw Total carbon C:N u Overall t u Overall t u Overall t u Overall t u Overall t u Overall t u Overall t u Overall 3 48.81 8 5 55.22 3 16.57 8 5 1.44 3 3.52 8 5 43.97 3 45.43 8 5 30.63 3 12.82 8 6 1.525 2 1.87 8 6 39.845 2 46.15 8 6 26.225 2 30.765 8 4.7 4.5 5 3.7 4.5 4.2 5 4.5 3 7 4.5 4.6 4.3 4.5 4.8 3.5 4.5 3.5 7.5 4.5 4.2 5.5 4.5 0.15 1 0.02 0.881 1 0.56 0.456 1 0.2 0.655 1 5 0.025*** 1 0.02 0.881 1 0.44 0.505 1 4 0.046*** 1 0.44 0.505 0.75 -0.75 -0.45 0.45 -2.24 2.24 0.15 -0.15 0.67 -0.67 -2 2 -0.67 0.67 Pre- and post-cultivation comparisons Table C-3. Results of Wilcoxon Signed Rank Confidence Interval test evaluating changes in pre- and post-cultivation substrate carbon, nitrogen (expressed as a percentage of oven dry sample mass) and C:N. P = pre-cultivation, C = post-cultivation. Note: Minitab statistical software will not perform the Wilcoxon test on sample sizes ≤3. Median CI for η, CI for η, Response change (P - lower upper Achieved Substrate Treatment variable N C) limit limit confidence % Interpretation Total Probable nitrogen 5 0.9 -0.07 2.43 94.09 increase Alder Total Treated chips carbon 5 -0.48 -2.05 1.33 94.09 No trend Probable C:N 5 -38.84 -53.12 5.78 94.09 decrease 155 Barley straw Hemp straw Alder chips Barley straw Hemp straw Treated Treated Untreated Untreated Untreated Total nitrogen Total carbon C:N Total nitrogen Total carbon C:N Total nitrogen Total carbon C:N Total nitrogen Total carbon C:N Total nitrogen Total carbon C:N 5 1.465 0.09 2.96 94.09 Increase 5 5 -1.75 -32.93 -2.47 -42.12 -1.46 -3.28 94.09 94.09 Decrease Decrease 6 1.55 0.42 2.76 94.08 Increase 6 6 -5.26 -34.055 -6.97 -52.54 -3.455 -17.3 94.08 94.08 Decrease Decrease 3 2.16 Insufficient data to perform test 3 3 -0.36 -46.38 Insufficient data to perform test Insufficient data to perform test 3 2.72 Insufficient data to perform test 3 3 0.49 -42.55 Insufficient data to perform test Insufficient data to perform test 2 1.16 Insufficient data to perform test 2 2 1.44 -32.56 Insufficient data to perform test Insufficient data to perform test 156 Appendix D: Substrate macronutrient ICP-OES analysis data tables Substrate comparisons Table D-1 (a) – (d). Results of Kruskal-Wallace Multiple Comparisons test on the relationship between substrate type and macronutrient (Ca, K, Mg, P, S) content (expressed in mg/kg of substrate dry matter). Substrates: A = alder chips, B = barley straw, H = hemp straw. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. (a) Treated (inoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Ca K Mg P A 5 5025.2 6.2 -1.3 B 5 4867.7 4.8 -2.1 H 6 11314.1 13.5 3.25 Overall 16 8.5 2 10.8 P-value 0.005*** B vs. H 3.02 0.0025*** A vs. H 2.53 0.0113*** A 5 1483.7 3.4 -2.89 B 5 4127.1 14 3.12 H 6 2267.4 8.2 -0.22 Overall 16 8.5 2 12.44 0.002*** A vs. B 3.52 0.0004*** B vs. H 2.02 0.043*** A 5 735.1 3 -3.12 B 5 1807.2 8 -0.28 H 6 2358.5 13.5 3.25 Overall 16 8.5 2 13.35 0.001*** A vs. H 3.64 0.0003*** B vs. H 1.91 0.0564* A 5 442.3 4.2 -2.44 B 5 1269.2 12.8 2.44 H 6 8.5 0 Overall 16 A vs. B 547.8 8.5 2 2.86 8.16 0.017*** 0.0043*** 157 S A 5 434.4 3 -3.12 B 5 1120.5 13.8 3 H 6 706.55 8.7 0.11 Overall 16 8.5 2 12.88 0.002*** A vs. B 3.59 0.0003*** A vs. H 1.97 0.0493*** (b) Untreated (uninoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Ca A 3 5537.6 4.7 0.15 B 3 4495.6 2.3 -1.94 H 2 9249.95 7.5 2 Overall 8 4.5 B vs. H K A 3 1428 3.7 -0.75 B 3 4077.5 7 2.24 H 2 1341.55 2 -1.67 4.5 B vs. H 3 736.9 2 -2.24 B 3 1778.9 5 0.45 H 2 2170.15 7.5 2 4.5 A vs. H 5.56 2 A 3 420.1 3 -1.34 B 3 1252.5 7 2.24 H 2 3 -1 446.6 4.5 A vs. B 6.25 A 3 400.1 2 -2.24 B 3 1098 7 2.24 H 2 663.5 4.5 0 0.044*** 0.0139*** 2 2 0.062* 0.0253*** 2.46 Overall 8 S 2 A 0.069 0.0209*** 2.24 Overall 8 P 5.36 2.31 Overall 8 Mg 2 P-value 5 0.082 0.0455*** 158 Overall 8 4.5 A vs. B 2 6.25 2.5 0.044*** 0.0124*** (c) Treated (inoculated) post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Ca K Mg P S A 5 6557.9 3.8 -2.66 B 5 9524.8 7.2 -0.74 H 6 13.5 3.25 28422 Overall 16 8.5 2 11.86 P-value 0.003*** A vs. H 3.36 0.0008*** B vs. H 2.19 0.0289*** A 5 1379.5 3 -3.12 B 5 4113.1 12 1.98 H 6 2893.4 10.2 1.08 Overall 16 8.5 2 10.11 0.006*** A vs. B 2.99 0.0028*** A vs. H 2.49 0.0129*** A 5 1027.9 3 -3.12 B 5 3089.4 8 -0.28 H 6 6443.6 13.5 3.25 Overall 16 8.5 2 13.35 0.001*** A vs. H 3.64 0.0003*** B vs. H 1.91 0.0564* A 5 429.1 3 -3.12 B 5 1608.7 13 2.55 H 6 9.3 0.54 992.6 Overall 16 8.5 2 11.32 0.003*** A vs. B 3.32 0.0009*** A vs. H 2.2 0.028*** A 5 504.6 3 -3.12 B 5 1860.8 13 2.55 H 6 1656.95 9.3 0.54 159 Overall 16 8.5 2 11.32 0.003*** A vs. B 3.32 0.0009*** A vs. H 2.2 0.028*** (d) Untreated (uninoculated) post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Ca A 3 6292.7 2 -2.24 B 3 7984 5 0.45 H 2 12921.6 7.5 2 Overall 8 4.5 A vs. H K A 3 1660 2 -2.24 B 3 7848.3 7 2.24 H 2 2915.85 4.5 0 4.5 A vs. B 3 875 2 -2.24 B 3 3657.2 6 1.34 H 2 3489.65 6 1 4.5 A vs. B 3 525 2 -2.24 B 3 2580.7 7 2.24 H 2 4.5 0 919.5 4.5 A vs. B 5 6.25 2.5 3 522.5 2 -2.24 B 3 2337 7 2.24 H 2 1153.85 4.5 0 Overall 8 4.5 0.044*** 0.0124*** 2 2.5 0.082 0.0455*** 2 A 0.044*** 0.0124*** 2 A A vs. B 6.25 2 Overall 8 S 2 A 0.044*** 0.0139*** 2.5 Overall 8 P 6.25 2.46 Overall 8 Mg 2 P-value 6.25 0.044*** 0.0124*** 160 Treatment comparisons Table D-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and substrate macronutrient (Ca, K, Mg, P, S) content (expressed in mg/kg of substrate dry matter). Treatment: t = treated (inoculated), u = untreated (uninoculated). P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation stage Substrate Response Treatment N Median Ca Alder chips Mg P Precultivation S Ca 5 5025.2 3.8 -1.04 u 3 5537.6 5.7 1.04 8 4.5 t 5 1483.7 4.6 0.15 u 3 4.3 -0.15 Overall 8 t 5 735.1 4.2 -0.45 u 3 736.9 5 0.45 Overall 8 t 5 442.3 4.2 -0.45 u 3 420.1 5 0.45 Overall 8 t 5 434.4 4.6 0.15 u 3 400.1 4.3 -0.15 Overall 8 4.5 t 5 4867.7 5.2 1.04 u 3 4495.6 3.3 -1.04 8 4.5 t 5 4127.1 4.4 -0.15 u 3 4077.5 4.7 0.15 8 4.5 t 5 1807.2 5 0.75 u 3 1778.9 3.7 -0.75 8 4.5 t 5 1269.2 4.2 -0.45 u 3 1252.5 5 0.45 Overall K Barley straw Overall Mg Overall P ZHValue DF value t Overall K Mean Rank 1428 4.5 4.5 4.5 P-value 1 1.09 0.297 1 0.02 0.881 1 0.20 0.655 1 0.20 0.655 1 0.02 0.881 1 1.09 0.297 1 0.02 0.881 1 0.56 0.456 161 Overall S Ca K 8 4.5 t 5 1120.5 4.6 0.15 u 3 4.3 -0.15 Overall 8 4.5 t 6 11314.1 5.5 2 u 2 1.5 -2 Overall 8 4.5 t 6 2267.4 5.5 2 u 2 1341.55 1.5 -2 8 4.5 t 6 2358.5 5.5 2 u 2 2170.15 1.5 -2 Overall 8 4.5 t 6 547.8 5 1 u 2 446.6 3 -1 Overall 8 4.5 t 6 706.55 5.3 1.67 u 2 2 -1.67 Overall 8 4.5 t 5 6557.9 5.4 1.34 u 3 6292.7 3 -1.34 8 4.5 t 5 1379.5 3.6 -1.34 u 3 6 1.34 Overall 8 4.5 t 5 1027.9 5.2 1.04 u 3 3.3 -1.04 Overall 8 t 5 429.1 3.4 -1.64 u 3 525 6.3 1.64 Overall 8 t 5 504.6 4.8 0.45 u 3 522.5 4 -0.45 Overall Hemp straw Mg P S Ca Overall K Postcultivation Alder chips Mg P S 1098 9250 663.5 1660 875 4.5 4.5 1 0.20 0.655 1 0.20 0.881 1 4.00 0.046*** 1 4.00 0.046*** 1 4.00 0.046*** 1 1.00 0.317 1 2.78 0.96 1 1.80 0.180 1 1.80 0.180 1 1.09 0.297 1 2.69 0.101 162 Overall Ca K 8 4.5 t 5 9524.8 4.6 0.15 u 3 4.3 -0.15 Overall 8 4.5 t 5 4113.1 3 -2.24 u 3 7848.3 7 2.24 8 4.5 t 5 3089.4 3.8 -1.04 u 3 3657.2 5.7 1.04 8 4.5 t 5 1608.7 3 -2.24 u 3 2580.7 7 2.24 8 4.5 t 5 1860.8 3.8 -1.04 u 3 5.7 1.04 Overall 8 4.5 t 6 28422 5.5 2 u 2 12921.6 1.5 -2 8 4.5 t 6 2893.4 4.5 0 u 2 2915.85 4.5 0 8 4.5 t 6 6443.6 5.5 2 u 2 3489.65 1.5 -2 Overall 8 4.5 t 6 992.6 4.8 0.67 u 2 919.5 3.5 -0.67 Overall 8 4.5 t 6 1656.95 5.5 2 u 2 1153.85 1.5 -2 8 4.5 Overall Barley straw Mg Overall P Overall S Ca Overall K Overall Hemp straw Mg P S Overall 7984 2337 1 0.2 0.655 1 0.02 0.881 1 5.00 0.025*** 1 1.09 1 5.00 0.025*** 1 1.09 1 4.00 0.046*** 1 0.00 1 4.00 0.046*** 1 0.44 1 4.00 0.046*** 0.297 0.297 1.000 0.505 163 Pre- and post-cultivation comparisons Table D-3. Results of Wilcoxon Signed Rank Confidence Interval test evaluating changes in pre- and post-cultivation substrate macronutrient (Ca, K, Mg, P, S) content (expressed in mg/kg of substrate dry matter). P = pre-cultivation, C = post-cultivation. Note: Minitab statistical software will not perform the Wilcoxon test on sample sizes ≤3. Treatment Median change Substrate Response N (C-P) Alder chips Treated (inoculated) Barley straw Hemp straw Alder chips Untreated (ininoculated) Barley straw CI for η, CI for η, lower upper limit limit Achieved confidence % Interpretation Ca 5 1585.85 1447 1724.7 94.09% Increase K 5 -101.65 -725.6 522.3 94.09% No trend Mg 5 276.2 126.3 415.9 94.09% Increase P 5 -3.95 -48.9 171.8 94.09% No trend S 5 95.8 50.2 172.8 94.09% Increase Ca 5 4143.2 1420.2 5020.1 94.09% Increase K 5 260.55 -1970.7 1361.3 94.09% No trend Mg 5 1352.95 506.1 3051.6 94.09% Increase P 5 216.9 -341.4 575.5 94.09% No trend S 5 782.4 597.6 967.2 94.09% Increase Ca 6 15875.5 13582 22510.8 94.08% Increase K 6 1024.2 280 2691.45 94.08% Increase Mg 6 4119.4 3329.4 4563.2 94.08% Increase P 6 242.3 -322.95 582.3 94.08% No trend S 6 829.1 569.3 1048.45 94.08% Increase Ca 3 983.8 Insufficient data to perform test K 3 241.2 Insufficient data to perform test Mg 3 137.0 Insufficient data to perform test P 3 34.7 Insufficient data to perform test S 3 94.3 Insufficient data to perform test Ca 3 3506.9 Insufficient data to perform test K 3 3770.8 Insufficient data to perform test Mg 3 1998.0 Insufficient data to perform test P 3 1336.7 Insufficient data to perform test S 3 1239.0 Insufficient data to perform test 164 Hemp straw Ca 2 3671.7 Insufficient data to perform test K 2 1574.3 Insufficient data to perform test Mg 2 1319.5 Insufficient data to perform test P 2 472.9 Insufficient data to perform test S 2 490.4 Insufficient data to perform test 165 Appendix E: Substrate micronutrient ICP-OES analysis data tables Substrate comparisons Table E-1 (a) – (d). Results of Kruskal-Wallace Multiple Comparisons test on the relationship between substrate type and micronutrient (B, Cu, Fe, Mn, Zn) content (expressed in mg/kg of substrate dry matter). Substrates: A = alder chips, B = barley straw, H = hemp straw. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. (a) Treated (inoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value B Cu A 5 5.1 5.5 -1.7 B 5 5 5.5 -1.7 H 6 13.6 13.5 3.25 Overall 16 8.5 Mn Zn 10.60 0.005*** A vs. H 2.78 0.0055*** B vs. H 2.78 0.0055*** A 5 5.4 5.9 -1.47 B 5 5.5 8.1 -0.23 H 6 6.5 11 1.63 Overall 16 Fe 2 P-value 8.5 A 5 42.7 3 -3.12 B 5 161.2 9.8 0.74 H 6 251.95 12 2.28 Overall 16 8.5 2 3.19 0.203 2 10.29 0.006*** A vs. H 3.12 0.0018*** A vs. B 2.26 0.0239*** A 5 67.5 8 -0.28 B 5 27.3 3 -3.12 H 6 103.8 13.5 3.25 Overall 16 8.5 2 13.35 0.001*** B vs. H 3.64 0.0003*** A vs. H 1.91 0.0564* A 5 23.2 8.8 0.17 166 B 5 26.8 14 3.12 H 6 13.5 3.7 -3.15 Overall 16 8.5 B vs. H 2 12.88 3.58 0.002*** 0.0003*** (b) Untreated (uninoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value B A 3 5.1 3.5 -0.89 B 3 5.1 3.5 -0.89 H 2 12.2 7.5 2 Overall 8 Cu 4.5 A 3 4.5 3 -1.34 B 3 6.3 5.7 1.04 H 2 5.5 5 0.33 Overall 8 Fe 4.5 A 3 50 2 -2.24 B 3 113.2 5.3 0.75 H 2 172.9 7 1.67 Overall 8 4.5 A vs. H Mn 3 62.5 5.3 0.75 B 3 20 2 -2.24 H 2 82.15 7 1.67 Overall 8 4.5 B vs. H Zn 4.10 0.129 2 1.89 0.389 2 5.56 0.062* 0.0253*** 2 5.56 2.24 A 3 24.2 4.3 -0.15 B 3 25.6 6.7 1.94 H 2 11.9 1.5 -2 Overall 8 B vs. H 2 2.24 A 4.5 0.062* 0.0253*** 2 2.32 P-value 5.43 0.066* 0.0201*** (c) Treated (inoculated) post-cultivation substrate samples. 167 Response variable Substrate N Median Mean rank Z-value DF H-value B Cu Fe Mn Zn A 5 6.6 3.4 -2.89 B 5 8.7 7.6 -0.51 H 6 34.55 13.5 3.25 Overall 16 8.5 2 12.53 P-value 0.002*** A vs. H 3.50 0.0005*** B vs. H 2.05 0.0407*** A 5 4.9 3 -3.12 B 5 8.4 8 -0.28 H 6 13.05 13.5 3.25 Overall 16 8.5 2 13.38 0.001*** A vs. H 3.65 0.0003*** B vs. H 1.91 0.0561* A 5 88.3 3 -3.12 B 5 395.2 9.2 0.4 H 6 981.5 12.5 2.6 Overall 16 8.5 2 11.02 0.004*** A vs H 3.30 0.001*** A vs. B 2.06 0.0395*** A 5 91.7 7 -0.85 B 5 54.1 4 -2.55 H 6 326.75 13.5 3.25 Overall 16 8.5 2 11.58 0.003*** B vs. H 3.30 0.001*** A vs. H 2.25 0.0242*** A 5 31.6 4.8 -2.1 B 5 44.9 13.6 2.89 H 6 34.1 7.3 -0.76 Overall 16 8.5 2 9.12 0.01*** A vs. B 2.92 0.0035*** B vs. H 2.17 0.0297*** (d) Untreated (uninoculated) post-cultivation substrate samples. 168 Response variable Substrate N Median Mean rank Z-value DF H-value B A 3 5.4 2 -2.24 B 3 8.6 5 0.45 H 2 18.9 7.5 2 Overall 8 4.5 A vs. H Cu Fe A 3 4.8 2 -2.24 B 3 9.5 6 1.34 H 2 9.75 6 1 Overall 8 4.5 A 3 139.6 2 -2.24 B 3 513.6 5 0.45 H 2 7.5 2 1352 4.5 A vs. H A 3 77.5 5 0.45 B 3 53.1 2 -2.24 H 2 173.55 7.5 2 4.5 B vs. H 5.00 0.082 2 6.25 0.044*** 0.0139*** 6.25 2.46 3 29 4 -0.45 B 3 48.6 7 2.24 H 2 24.45 1.5 -2 Overall 8 B vs. H 2 2 A 4.5 0.044*** 0.0139*** 2.46 Overall 8 Zn 6.25 2.46 Overall 8 Mn 2 P-value 0.044*** 0.0139*** 2 6.25 2.46 0.044*** 0.0139*** Treatment comparisons Table E-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and substrate micronutrient (B, Cu, Fe, Mn, Zn) content (expressed in mg/kg of substrate dry matter). Treatment: t = treated (inoculated), u = untreated (uninoculated). P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation stage Substrate Response Treatment N Median Mean Rank ZHValue DF value P-value 169 B Cu Alder chips Fe Mn Zn Pre cultivation B Cu Barley straw Fe Mn Zn Hemp straw B t 5 5.1 4.1 -0.6 u 3 5.1 5.2 0.6 Overall 8 t 5 5.4 5.4 1.34 u 3 4.5 3 -1.34 Overall 8 t 5 42.7 3.8 -1.04 u 3 50 5.7 1.04 Overall 8 t 5 67.5 4.6 0.15 u 3 62.5 4.3 -0.15 Overall 8 t 5 23.2 3.1 -2.09 u 3 24.2 6.8 2.09 Overall 8 t 5 5 4.2 -0.45 u 3 5.1 5 0.45 Overall 8 t 5 5.5 4.6 0.15 u 3 6.3 4.3 -0.15 Overall 8 4.5 t 5 161.2 5.6 1.64 u 3 113.2 2.7 -1.64 Overall 8 4.5 t 5 27.3 5.1 0.89 u 3 20 3.5 -0.89 Overall 8 t 5 26.8 4.8 0.45 u 3 25.6 4 -0.45 Overall 8 t 6 13.6 4.8 0.67 u 2 12.2 3.5 -0.67 Overall 8 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 4.5 1 0.36 0.549 1 1.80 0.180 1 1.09 0.297 1 0.02 0.881 1 4.41 0.036*** 1 0.20 0.655 1 0.02 0.881 1 2.69 0.101 1 0.81 0.368 1 0.20 0.655 1 0.45 0.502 170 Cu Fe t 6 6.5 4.9 0.83 u 2 5.5 3.3 -0.83 Overall 8 4.5 t 6 251.95 5.3 1.67 u 2 172.9 2 -1.67 8 4.5 t 6 103.8 5.2 1.33 u 2 82.15 2.5 -1.33 Overall 8 4.5 t 6 13.5 5.5 2 u 2 11.9 1.5 -2 Overall 8 t 5 6.6 5.1 0.89 u 3 5.4 3.5 -0.89 Overall 8 t 5 4.9 4.2 -0.45 u 3 4.8 5 0.45 Overall 8 t 5 88.3 3.6 -1.34 u 3 139.6 6 1.34 Overall 8 4.5 t 5 91.7 5.8 1.94 u 3 77.5 2.3 -1.94 Overall 8 t 5 31.6 5.8 1.94 u 3 29 2.3 -1.94 Overall 8 t 5 8.7 4.8 0.45 u 3 8.6 4 -0.45 Overall 8 t 5 8.4 3.1 -2.09 u 3 9.5 6.8 2.09 Overall 8 Overall Mn Zn B Cu Alder chips Postcultivation Fe Mn Zn B Barley straw Cu 4.5 4.5 4.5 4.5 4.5 4.5 4.5 1 0.71 0.399 1 2.78 0.096 1 1.78 0.182 1 4.05 0.044*** 1 0.81 0.368 1 0.20 0.653 1 1.80 0.180 1 3.76 0.053* 1 3.76 0.053* 1 0.20 0.655 1 4.46 0.035*** 171 t 5 395.2 4.4 -0.15 u 3 513.6 4.7 0.15 Overall 8 4.5 t 5 54.1 4.8 0.45 u 3 53.1 4 -0.45 Overall 8 t 5 44.9 4.2 -0.45 u 3 48.6 5 0.45 Overall 8 4.5 t 6 34.55 5.5 2 u 2 1.5 -2 Overall 8 4.5 t 6 13.05 5.5 2 u 2 1.5 -2 Overall 8 4.5 t 6 981.5 4.5 0 u 2 4.5 0 Overall 8 4.5 t 6 326.75 5.5 2 u 2 173.55 1.5 -2 Overall 8 4.5 t 6 34.1 5.2 1.33 u 2 24.45 2.5 -1.33 8 4.5 Fe Mn Zn B Cu Hemp straw Fe Mn Zn Overall 4.5 18.9 9.75 1352 1 0.02 0.881 1 0.20 0.655 1 0.20 0.655 1 4.00 0.046*** 1 4.00 0.046*** 1 0.00 1 4.00 0.046*** 1 1.78 1.000 0.182 Pre- and post-cultivation comparisons Table E-3. Results of Wilcoxon Signed Rank Confidence Interval test evaluating changes in pre- and post-cultivation substrate micronutrient (B, Cu, Fe, Mn, Zn) content (expressed in mg/kg of substrate dry matter). P = pre-cultivation, C = post-cultivation. Note: Minitab statistical software will not perform the Wilcoxon test on sample sizes ≤3. Treatment Median CI for η, CI for η, change lower upper Substrate Response N (C-P) limit limit Achieved confidence % Interpretation 172 Alder chips Treated (inoculated) Barley straw Hemp straw Alder chips Untreated (uninoculated) Barley straw Hemp straw B 5 2.2 -0.4 5.4 94.09% Probable increase Cu 5 -0.45 -2 1.4 94.09% No trend Fe 5 56.3 20.8 220.4 94.09% Increase Mn 5 26.9 12.2 46.1 94.09% Increase Zn 5 8.8 5.6 12 94.09% Increase B 5 3.65 2.6 4.7 94.09% Increase Cu 5 2.3 0.2 4.5 94.09% Increase Fe 5 240.4 31.5 486.4 94.09% Increase Mn 5 27.9 25.2 60.6 94.09% Increase Zn 5 18.6 9.1 20.9 94.09% Increase B 6 21.15 17.85 27.4 94.08% Increase Cu 6 5.95 5.1 7.35 94.08% Increase Fe 6 809 266.35 1868.02 94.08% Increase Mn 6 219.65 141.95 298.7 94.08% Increase Zn 6 17.15 10.85 22.7 94.08% Increase B 3 0.2 Insufficient data to perform test Cu 3 0.2 Insufficient data to perform test Fe 3 89.6 Insufficient data to perform test Mn 3 15.4 Insufficient data to perform test Zn 3 4.8 Insufficient data to perform test B 3 2.4 Insufficient data to perform test Cu 3 3.2 Insufficient data to perform test Fe 3 382.3 Insufficient data to perform test Mn 3 33.0 Insufficient data to perform test Zn 3 23.7 Insufficient data to perform test B 2 6.7 Insufficient data to perform test Cu 2 4.25 Insufficient data to perform test Fe 2 1179.15 Insufficient data to perform test Mn 2 91.4 Insufficient data to perform test Zn 2 12.6 Insufficient data to perform test 173 Appendix F: Substrate Al and Na ICP-OES analysis data tables Substrate comparisons Table F-1 (a) – (d). Results of Kruskal-Wallace Multiple Comparisons test on the relationship between substrate type and Al & Na content (expressed in mg/kg of substrate dry matter). Substrates: A = alder chips, B = barley straw, H = hemp straw. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. (a) Treated (inoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Al A 5 17.5 3 -3.12 B 5 106.3 8.2 -0.17 H 6 202.95 13.3 3.15 Overall 16 8.5 A vs. H Na 2 12.88 3.58 A 5 76.7 3.4 -2.89 B 5 588.1 14 3.12 H 6 141.8 8.2 -0.22 Overall 16 8.5 P-value 0.002*** 0.0003*** 2 12.44 0.002*** A vs. B 3.52 0.0004*** B vs. H 2.02 0.0430*** (b) Untreated (uninoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Al A 3 18.8 2 -2.24 B 3 67.7 5 0.45 H 2 133.65 7.5 2 Overall 8 4.5 A vs. H Na 2 6.25 2.46 A 3 64.5 2 -2.24 B 3 567.3 7 2.24 H 2 141.1 4.5 0 Overall 8 4.5 P-value 0.044*** 0.0139 2 6.25 0.044*** 174 A vs. B 2.5 0.0124*** (c) Treated (inoculated) post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Al Na A 5 48.7 3.2 -3 B 5 266.7 9 0.28 H 6 659.25 12.5 2.6 Overall 16 8.5 2 10.49 P-value 0.005*** A vs. H 3.22 0.0013*** A vs. B 1.93 0.0541* A 5 81.8 3 -3.12 B 5 776 14 3.12 H 6 214.55 8.5 0 Overall 16 8.5 2 13.35 0.001*** A vs. B 3.65 0.0003*** A vs. H 1.91 0.0564* B vs. H 1.91 0.0564* (d) Untreated (uninoculated) post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value Al A 3 83.8 2 -2.24 B 3 338.1 5 0.45 H 2 828.2 7.5 2 Overall 8 4.5 A vs. H Na 2 2.46 A 3 115.8 2 -2.24 B 3 1112.5 7 2.24 H 2 217.1 4.5 0 Overall 8 A vs. B 6.25 4.5 0.044*** 0.0139*** 2 2.5 P-value 6.25 0.044*** 0.0124*** Treatment comparisons 175 Table F-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and substrate Al & Na content (expressed in mg/kg of substrate dry matter). Treatment: t = treated (inoculated), u = untreated (uninoculated). P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation stage Substrate Response Treatment N Median Al Alder chips Na Al Pre-cultivation Barley straw Na 5 17.5 4.4 -0.15 u 3 18.8 4.7 0.15 Overall 8 t 5 76.7 4.6 0.15 u 3 64.5 4.3 -0.15 Overall 8 4.5 t 5 106.3 5.6 1.64 u 3 2.7 -1.64 Overall 8 4.5 t 5 588.1 4.4 -0.15 u 3 567.3 4.7 0.15 8 4.5 t 6 202.95 5.3 1.67 u 2 133.65 2 -1.67 8 4.5 t 6 141.8 4.5 0 u 2 141.1 4.5 0 Overall 8 4.5 t 5 48.7 3.6 -1.34 u 3 83.8 6 1.34 Overall 8 t 5 81.8 3.8 -1.04 u 3 115.8 5.7 1.04 8 4.5 t 5 266.7 4.4 -0.15 u 3 338.1 4.7 0.15 Overall 8 4.5 t 5 Overall Hemp straw Na Al Alder chips Na Postcultivation Overall Barley straw Al Na ZHValue DF value t Overall Al Mean Rank 4.5 67.7 4.5 776 4.6 Pvalue 1 0.02 0.881 1 0.02 0.881 1 2.69 0.101 1 0.02 0.881 1 2.78 0.096 1 0.00 1.000 1 1.80 0.180 1 1.09 0.297 1 0.02 0.881 0.15 176 u 3 1112.5 4.3 8 4.5 t 6 659.25 4.5 0 u 2 828.2 4.5 0 8 4.5 t 6 214.55 4.7 0.33 u 2 217.1 4 -0.33 8 4.5 Overall Al Overall Hemp straw Na Overall -0.15 1 0.02 0.881 1 0.00 1.000 1 0.11 0.739 Pre- and post-cultivation comparisons Table F-3. Results of Wilcoxon Signed Rank Confidence Interval test evaluating changes in pre- and post-cultivation substrate Al & Na content (expressed in mg/kg of substrate dry matter). P = precultivation, C = post-cultivation. Note: Minitab statistical software will not perform the Wilcoxon test on sample sizes ≤3. Median CI for η, Treatment Substrate Response N change (C- lower P) limit Alder chips Treated Barley straw Hemp straw Alder chips Untreated Barley straw Hemp straw CI for η, Achieved upper Interpretation confidence % limit Al 5 35.4 12.5 162.5 94.09 Increase Na 5 2.8 -37.3 42.9 94.09 No trend Al 5 247 61.8 432.2 94.09 Increase Na 5 412.25 151.4 1281.5 94.09 Increase Al 6 513.7 198.25 1136.9 94.08 Increase Na 6 73.95 38.4 112.55 94.08 Increase Al 3 64.8 Insufficient data to perform test Na 3 51.3 Insufficient data to perform test Al 3 270.3 Insufficient data to perform test Na 3 586 Insufficient data to perform test Al 2 694.6 Insufficient data to perform test Na 2 76 Insufficient data to perform test 177 Appendix G: Van Soest fiber analysis data tables Substrate comparisons Table G-1 (a) – (d). Results of Kruskal-Wallace Multiple Comparisons test on the relationship between substrate type and lignin, cellulose, hemicellulose, and total lignocellulosic biomass content of substrate samples, expressed as a percentage of oven dry sample mass. Substrates: A = alder chips, B = barley straw, H = hemp straw. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. (a) Treated (inoculated) pre-cultivation substrate samples. Response variable Lignin (% of O.D. sample mass) Substrate N Median Mean rank Z-value DF A 5 17.65 14 3.12 B 5 3.95 3 -3.12 H 6 12.185 8.5 0 Overall 16 Total lignocellulosic biomass (% of O.D. sample mass) 2 13.35 0.001*** A vs. B 3.65 0.0003*** B vs. H 1.91 0.0564* A vs. H 1.91 0.0564* A 5 46.53 8.8 0.17 B 5 41.96 3 -3.12 6 54.71 12.8 2.82 Cellulose (% of O.D. H sample mass) Overall Hemicellulose (% of O.D. sample mass) 8.5 H-value P-value 16 8.5 2 11.66 0.003*** B vs. H 3.42 0.0006*** A vs. B 1.93 0.0541* A 5 19.3 7.6 -0.51 B 5 37.57 14 3.12 H 6 17.855 4.7 -2.49 Overall 16 8.5 2 10.74 0.005*** B vs. H 3.24 0.0012*** A vs. B 2.13 0.0335*** A 5 83.44 8.2 -0.17 B 5 82.41 7.2 -0.74 H 6 84.71 9.8 0.87 Overall 16 8.5 2 0.86 0.649 178 (b) Untreated (uninoculated) pre-cultivation substrate samples. Response variable Lignin (% of O.D. sample mass) Substrate N Median Mean rank Z-value A 3 17.1 7 2.24 B 3 4.28 2 -2.24 H 2 13.25 4.5 0 Overall 8 4.5 A vs. B Cellulose (% of O.D. sample mass) A 3 44.69 4.7 0.15 B 3 41.52 2.3 -1.94 H 2 52.75 7.5 2 Overall 8 6.25 2 5.36 2.31 A 3 18.94 2.7 -1.64 B 3 38.25 7 2.24 H 2 19.595 3.5 -0.67 Overall 8 2 5.14 2.17 3 80.97 3 -1.34 B 3 86.26 4.7 0.15 H 2 85.595 6.5 1.33 Overall 8 0.069* 0.0209*** 4.5 A 0.044*** 0.0124*** 4.5 A vs. B Total lignocellulosic biomass (% of O.D. sample mass) 2 2.5 B vs. H Hemicellulose (% of O.D. sample mass) DF H-value P-value 0.077 0.0303*** 4.5 2 2.47 0.291 (c) Treated (inoculated) post-cultivation substrate samples. Response variable Lignin (% of O.D. sample mass) Substrate N Median Mean rank Z-value DF H-value P-value A 5 17.4 14 3.12 B 5 5.65 7.8 -0.4 H 6 4.885 4.5 -2.6 Overall 16 8.5 2 11.02 0.004*** A vs. H 3.3 0.001*** A vs. B 2.06 0.0395*** 179 A 5 39.8 13.4 2.78 B 5 26.38 3 -3.12 6 31.68 9 0.33 Cellulose (% of O.D. H sample mass) Overall Hemicellulose (% of O.D. sample mass) Total lignocellulosic biomass (% of O.D. sample mass) 16 8.5 2 12.04 0.002*** A vs. B 3.45 0.0006*** B vs. H 2.08 0.0374*** A 5 14.74 9.4 0.51 B 5 22.81 13.6 2.89 H 6 6.985 3.5 -3.25 Overall 16 8.5 2 12.53 0.002*** B vs. H 3.5 0.0005*** A vs. H 2.05 0.0407*** A 5 71.75 14 3.12 B 5 55.73 8.4 -0.06 H 6 43.555 4 -2.93 Overall 16 8.5 2 12.04 0.002*** A vs. H 0.0005*** A vs. B 0.0629* (d) Untreated (uninoculated) post-cultivation substrate samples. Response variable Lignin (% of O.D. sample mass) Substrate N Median Mean rank Z-value DF H-value P-value A 3 21.18 6 1.34 B 3 11.85 2 -2.24 H 2 22.1 6 1 Overall 8 4.5 A vs. B A B Cellulose (% of O.D. H sample mass) Overall 5 2 3 46.02 6 1.34 3 26.91 2 -2.24 2 43.815 6 1 8 4.5 A vs. B A 2 3 17.11 2 0.0455*** 2 2 0.082 5 0.082 0.0455*** -2.24 180 Hemicellulose (% of O.D. sample mass) B 3 32.06 7 2.24 H 2 18.895 4.5 0 Overall 8 4.5 2 A vs. B Total lignocellulosic biomass (% of O.D. sample mass) 6.25 2.5 0.0124*** A 3 83.53 5.7 1.04 B 3 69.57 2 -2.24 H 2 84.81 6.5 1.33 Overall 8 4.5 2 B vs. H 0.044*** 5.14 2.01 0.077 0.0442*** Treatment comparisons Table G-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and lignin, cellulose, hemicellulose, and total lignocellulosic biomass content of substrate samples, expressed as a percentage of oven dry sample mass. Treatment: t = treated (inoculated), u = untreated (uninoculated). P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation Mean ZHSubstrate stage Response variable Treatment N Median Rank value DF value P-value Alder chips Barley straw t Lignin (% of O.D. u sample mass) overall 5 17.65 4.6 0.15 3 17.1 4.3 -0.15 8 4.5 t Cellulose (% of u O.D. sample mass) overall 5 46.53 5.4 1.34 3 44.69 3 -1.34 8 4.5 Hemicellulose (% t of O.D. sample u mass) overall 5 19.3 4.6 0.15 3 18.94 4.3 -0.15 8 4.5 Total t lignocellulosic u biomass (% of O.D. sample mass) overall 5 83.44 5.4 1.34 3 80.97 3 -1.34 8 4.5 t PreLignin (% of O.D. u cultivation sample mass) overall 5 3.95 4.2 -0.45 3 4.28 5 0.45 8 4.5 Precultivation 1 0.02 0.881 1 1.8 1 0.02 0.881 1 1.8 0.18 1 0.2 0.655 0.18 181 Hemp straw Alder chips Precultivation Postcultivation t Cellulose (% of u O.D. sample mass) overall 5 41.96 4.4 -0.15 3 41.52 4.7 0.15 8 4.5 Hemicellulose (% t of O.D. sample u mass) overall 5 37.57 3.6 -1.34 3 38.25 6 1.34 8 4.5 Total t lignocellulosic u biomass (% of O.D. sample mass) overall 5 82.41 3.8 -1.04 3 86.26 5.7 1.04 8 4.5 t Lignin (% of O.D. u sample mass) overall 6 12.185 3.8 -1.33 2 13.25 6.5 1.33 8 4.5 t Cellulose (% of u O.D. sample mass) overall 6 54.71 4.8 0.67 2 52.75 3.5 -0.67 8 4.5 Hemicellulose (% t of O.D. sample u mass) overall 6 17.855 4 -1 2 19.595 6 1 8 4.5 Total t lignocellulosic u biomass (% of O.D. sample mass) overall 6 84.71 4 -1 2 85.595 6 1 8 4.5 t Lignin (% of O.D. u sample mass) overall 5 17.4 3.2 -1.94 3 21.18 6.7 1.94 8 4.5 t Cellulose (% of u O.D. sample mass) overall 5 39.8 3.2 -1.94 3 46.02 6.7 1.94 8 4.5 Hemicellulose (% t of O.D. sample u mass) overall 5 14.74 3.4 -1.64 3 17.11 6.3 1.64 8 4.5 t 5 71.75 3 -2.24 u 3 83.53 7 2.24 Total lignocellulosic 1 0.02 0.881 1 1.8 1 1.09 0.297 1 1.78 0.182 1 0.44 0.505 1 1 0.317 1 1 0.317 1 3.76 0.053* 1 3.76 0.053* 1 2.69 0.101 0.18 182 biomass (% of O.D. sample mass) overall Barley straw Hemp straw Postcultivation Postcultivation 8 4.5 t Lignin (% of O.D. u sample mass) overall 5 5.65 3 -2.24 3 11.85 7 2.24 8 4.5 t Cellulose (% of u O.D. sample mass) overall 5 26.38 4.2 -0.45 3 26.91 5 0.45 8 4.5 Hemicellulose (% t of O.D. sample u mass) overall 5 22.81 3 -2.24 3 32.06 7 2.24 8 4.5 Total t lignocellulosic u biomass (% of O.D. sample mass) overall 5 55.73 3 -2.24 3 69.57 7 2.24 8 4.5 t Lignin (% of O.D. u sample mass) overall 6 4.885 3.5 -2 2 22.1 7.5 2 8 4.5 t Cellulose (% of u O.D. sample mass) overall 6 31.68 3.7 8 4.5 Hemicellulose (% t of O.D. sample u mass) overall 6 6.985 3.5 -2 2 18.895 7.5 2 Total t lignocellulosic u biomass (% of O.D. sample mass) overall 6 43.555 3.5 -2 2 84.81 7.5 2 8 4.5 2 43.815 7 8 1 5 0.025*** 1 5 0.025*** 1 0.2 0.655 1 5 0.025*** 1 0.025*** 1 4 0.046*** 1 2.78 0.096 1 4 0.046*** 1 4 0.046*** -1.67 1.67 4.5 Pre- and post-cultivation comparisons Table G-3. Results of Wilcoxon Signed Rank Confidence Interval test evaluating changes in pre- and post-cultivation substrate sample content of lignin, cellulose, hemicellulose, and total lignocellulosic biomass, expressed as a percentage of oven dry sample mass. P = pre-cultivation, C = postcultivation. Note: Minitab statistical software will not perform the Wilcoxon test on sample sizes ≤3. 183 Treatment Substrate Alder chips Treated (inoculated) Barley straw Hemp straw Alder chips Untreated (uninoculated) Barley straw Hemp straw Response variable N Median change CI for η, CI for η, Achieved (C – P) lower limit upper limit confidence % Lignin 5 0.22 -1.48 2.22 94.09 Cellulose 5 -6.02 -7.97 -2.88 94.09 Hemicellulose 5 -4.14 -7.25 -2.52 94.09 Total lignocellulosic 5 -9.26 -15.25 -3.18 94.09 Lignin 5 2.41 1.36 4.01 94.09 Cellulose 5 -15.26 -18.22 -12.98 94.09 Hemicellulose 5 -14.135 -22.32 -11.86 94.09 Total lignocellulosic 5 -27.255 -36.53 -23.2 94.09 Lignin 6 -7.28 -9.085 -5.775 94.08 Cellulose 6 -19.795 -25.565 -13.615 94.08 Hemicellulose 6 -10.72 -12.2 -9.13 94.08 Total lignocellulosic 6 -38.085 -44.805 -31.11 94.08 Lignin 3 4.47 Insufficient data to perform test Cellulose 3 2.05 Insufficient data to perform test Hemicellulose 3 -1.83 Insufficient data to perform test Total lignocellulosic 3 2.63 Insufficient data to perform test Lignin 3 8.27 Insufficient data to perform test Cellulose 3 -14.61 Insufficient data to perform test Hemicellulose 3 -7.87 Insufficient data to perform test Total lignocellulosic 3 -16.69 Insufficient data to perform test Lignin 2 8.85 Insufficient data to perform test Cellulose 2 -8.935 Insufficient data to perform test Hemicellulose 2 -0.7 Insufficient data to perform test Total lignocellulosic 2 -0.785 Insufficient data to perform test 184 Appendix H: pH and EC data tables Substrate comparisons Table H-1 (a) – (d). Results of Kruskal-Wallace Multiple Comparisons test on the relationship between substrate type and pH and electrical conductivity (EC) of substrate samples. Substrates: B = barley straw, A = alder chips, H = hemp straw. P-values < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. (a) Treated (inoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean Rank Z-Value DF H-value P-value pH EC (mS/cm) A 5 5.2 3 -3.12 B 5 7.1 10.3 1.02 H 6 6.95 11.6 2.01 Overall 16 8.5 2 9.99 0.007*** A vs. H 2.99 0.00028*** A vs. B 2.44 0.0149*** A 5 0.34 3 -3.12 B 5 1.06 11.9 1.93 H 6 0.825 10.3 1.14 Overall 16 8.5 2 10.05 0.007*** A vs. B 2.96 0.0031*** A vs. H 2.52 0.0118*** (b) Untreated (uninoculated) pre-cultivation substrate samples. Response variable Substrate N Median Mean Rank Z-Value DF H-value pH A 3 5.3 2 -2.24 B 3 7.3 6 1.34 H 2 7.2 6 1 Overall 8 4.5 A vs. B EC (mS/cm) 2 2.01 A 3 0.44 2 -2.24 B 3 1.44 7 2.24 H 2 0.73 4.5 0 5.06 P-value 0.08 0.0442*** 185 Overall 8 4.5 2 A vs. B 6.25 0.044*** 2.5 0.0124 (c) Treated (inoculated) post-cultivation substrate samples. Response variable Substrate N Median Mean rank Z-value DF H-value pH A 5 3.5 5.2 -1.87 B 5 4 8.6 0.06 H 6 4.2 11.2 1.74 Overall 16 8.5 2 A vs. H EC (mS/cm) 4.44 2.12 A 5 0.69 3 -3.12 B 5 2.02 9 0.28 H 6 2.515 12.7 2.71 Overall 16 8.5 P-value 0.109 0.0352*** 2 11.32 0.003*** A vs. H 3.35 0.0008*** A vs. B 1.99 0.0463*** (d) Untreated (uninoculated) post-cultivation substrate sample. Response variable Substrate N Median Mean Rank Z-Value DF H-value pH A 3 5.7 2 -2.24 B 3 7 5 0.45 H 2 7.35 7.5 2 Overall 8 4.5 A vs. H EC (mS/cm) 2 2.47 A 3 0.37 2 -2.24 B 3 1.33 7 2.24 H 2 0.585 4.5 0 Overall 8 A vs. B 6.33 4.5 0.042*** 0.0133*** 2 2.5 P-value 6.25 0.044*** 0.0124*** Treatment comparisons 186 Table H-2: Results of Kruskal-Wallace test on the relationship between treatment (inoculation) and pH and EC of substrate samples. Treatment: t = treated (inoculated), u = untreated (uninoculated). Pvalues < 0.05 are indicated by *** and P-values 0.05 < p < 0.067 are indicated by *. Cultivation stage Substrate Mean Response Treatment N Median Rank pH Alder chips EC (mS/cm) pH Precultivation Barley straw EC (mS/cm) pH Hemp straw EC (mS/cm) pH Alder chips EC Postcultivation Barley straw pH EC ZHValue DF value t 5 5.2 4.5 0 u 3 5.3 4.5 0 Overall 8 t 5 0.34 3 -2.24 u 3 0.44 7 2.24 Overall 8 t 5 7.1 3.6 -1.34 u 3 7.3 6 1.34 Overall 8 t 5 1.06 4.2 -0.45 u 3 1.44 5 0.45 Overall 8 t 6 6.95 4.1 -0.83 u 2 7.2 5.8 0.83 Overall 8 4.5 t 6 0.825 5.2 1.33 u 2 2.5 -1.33 Overall 8 t 5 3.5 3 -2.24 u 3 5.7 7 2.24 Overall 8 t 5 0.69 5.9 2.09 u 3 0.37 2.2 -2.09 Overall 8 t 5 4.0 3 -2.24 u 3 7.0 7 2.24 Overall 8 t 5 4.5 4.5 4.5 4.5 0.73 4.5 4.5 4.5 4.5 2.02 6 P-value 1 0.00 1.000 1 5 0.025*** 1 1.84 0.174 1 0.2 0.655 1 0.74 0.39 1 1.78 0.182 1 5.06 0.024*** 1 4.41 0.036*** 1 5.75 0.016*** 2.24 187 u 3 Overall 8 t 6 4.2 3.5 -2 u 2 7.35 7.5 2 Overall 8 4.5 t 6 2.515 5.5 2 u 2 0.585 1.5 -2 8 4.5 pH Hemp straw EC Overall 1.33 2 -2.24 4.5 1 5 0.025*** 1 4.1 0.043*** 1 4 0.046*** Pre- and post-cultivation comparisons Table H-3. Results of Wilcoxon Signed Rank Confidence Interval test evaluating changes in pre- and post-cultivation substrate pH and EC. P = pre-cultivation, C = post-cultivation. Note: Minitab statistical software will not perform the Wilcoxon test on sample sizes ≤3. Substrate Treatment Alder chips Barley straw Hemp straw Treated (inoculated) Median CI for η, CI for η, Achieved change lower upper Interpretation confidence % (C - P) limit limit Response N pH 5 -1.8 -2.9 -1.1 94.09 decrease EC (mS/cm) 5 0.27 -0.53 0.81 94.09 no change pH 5 -2.4 -3.5 -1.7 94.09 decrease EC (mS/cm) 5 1.335 0.45 2.28 94.09 increase pH 6 -2.95 -3.25 -2.55 94.08 decrease EC (mS/cm) 6 1.46 1.24 1.95 94.08 increase Alder chips pH 3 0.9 Insufficient data to perform test EC (mS/cm) 3 -0.07 Insufficient data to perform test Barley straw pH Untreated (uninoculated) EC (mS/cm) 3 -0.2 Insufficient data to perform test 3 -0.03 Insufficient data to perform test pH 2 0.15 Insufficient data to perform test EC (mS/cm) 2 -0.145 Insufficient data to perform test Hemp straw 188