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The Pennsylvania State University the Graduate School Department The Pennsylvania State University The Graduate School Department of Plant Pathology REUSE OF SPENT MUSHROOM COMPOST FOR PRODUCTION OF AGARICUS BISPORUS A Thesis in Plant Pathology by Emmie L. Warnstrom © 2013 Emmie L. Warnstrom Submitted in Partial Fulfillment of the Requirements for the Degree of Master of Science August 2013 ii The thesis of Emmie L. Warnstrom was reviewed and approved* by the following: Donald D. Davis Professor of Plant Pathology and Environmental Microbiology Thesis Co-Adviser Daniel J. Royse Professor Emeritus of Plant Pathology Thesis Co-Adviser John A. Pecchia Associate Professor of Plant Pathology and Environmental Microbiology Timothy W. McNellis Professor of Plant Pathology and Environmental Microbiology Michael A. Fidanza Professor of Plant and Soil Sciences Fredrick E. Gildow Head of the Department of Plant Pathology and Environmental Microbiology *Signatures are on file in the Graduate School iii ABSTRACT Annual (2011-2012) revenue from production of Agaricus bisporus in the United States has risen to 1.1 billion dollars. In southeastern PA, more than 2.7 M m3 of spent mushroom compost, the nitrogen-rich byproduct of a mushroom crop, is produced annually. Disposal of SMC off the farm is relatively eXpensive and growers often seek beneficial uses of SMC to help offset the cost of disposal. The objective of this study was to investigate the reuse of SMC as an ingredient in the preparation of fresh mushroom compost. Three mushroom crops (Crops 1,2,3) were grown from compost made with and without SMC as an ingredient at the Mushroom Research Center of The Pennsylvania State University. Three compost types were prepared for each crop as follows: 1) 100% control formula (no SMC, Ctl), 2) 20% SMC + 80% control formula (80Ctl), and 3) 20% SMC + 80% lignocellulose formula (80LC). Compost types 1 and 2 were based on straw-bedded horse manure while the third formula was prepared with comparatively higher lignocellulose-rich materials including corn stover, corn cobs, cottonseed hulls and wheat straw (80LC). At spawning, the three composts were supplemented with various nutrients, including corn gluten feed, Grit-O’Cobs® 80, corn bran, and Lambert Full House® T6 (a commercial nutrient), to determine their single or interactive effect with compost type on mushroom yield and biological efficiency (BE). Bulk densities of the three composts were also iv measured at spawning to determine if SMC addition to formulations significantly affected the mass to volume ratio of the compost types. Compost type was a significant factor for BE of mushroom production in two out of three crops (Crops 1,2). In Crop 1, BE was equivalent for Ctl and 80LC, while the BE of the 80Ctl was significantly lower. In Crop 2, BE was significantly higher for the 80LC compost compared to the Ctl and 80Ctl. These results suggest that at least 20% SMC, depending on the formulation, may be incorporated as a bulk ingredient in the preparation of compost without adversely affecting mushroom yields. This could be an advantage to commercial growers who are eXperiencing increasing costs for compost raw materials and SMC disposal. Since SMC is relatively high in nitrogen (N), organic content and minerals, reclaiming nutrients left over in SMC for compost preparation might be an economical prospect for growers. Type of nutrient supplement added at spawning was a significant factor for all three Crops. When used at the same rate (approXimately 3.7% of compost dry wt), none of the supplements stimulated yield more than the commercial supplement T6. However, supplementation of compost at spawning with Grit-O’Cobs® 80, consistently resulted in lower yields and BEs compared to T6. SMC addition to phase I compost significantly increased bulk density of phase II compost. Mean bulk density (kg/m3) increased by 19.2% (from 110.8 to 132.1 kg/m3) in the 80Ctl compared to the Ctl compost. There was no significant difference v between the 80LC and 80Ctl formula in relation to bulk density, suggesting no significant major structural differences between the 80Ctl and 80LC formulations. Analysis of bacterial populations of two-phase II composts (Ctl, 80LC) revealed greater variability among crops than among compost types. Of the 15 known bacterial phyla detected in Ctl and 80LC formulas, 72% of amplifiable DNA from Ctl compost (Crop 1- 73.5%; Crop 2- 72.5%; Crop 3- 70.1%) and 69.6% of amplifiable DNA from 80LC compost (Crop 1- 76.3%; Crop 2- 66.8%; Crop 3- 65.6%) were from four phyla: Firmicutes, Actinobacteria, Proteobacteria, and ChlorofleXi. The Gemmatimonadetes was the only phylum of bacteria statistically different according to metagenomic analysis of the two compost types, comprising only 0.03% of the bacterial populations in the Ctl treatment and 0.002% of populations in the 80LC treatment. Bacterial populations of the genus Truepera showed the largest differences in amplifiable DNA eXtracted from the composts, but still accounted for only 2% of the total population in the Ctl compost. This suggests the post- pasteurization microbial communities may not be different in compost containing SMC vs the control. If this is the case, then selection of raw materials to complement SMC would be more important than particular profiles of microbial populations in the compost. More research is needed in this area, and our work with quantification of bacterial populations may be a useful beginning. vi TABLE OF CONTENTS List of Figures ............................................................................................................. vii List of Tables ............................................................................................................... X Acknowledgements ................................................................................................... xi 1. Introduction ............................................................................................................ 1 2. Methods .................................................................................................................. 5 2.1 Spent Mushroom Compost .................................................................... 5 2.2 Crops and Compost Formulation ........................................................... 5 2.3 Prewet ..................................................................................................... 6 2.4 Phase I Compost ..................................................................................... 7 2.5 Phase II Compost .................................................................................... 7 2.6 Spawning ................................................................................................ 8 2.7 Harvesting ............................................................................................... 9 2.8 Compost Sampling for Fiber, Ash, and Protein Analyses ..................... 10 2.9 Bulk Density Analysis .............................................................................. 11 2.10 DNA EXtraction - Compost Sampling ................................................... 12 2.11 DNA EXtraction - Extraction Methods .................................................. 13 3. Results ..................................................................................................................... 16 3.1 Phase I Temperatures ............................................................................. 16 3.2 Yield and Biological Efficiency (BE) ........................................................ 18 3.3 Simple Correlation Coefficients ............................................................. 26 3.4 Bulk Density ............................................................................................ 27 3.5 Metagenomic Analysis ........................................................................... 28 4. Discussion ............................................................................................................... 31 References ........................................................................................................... 36 AppendiX A Significant Correlations (complete) .............................................. 41 AppendiX B Metagenomic Data (raw) ............................................................... 44 vii LIST OF FIGURES Figure 2-1. Straight-sided 18.9 L bucket used for bulk density test of phase II compost. Bucket was filled 1/3 of the volume at a time, dropped 10 times from 30 cm off the ground onto a rubber mat, and topped off before weighing (U.S. Composting Council, 2001). ............................................................................... 12 Figure 3-1. Phase I temperatures (recorded at 10 min intervals) of Ctl (control), 80Ctl (20% SMC amended control), and 80LC (20% SMC amended lignocellulose) compost types contained in bunkers (Crop 1; MRC 1214). ............... 17 Figure 3-2. Phase I temperatures (recorded at 10 min intervals) of Ctl (control), 80Ctl (20% SMC amended control), and 80LC (20% SMC amended lignocellulose) compost types contained in bunkers (Crop 2; MRC 1215). .............. 17 Figure 3-3. Phase I temperatures of Ctl (control), 80Ctl (20% SMC amended control), and 80LC (20% SMC amended lignocellulose) compost types contained in bunkers (Crop 3; MRC 1302). ................................................................ 18 Figure 3-4. Groupings from analysis of variance for compost type for mushroom yield and biological efficiency (BE). Ctl= Control, 80C= 20% SMC + 80% Control, 80LC= 20% SMC + 80% Lignocellulose
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