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

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.

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 formula. Bars labeled with the same letter are not significantly different (P=0.05) according to Tukey’s HSD (Crop 1; MRC 1214)...... 21

Figure 3-5. Groupings from analysis of variance for supplement added at spawning for yield and biological efficiency (% BE). Supplements: CGF = corn gluten feed, CR = Grit-O’Cobs 80, CB =corn bran, T6M= Lambert’s commercial delayed release supplement treated with Mertect. Corn supplements (18g) were complemented with T6M (18 g) for a total of 36 g supplement. Bars labeled with the same letter are not significantly different (P=0.05) according to Tukey’s HSD (Crop 1; MRC 1214)...... 21

Figure 3-6. Groupings from analysis of variance for compost type for mushroom yield and biological efficiency (BE). Ctl= Control, 80Ctl= 20% SMC + 80% Control, 80LC= 20% SMC + 80% Lignocellulose formula. Columns with the same letter are not significantly different according to Tukey’s HSD significant at 0.05 (Crop 2; MRC 1215)...... 23

viii supplement. Columns with the same letter are not significantly different according to Tukey’s HSD (Crop 2; MRC 1215)...... 24

Figure 3-8. Groupings from analysis of variance for supplement added at spawning for yield and biological efficiency (BE). Supplements: CGF = corn gluten feed, CR = Grit-O’Cobs® 80, CB =corn bran. T6M = Lambert commercial supplement. Columns with the same letter are not significantly different according to Tukey’s HSD (Crop 3; MRC 1302)...... 25

Figure 3-9. Mean bulk density of three phase II compost types at spawning for three crops (Crops 1,2,3). Ctl= Control, 80Ctl= 20% SMC + 80% Control, 80LC= 20% SMC + 80% lignocellulose formula. Bars with the same letter are not significantly different...... 27

Figure 3-10. Percentage of bacteria detectable by phyla at spawning: a) Crop 1, Ctl b) Crop 1, 80LC c) Crop 2, Ctl d) Crop 2, 80LC e) Crop 3, Ctl and f) Crop 3, 80LC ...... 29

Figure 3-11. Percentage of total bacterial populations (estimated by DNA analysis) of the genus Truepera from two compost types sampled after phase II (Ctl, 80LC) used to produce Agaricus bisporus. Bars with the same letter are not significantly different (P=0.05) according to Tukey’s pairwise comparison. .... 30

LIST OF TABLES

Table 3-1. Probabilities >F from analysis of variance for two factors (compost, supplement) tested for yield and biological efficiency (BE) of A. bisporus for 3 crops (Crops 1,2,3)...... 19

Table 3-3. Mushroom yield (kg/m2) and biological efficiency of Agaricus bisporus produced on three compost types not supplemented or supplemented with T6M plus Grit-O’Cobs® 80, xylan, or corn bran nutrient at time of spawning (Crop 2; MRC 1215)...... 22

Table 3-5. Simple correlation coefficients (r) with r-values >0.4 of compost factors, mushroom yields and BE, ranked from high to low...... 26

ACKNOWLEDGEMENTS Special thanks to…

 Dr. Dan Royse for taking me on as a Master’s candidate in his lab

 My Committee members for their help and guidance on this project

 The Plant Pathology and Environmental Microbiology Department for a 2 year

Assistantship

 Giorgi Mushroom Co. for providing research funding, spent mushroom

compost and corncobs for my research

 Matt Michonski for the time he spent analyzing and reanalyzing over 300

compost samples at Cumberland Valley Analytical Services

 Doug Keith for all his hard work in the pre-wetting and composting of

materials; retrieval of SMC from Giorgi; spawning and harvest assistance for

my crops, and maintenance of the MRC

 Dr. John Pecchia for providing insight and assistance in the preparation of

compost formulas

 Vija Wilkinson for frequent assistance in the lab

 Michelle Carlson and Suzanne Kennedy from MoBio for sharing their technical

expertise of DNA extraction of samples containing high inhibitors

 Deb Grove and the Huck Institute for answering questions and processing my

DNA on the 454 Pyrosequencer

 Michelle Mansfield and Marlena Sheridan for lyophilizing my compost samples

for DNA extraction

1. Introduction

Maximizing production efficiency of Agaricus bisporus is economically important for growers. Annual revenue from production of A. bisporus in the U.S. has risen to 1.1 billion dollars in 2012 (USDA, 2012). With increased production comes increased output of spent mushroom compost (SMC), the byproduct of a mushroom crop.

SMC continues to be a disposal burden for growers, i.e. southeastern PA annually produces more than 2.7 M m3 SMC (Davis and Fidanza, 2011).

Within the industry, there is interest in reclaiming SMC for use as a compost raw material. SMC may contain up to 3% N, in the form of mushroom mycelium, unused supplement, and non-decomposed fibrous components. However, concern over partial nutrient depletion (Flegg et al., 1985), structural loss through decomposition

(Schisler, 1990), and possible accumulation of toxic metabolites that may restrict growth of A. bisporus (Giovannozzi et al., 1978), may have hindered efforts by the industry to pursue more vigorously using SMC in this manner.

Major components of compost used in A. bisporus production are lignocellulose- rich materials such as hay and straw (Bonnen et al., 1994). The mycelium secretes specialized enzymes to degrade cellulose, hemicellulose, and lignin, the major fibrous components of compost (Hayes, 1977). Cellulose is part of a matrix structure in which

30 cellulose molecules, held together by β 1-4-linked glucose molecules, form a protofibril (Lynd et al., 2002). Protofibrils are further assembled in an un-branched

2 arrangement called a microfibril that is held together by hydrogen bonding and Van der Waals forces. While hemicelluloses are thought to regulate the aggregation of these cellulose molecules to form its crystalline structure (Atalla et al., 1993), lignin, the other molecule of lignocellulose, strongly binds to hemicellulose and cellulose

(Philippoussis, 2009). The lignin is the shield that protects cellulose from hydrolase attack (Raimbault, 1998).

Gene studies in A. bisporus indicate that cellulases (cellulose degrading enzymes), xylanases (hemicellulose degrading enyzymes) and laccases (lignin degrading enzymes) responsible for lignocellulosic waste degradation are developmentally regulated (Patyshakuliyeva and de Vries 2011). Laccase was shown to increase during vegetative growth in association with manganese peroxidase to degrade lignin, while cellulase and xylanase did not accumulate until fruiting. Although lignin could be degraded more efficiently by A. bisporus than by other microflora, lignin is bound up with proteins whereas cellulose and hemicellulose are converted into microbial biomass (Patyshakuliyeva and de Vries 2011). The byproduct of this degradation process by termination of the crop is SMC.

Preparation of raw materials for A. bisporus production involves two steps: decomposition (phase I) and pasteurization/conditioning (phase II). Selectivity of the compost, a direct result of phase II, determines what if any organisms may persist or invade during a crop cycle.

3 Microbial biomass is suggested to be an important element of nutrition for A. bisporus (Waksman et al. 1939, Gerrits et al. 1967, Hayes 1968, Eddy and Jacobs 1976 and Ross 1978). Reuse of SMC sparks interest in determining what impact SMC may have on the microbial populations during phase II that may adversely affect substrate selectivity. During composting and conditioning, a polysaccharide matrix full of microbial cells and debris coats the straw surfaces (Eddy and Jacobs 1976; Atkey and

Wood 1983), this matrix was not only shown to disappear during A. bisporus colonization (Flegg et al. 1985), but it was shown that A. bisporus grew better on this polysaccharide layer than on a glucose carbon-source medium alone (Stanek, 1968;

Stanek, 1972). This microbial matrix has water-holding power, and may also regulate nutrient availability within the compost.

Although reuse of SMC poses some potential challenges, optimizing raw material use through the incorporation of SMC into fresh compost is not a new concept. As early as 1962, autoclaved, supplemented SMC was shown to produce equivalent or higher A. bisporus yields than the original compost (Till, 1962). Till (1962) concluded that substantial nutrients remain in SMC after the crop has been terminated. By 1972,

Murphy produced the first SMC-based formula, surpassing yields of those obtained from common horse manure formulae. Murphy (1972) accomplished this with the addition of corncobs and cottonseed meal to SMC prior to pasteurization in phase II composting, suggesting that corncobs and cottonseed meal may complement SMC nutritional deficiencies.

4 Use of near-infrared spectroscopy (De Leeuw, 2011) has recently allowed for more accurate monitoring of fiber, protein, ash, and biomass from initiation to the end.

Carbohydrates found at the beginning of the composting process (phase I), were as high as 70% dry matter, but diminished to 40% by the end of the crop (De Leeuw,

2011). Comparison of U.S. compost to those of Poland and the Netherlands revealed that the U.S. uses a more highly composted product, resulting in lower cellulose and hemicellulose available to the mushroom. In addition, ash content in the U.S. compost is higher, even though protein is more readily available (De Leeuw, 2011).

Research reported herein focuses on the reuse of SMC as an ingredient in fresh compost. We incorporated SMC at the beginning of phase I composting since this has shown the greatest promise for increasing mushroom yield (Royse, 2012). In addition, we wanted to examine and compare, using DNA analysis, populations of bacteria found in a control substrate containing no SMC versus an SMC (20% dwt) formulated compost.

5 2. Methods

2.1 Spent Mushroom Compost

Fresh 3 break harvested, post-crop steamed SMC with the casing (pH buffered sphagnum peat moss layer) removed was obtained from Giorgi Mushroom Company

(Temple, PA). Compost treatments incorporating SMC were formulated at the

Pennsylvania State University Mushroom Test Demonstration Facility (MTDF), while cropping was done at the Mushroom Research Center (MRC) located at the

Pennsylvania State University, University Park campus. Depending on the treatment,

SMC was incorporated into compost on a 20% dry weight basis.

2.2 Crops and Compost Formulation

Three composts (for each of three crops) were prepared as follows: 1) 100% control formula (Ctl), 2) 20% SMC + 80% control formula (80Ctl), and 3) 20% SMC + 80% lignocellulose formula (80LC). The Ctl was a standard MTDF formulation that has been used to successfully produce excellent crops of mushrooms for several years. It contained 85.4% straw-bedded horse manure, 5.1% poultry manure, 4.7% distiller’s grain, and 4.0% gypsum by dry weight. The 80Ctl formula contained 20% SMC, 71.8% straw-bedded horse manure, 4% gypsum, 2.2% distillers grain and 2.1% poultry manure.

All three composts were formulated to contain 1.45% N dwt at compost build (start of

6 phase I composting). Nitrogen additives in the form of distillers grain and poultry manure were less than the Ctl formula due to the relatively high N content of SMC.

Gypsum was added at 4% formula dry weight (dwt). In an attempt to increase the relative amounts of hemicellulose and cellulose in the 80LC formulation, ingredients were mixed as follows: 20.0% SMC, 19.0% wheat straw, 18.8% corn stover, 14.9% corn cobs, 8.3% distiller’s grain, 7.8% poultry manure, 4.5% cottonseed hull pellets, 4.0% gypsum, and 2.7% straw-bedded horse manure.

2.3 Prewet

One objective was to prepare a compost formula (80LC) that was able to complement the relatively high N and low-lignocellulose content of SMC. To accomplish this, pre-wet (period of hydration and passive composting with temperatures reaching 60-70°C preceding phase I) ingredients were: corncobs, cottonseed hulls, corn stover, and wheat straw. Cottonseed hull pellets were hydrated 7 days before build with 1.5 parts water to 1-part hulls by weight. After 3 days, the hulls were added to 1.9 cm ground corn cobs, WIC® (bale chopper) bale- chopped corn stover, wheat straw, poultry manure, water, 43% of the formula’s distiller’s grain and 39% of the formula’s gypsum for an additional 4 day pre-wet. All pre-wet materials were added with water to a Jaylor® mixer to uniformly mix them together.

7 2.4 Phase I Compost

For phase I, the 80LC pre-wet pile was supplemented and mixed with a Jaylor® mixer with 30% of the formula’s distiller’s grain, the remaining gypsum, SMC and horse manure, and hydrated to the point that water could be squeezed from the material. For the Ctl and 80Ctl formulas, all materials were mixed together simultaneously with exception of distiller’s grain. The materials were stacked in a bunker to promote even airflow throughout the pile. Bunkers fans below the pile aerate the materials and exhaust heat and CO2 produced by the microflora.

Bunker fans were set for 2 min. on, 18 min. off. At build, compost fan speed was set at 2700 rpm and lowered as needed (≅2100 rpm) to meet the oxygen demand of the microorganisms within the pile. Three days into phase I, compost was mixed again with the remaining N (distiller’s grain) and water added.

2.5 Phase II Compost

Phase II composting is used to pasteurize and condition compost for growth and development of A. bisporus. To pasteurize compost, phase I substrate was placed into 1.2 m x 1.2 m trays, stacked, and placed in phase II rooms at the MRC. Air temperature in the phase II room was raised from room temperature to 60°C. Once compost temperatures reached 60°C (often compost temps reach 65°C+), the temperature was held for 1.5 hours. Pasteurization kills pathogens, weed seeds, and most vegetative bacteria that may be detrimental to the crop. Post-pasteurization,

8 the phase II program begins cooling the compost to 55°C immediately (Conditioning

1). The average time required to drop compost temperatures to 55°C was 10 hours.

Conditioning 2, cooling to 48°C, was set to last 4 days. Phase II conditioning utilizes de-ammonifying organisms for the stabilization of N (Flegg et al., 1985). Once the ammonia (gaseous N phase) was no longer detected, the program was set to cool compost with 100% fresh air prior to spawning.

2.6 Spawning

Compost (3.63 kg wet weight) was spawned (30 g) and supplemented in 22.8 x

17.8 x 23.5 cm plastic bins. For Crops 1 and 2, Amycel® XXX spawn was used while for

Crop 3, Sylvan® A15 spawn was used. Both types are commercially available similar off-white hybrid spawns. Supplements were added at spawning to increase fiber and protein content of the substrate. Three corn-based supplements (% cellulose, % hemicellulose, % lignin; % protein): Grit-O’Cobs® 80 (CR) (31.4, 35.6, 4.3; 3.9), corn bran

(CB) (13.5, 48.0, 0.81; 6.7) and corn gluten feed (CGF)(8.9, 24.6, 2.9; 23.8), each with varying lignocellulose contents were added to the compost. Supplement (36 g total) was added to each bin: Lambert® T6M (18 g) and corn substrate (18 g), or corn substrate (36 g). T6M is a slow release nitrogen supplement and mineral source.

Slow release nitrogen supplements are typically used in commercial mushroom production to increase yields. Crop 1 used a 3 (Ctl, 80Ctl, 80LC) x 7 (36T6M, 18T6M +

18CB, 36CB, 18T6M + 18CGF, 36CGF, 18T6M + 18CR, 36CR) factorial design (Crop 1 T6M

9 treatments only contained 18 g). In Crop 2, CGF reps were replaced with Xylan, and due to cost limitations, xylan quantities were modified to (9 g, 18 g) with T6M (27, 18 g). Xylan, considered a pure hemicellulose, was used to evaluate if hemicellulose depletion over the course of the crop might be a limiting factor in reuse of SMC

(personal communication, Bart de Leeuw, MC Substradd, Graanhandel).

After spawning, bins within the room were covered with plastic to maintain adequate moisture content in the compost and increase CO2 to promote vegetative growth during the spawn run. Air temperature was set to 18 °C and was adjusted to maintain compost temperatures in the 20-25 °C range. Plastic was removed 2-3 days before casing, approximately 13 days after spawning, to allow condensation to evaporate. The casing layer, composed of sphagnum peat moss and crushed agricultural lime, was hydrated to approximately 80% moisture and supplemented with casing inoculum (CI). Casing (5 cm) was added to the compost surface to stimulate fruiting and provide moisture for the developing mushrooms.

2.7 Harvesting

Closed mushrooms were harvested daily for 3 weeks (breaks). For each compost treatment, mushrooms from 10-12 bins per crop (Figure 2.2) were harvested, counted, and weighed. Average yield (g mushrooms) per bin by treatment was used to calculate biological efficiency as follows: BE= yield / [dwt (compost + supplement +

10 spawn)]*100. Each compost type by crop had a T6M-only treatment for yield comparison (Crop 1- 18 g; Crops 2,3- 36 g).

2.8 Compost Sampling for Fiber, Ash, and Protein Analyses

For each of 21 treatments, 0.91 kg (ww) phase II compost for Crops 2 (1 rep) and 3

(3 reps) were spawned, supplemented, dried (3-6 days in a drying oven at 55°C), and submitted to Cumberland Valley Analytical Services (CVAS), Maugansville, Maryland for analysis at spawning. Wet lab chemistry was used to determine N, ash, and relative fiber ratios of cellulose, hemicellulose, and lignin by dry weight before and after A. bisporus production (at the completion of 3rd break). Nutrient detergent fiber

(NDF) represented all fractions of cellulose, hemicellulose and lignin, but the acid detergent fiber (ADF) represented only the cellulose and lignin fractions (Sharma and

Kilpatrick, 2000). Therefore, the differences between the ADF and lignin, and NDF and ADF fractions represented percent cellulose and hemicellulose, respectively, by dry weight. An ash-free organic matter and sequential method was used to determine ADF and NDF values (Matt Michonski, CVAS personal communication).

Due to the complexity of the composted substrate, NDF was performed on the sample first, which was then followed by an ADF assessment (sADFom) using the sequential method to remove pectin that obstructed accurate assessment of the hemicellulose content in the sample (Matt Michonski, CVAS, personal communication). Simple correlation coefficients (r) were calculated between

11 fibrous, N, and ash contents at spawning and after crop termination, in addition to yield and BE.

2.9 Bulk Density Analysis

Bulk density of compost was assessed using an 18.9 L bucket at spawning according to procedure 3.01C in the Test Methods for the Examination of Composting and Compost (US Composting Council, 2001). Pasteurized compost was sampled randomly and filled into a straight-sided 18.9 L bucket for bulk density determination

(Fig 2-1). The bulk density test was utilized to determine if fill weight would increase with the addition of SMC. Compost bins were filled at spawn by weight. Three reps for each compost type by crop were evaluated, where the bulk density estimate

(kgm-3) was equal to: [(mass of the filled bucket - mass of the empty bucket)/(volume of the water filled bucket – mass of the empty bucket)] x 100.

3/3

⅔

⅓

2.10 DNA Extraction - Compost Sampling

Conditioned compost was collected, prior to spawning, for bacterial DNA extraction. For each crop, 3-L bags of each compost type were collected and refrigerated until subsampled (within 24 hr of refrigeration). From each bag, one 20- ml scintillation vial was filled aseptically with compost and frozen at -20°C until lyophilized. After lyophilization, all scintillation vials were stored at -80°C until used.

13 2.11 DNA Extraction - Extraction Methods

PowerLyzer Powersoil® (MoBio™; Carlsbad, CA) Extraction kit #12855 was used to extract DNA from the lyophilized compost. However, the presence of inhibitors in the compost required use of an alternative Powersoil® protocol provided by MoBio

(Michelle Carlson, phone communication). Supplementary materials, not in the extraction kit: phenol: chloroform: isoamyl alcohol (25:24:1) pH 8, RNAse A, and absolute ethanol, were purchased to complete the extraction.

One-third of the lyophilized frozen compost from each vial was ground in a chilled mortar and pestle per extraction. Fifty milligrams of compost and 700 μl bead solution were subsequently added to a bead tube and vortexed (5 seconds). In a fume hood, 200 μl of Affymetrix MB Grade phenol: chloroform: isoamyl alcohol

(25:24:1) pH 8, was added to each tube and vortexed (5 sec) followed by the addition of 60 μl Solution C1 (detergent solution) and 30 μl Solution C2 (Inhibitor Removal

Technology® used for removal of humic material). Compost was homogenized 6 bead tubes at a time using the MoBio Vortex-Genie® 2 vortex with adapter (set on high for

10 min.). The compost and cell polysaccharides were centrifuged out of solution

(14,000 g) at room temperature for 2 min. The supernatant was pipetted into a new tube followed by the addition of 200 ul Solution C3 (further inhibitor removal). Tubes were vortexed (5 sec) to mix followed by a 5 min. incubation at 4°C. After 5 min. the incubated tubes were centrifuged (14,000 g). Since phenol: chloroform: isoamyl

14 alcohol at pH 8 also extracts RNA in addition to DNA, lysate moved to clean tube was treated with 1 μl RNAse A (25mg/ml) for 0.5-1 hour at room temperature. To 650 μl lysate, 650 μl Solution C4 and 650 μl of 100% ethanol were added and mixed. Lysate was loaded onto a spin filter column, attached to a Promega® vacuum manifold using

PowerVac® spin filter adapters, 650 μl at a time. The spin column was rinsed with a mixture of 300 μl Solution C4 and 370 μl 100% ethanol, followed by two more washes:

500 μl Solution C5 and 800 μl 100% ethanol. A vacuum was applied for 1 minute after solutions had passed through the filter to dry the membrane. Each port was closed, an unused port was vented, and filters removed from adapters were placed in tubes.

The spin columns in the tubes were centrifuged for 2 min. at 14,000 g with open lids.

The spin filters were then transferred to new 2 ml collection tubes, and 60 μl C6 buffer was added and allowed to sit on membrane on ice for 5 minutes before elution. Tubes were spun for 30 seconds at 10,000 g to collect DNA.

DNA quality was tested for each extraction (54 total) using a nanodrop. Fractions of eluted DNA of the same crop and treatment with a 260/280 ratio greater than 1.7 and a 260/230 ratio greater than 1.4 respectively, were pooled together: 1.) Crop 1214

80LC, 2.) Crop 1215 80LC, 3.) Crop 1302 80LC, 4.) Crop 1214 Ctl, 5.) Crop 1215 Ctl, 6.)

Crop 1302 Ctl. DNA for each of the 6 treatments was diluted to 10-25 ng/μl and test- amplified using polymerase chain reaction (PCR) prior to DNA submission to the Huck

Institute’s Genomics Core Facility (Penn State University, University Park campus).

15 Primers provided by the Huck Institute used for the test amplification and the emulsion PCR were: 27 F ‘AGAGTTTGATCMTGGCTCAG’ (10 μM at 0.5 μl) and 907 R

‘CCCCGTCAATTCMTTTGAGTTT’ (10 μM at 0.5 μl). The master mix used for the test amplification contained 10 μl 2X Go Taq Green, 1 μl reverse primer, 1 μl forward primer, 4 μl DNA (10-25 ng/μl ) and 4 μl water. The PCR was run according to the Huck

Institute protocol: 1.) 94°C-3 minutes 2.) 94°C-15 seconds 3.) 55°C-45 seconds 4.) 72°C for 1 minute 5.) Repeat steps 2-4, 34 times 6.) Hold at 72°C for 8 minutes. After DNA was successfully amplified, non-amplified DNA was provided to the Huck Institute for emulsion PCR and 454 sequencing on a single quad according to Huck Institute protocols.

Dr. Istvan Albert, Associate Professor of Bioinformatics at Penn State, analyzed the data for statistical variation and abundance of detectable taxonomic groups by crop (1,2,3) and compost type (Ctl, 80LC). Statistical software including Mothur

(Schloss and Westcott, 2009), Classifier (Wang et al., 2007), and Metastats (White et al., 2009) were used in analyzing the data.

16 3. Results

3.1 Phase I Temperatures

The variability of phase I compost temperatures by crop and compost type is illustrated in Figures 3-1 to 3-3. For all crops (1,2 and 3) temperatures rose faster in the 80LC formulation than in the Ctl and 80Ctl formulas. Quick heating of the 80LC formula compared to the horse manure-based control was likely due to the addition of a large amount of already composting (hot) pre-wet to minimal other ingredients

(including SMC). For Crops 1 and 3, the control compost lagged behind the SMC- amended compost treatments. Slow heating of the 80Ctl and Ctl formulas was also due to the location of the bunkers, which were exposed to ambient conditions including subfreezing temperatures (Crop 3). Crops began phase I on 28 September

2012 (Crop 1), 19 October 2012 (Crop 2), and 24 January 2013 (Crop 3). Phase I for Crop

3 (Fig. 3-3) required an extra day due to slow heating of the compost pile, likely due to low outside air temperatures in January. Crops 1 and 2 took approximately 50% of the time of Crop 3 to reach peak temperatures (30 h). Greater variability by compost type was observed in Crop 3 phase I temperatures, compared to Crops 1 and 2 (Fig. 3-

1, 3-2), with the 80LC formulation maintaining high temperatures for a longer period of time (Fig. 3-3).

90 80 70 60 50 40 Ctl 30 80Ctl

Temperature (°C) 20 80LC 10 0

time (h)

Figure 3-1. Phase I temperatures (recorded at 10 min intervals) of Ctl (control), 80Ctl (20% SMC amended control), and 80LC (20% amended lignocellulose) compost types contained in bunkers (Crop 1; MRC 1214).

90 80 70 60 50 40 Ctl 30 80Ctl

Temperature (°C) 20 80LC 10 0

time (h)

Figure 3-2. Phase I temperatures (recorded at 10 min intervals) of Ctl (control), 80Ctl (20% SMC amended control), and 80LC (20% amended lignocellulose) compost types

18 contained in bunkers (Crop 2; MRC 1215). 80.00 70.00 60.00 50.00 40.00 Ctl 30.00 80Ctl 20.00 Temperature (°C) 10.00 80LC 0.00

time (h)

Figure 3-3. Phase I temperatures of Ctl (control), 80Ctl (20% SMC amended control), and 80LC (20% amended lignocellulose) compost types contained in bunkers (Crop 3; MRC 1302).

3.2 Yield and Biological Efficiency (BE)

Significant sources of variation from the analysis of variance were similar for all three crops (Table 3-1). Supplement significantly influenced yield and BE in all three crops, while compost type significantly influenced yield and BE in Crops 1 & 2. In

Crop 1, yields ranged from a low of 17.32 kg/m2 on 80Ctl compost supplemented with

Grit-O’Cobs® 80 (CR) at 36 g (Trt 14, Table 3-2) to a high of 27.12 kg/m2 on 80LC supplemented with corn bran (CB) at 18 g + T6 at 18 g (Trt 18, Table 3-2). In general, yields and BEs were highest from 80LC compost and lowest from 80Ctl compost (Fig.

4-4). Yields were also highest when composts were supplemented with corn gluten

19 feed (CGF) +18 g T6M (commercial supplement), followed by 18 g CB +18 g T6M and then 18 g CR +18 g T6M (Fig 4-5).

Table 3-1. Probabilities >F from analysis of variance for two factors (compost, supplement) tested for yield and biological efficiency (BE) of A. bisporus for 3 crops (Crops 1,2,3). Probability > Fa df Yield BE Crop I Compost (CST) 2 <0.0001 <0.0001 Supplement (SP) 6 <0.0001 <0.0001 CST x SP 12 0.7808 0.1954 Crop II CST 2 <0.0001 <0.0001 SP 6 0.0002 0.0003 CST x SP 12 0.7969 0.7962 Crop III CST 2 0.0915 0.0886 SP 5 <0.0001 <0.0001 CST x SP 10 0.5305 0.5278 aF Values < 0.05 are significant according to Fisher’s LSD.

Table 3-2. Mean mushroom yield (kg/m2) and biological efficiency of A. bisporus produced on three compost types non supplemented or supplemented with T6M, plus corn gluten feed, Grit-O’Cobs® 80, or corn bran nutrient at time of spawning (Crop 1; MRC 1214). Trt No. Supplementb Yield BE (%)e Compost typea T6M Corn Total (kg/m2)e (g)c substrate (g) (g)d 1 Ctlf 18 0 18 23.73 abcd 76.0 abcd 2 Ctl 18 18 CGF 36 26.1 ab 82.6 a 3 Ctl 0 36 CGF 36 22.61 bcde 66.6 bcdefg

20 4 Ctl 18 18 CB 36 24.5 abcd 77.5 abc 5 Ctl 18 36 CB 36 20.83 def 61.3 efgh 6 Ctl 18 18 CR 36 23.67 abcd 74.9 abcd 7 Ctl 0 36 CR 36 20.22 def 59.5 fgh 8 80Ctlf 18 0 18 20.92 def 63.8 defgh 9 80Ctl 18 18 CGF 36 24.45 abcd 73.6 abcde 10 80Ctl 0 36 CGF 36 20.77 def 61.1 efgh 11 80Ctl 18 18 CB 36 21.95 bcde 66.1 cdefg 12 80Ctl 0 36 CB 36 18.69 ef 55 gh 13 80Ctl 18 18 CR 36 21.64 cdef 65.1 cdefg 14 80Ctl 0 36 CR 36 17.32 f 50.9 h 15 80LCf 18 0 18 25.61 abc 76.2 abcd 16 80LC 18 18 CGF 36 27.08 a 79.6 ab 17 80LC 0 36 CGF 36 24.62 abcd 72.5 abcdef 18 80LC 18 18 CB 36 27.12 a 79.7 ab 19 80LC 0 36 CB 36 24.62 abcd 72.4 abcdef 20 80LC 18 18 CR 36 24.31 abcd 71.4 abcdef 21 80LC 0 36 CR 36 22.62 bcde 66.5 bcdefg aCompost types: C = control (no SMC addition), 80C = control formula containing 20% SMC, 80LC = lignocellullose formula containing 20% SMC. bSupplement rate: 18 g = approximately 1.7% on compost dry wt basis. Supplements added to phase II compost at time of spawning. cLambert’s commercial delayed release supplement treated with Mertect. dCGF= corn gluten feed, CR= Grit-O’Cobs 80, CB=corn bran. eMeans followed by the same letter are not significantly different according to Tukey’s HSD at P=0.05. fAdditional control treatments containing ½ supplement rate (18 g).

30" 100" A" 25" B C" 80" A" A" 20" B" 60" 15" %&BE& 40" 10" Yield&(kg/m2)& 5" 20"

0" 0" Ctl" 80Ctl" 80LC" Ctl" 80Ctl" 80LC"

30" 100" BC# A AB# A# 25" BC# BC# CD# 80" BC# BC# AB# 20" D# CD# E DE# 60" 15" %&BE& 40" 10"

Yield&(kg/m2)& 5" 20" 0" 0"

36CR" 36CB" 36CR" 36CB" 18T6M" 36CGF" 18T6M" 36CGF"

18CR+18T6M" 18CB+18T6M" 18CR+18T6M" 18CB+18T6M" 18CGF+18T6M" 18CGF+18T6M" Figure 3-5. Groupings from analysis of variance for supplement added at spawning for yield and biological efficiency (% BE). Supplements: CGF = corn gluten feed, CR = Grit- O’Cobs 80, CB =corn bran, T6M= Lambert’s commercial delayed release supplement treated with Mertect. Corn supplements (18g) were complemented with T6M (18 g) for a total of 36 g supplement. Bars labeled with the same letter are not significantly different (P=0.05) according to Tukey’s HSD (Crop 1; MRC 1214).

Overall biological efficiencies (BE) were higher in Crop 2 compared to Crop 1. For example, the highest BE in Crop 1 was 82.6% (Trt 2, Table 3-2) while in Crop 2, the highest BE was 97.6% (Trt 16, Table 3-3). In Crop 2, mushroom yields ranged from a low of 22.63 kg/m2 (Trt 12) to a high of 30.77 kg/m2 (Trt 16). Lowest yields for all three crops were observed in Crop 3. For Crop 3, lowest yield was observed from Ctl

22 compost supplemented with 36 g Grit-O’-Cobs® 80 (16.59 kg/m2, Trt 7) while the highest yield was observed from Ctl compost supplemented with 36 g T6 (21.68 kg/m2, Trt 1). There was no significant difference in compost types for yield or BE for

Crop 3.

T6M Corn Total (g) (g)c substrate (g)d

1 Ctl 36 0 36 29.25 ab 92.7 ab 2 Ctl 18 18 CB 36 27.4 abc 86.9 abc 3 Ctl 0 36 CB 36 26.34 abc 83.6 abc 4 Ctl 18 18 CR 36 27.86 abc 88.4 abc 5 Ctl 0 36 CR 36 24.16 bc 76.7 bc 6 Ctl 18 9 Xln 27 27.34 abc 86.7 abc 7 Ctl 18 18 Xln 36 27.21 abc 86.3 abc 8 80Ctl 36 0 36 27.45 abc 87.0 abc 9 80Ctl 18 18 CB 36 26.91 abc 85.4 abc 10 80Ctl 0 36 CB 36 24.53 bc 77.9 bc 11 80Ctl 18 18 CR 36 25.39 abc 80.5 abc 12 80Ctl 0 36 CR 36 22.63 c 71.9 c 13 80Ctl 18 9 Xln 27 26.65 abc 84.5 abc 14 80Ctl 18 18 Xln 36 25.74 abc 81.6 abc 15 80LC 36 0 36 28.46 ab 90.3 ab

23 16 80LC 18 18 CB 36 30.77 a 97.6 a 17 80LC 0 36 CB 36 27.96 abc 88.7 abc 18 80LC 18 18 CR 36 29.11 ab 92.4 ab 19 80LC 0 36 CR 36 27.06 abc 85.9 abc 20 80LC 18 9 Xln 27 27.66 abc 87.7 abc 21 80LC 18 18 Xln 36 28.31 ab 89.8 ab aCompost types: C = control (no SMC addition), 80C = control formula containing 20% SMC, 80LC = lignocellulose formula containing 20% SMC. bSupplement rate: 18 g = approximately 1.7% on compost dry wt basis. Supplements added to phase II compost at time of spawning. cLambert’s commercial delayed release supplement treated with Mertect. dCR= Grit-O’Cobs 80, CB=corn bran, Xln=Xylan. eMeans followed by the same letter are not significantly different according to Tukey’s HSD at P=0.05.

30" B# A 100" A C B# C 25" 80" 20" 60" 15" %&BE& 40" 10" Yield&(kg/m2)& 5" 20"

0" 0" Ctl" 80Ctl" 80LC" Ctl" 80Ctl" 80LC" Figure 3-6. Groupings from analysis of variance for compost type for mushroom yield and biological efficiency (BE). Ctl= Control, 80Ctl= 20% SMC + 80% Control, 80LC= 20% SMC + 80% Lignocellulose formula. Columns with the same letter are not significantly different according to Tukey’s HSD significant at 0.05 (Crop 2; MRC 1215).

30" A" A A" 100% A B AB" A AB" A A AB" AB" AB" 25" 80% B 20" 60% 15" %&BE& 40% 10"

Yield&(kg/m2)& 5" 20% 0" 0%

36CR% 36CB% 36CR% 36CB% 36T6M% 36T6M%

9Xln+27T6M% 9Xln+27T6M% 18CR+18T6M% 18CB+18T6M% 18Xln+18T6M% 18CR+18T6M% 18CB+18T6M% 18Xln+18T6M%

Figure 3-7. Groupings from analysis of variance for supplement added at spawning for yield and biological efficiency (%BE). Supplements: CGF = corn gluten feed, CR = Grit- O’Cobs 80, CB=corn bran, T6 = Lambert commercial supplement. Columns with the same letter are not significantly different according to Tukey’s HSD (Crop 2; MRC 1215).

Table 3-4. Mean mushroom yield (kg/m2) and biological efficiency of Agaricus bisporus produced on three compost types non supplemented or supplemented with T6M plus Grit-O’Cobs® 80, corn gluten feed or corn bran nutrient at time of spawning (Crop 3; MRC 1302). Trt Compost Supplementb Yield BE (%)c No. typea T6M (g) Corn Total (kg/m2)c substrate (g) (g) 1 Ctld 36 0 36 21.68 a 76.3 a 2 Ctl 18 18 CGF 36 21.15 ab 74.5 ab 3 Ctl 0 36 CGF 36 20.95 ab 73.9 ab 4 Ctl 18 18 CB 36 19.44 abc 68.4 abc 5 Ctl 0 36 CB 36 18.32 abc 64.5 abc 6 Ctl 18 18 CR 36 20.47 abc 72.0 abc 7 Ctld 0 36 CR 36 16.59 c 58.4 c 8 80Ctl 36 0 36 19.94 abc 70.1 abc 9 80Ctl 18 18 CGF 36 18.97 abc 66.8 abc

25 10 80Ctl 0 36 CGF 36 20.32 abc 71.6 abc 11 80Ctl 18 18 CB 36 18.84 abc 66.3 abc 12 80Ctl 0 36 CB 36 18.81 abc 66.2 abc 13 80Ctl 18 18 CR 36 19.41 abc 68.3 abc 14 80Ctl 0 36 CR 36 17.33 bc 61.0 bc 15 80LCd 36 0 36 20.53 abc 72.3 abc 16 80LC 18 18 CGF 36 20.62 abc 72.6 abc 17 80LC 0 36 CGF 36 20.69 abc 72.9 abc 18 80LC 18 18 CB 36 21.44 ab 75.6 ab 19 80LC 0 36 CB 36 19.65 abc 69.1 abc 20 80LC 18 18 CR 36 19.17 abc 67.5 abc 21 80LC 0 36 CR 36 18.10 abc 63.7 abc aCompost types: C = control (no SMC addition), 80C = control formula containing 20% SMC, 80LC = lignocellulose formula containing 20% SMC. bCGF=corn gluten feed, CR= Grit-O’Cobs 80, CB=corn bran, T6M=Lambert’s T6M 18 g= approximately 1.7% on compost dry weight basis. Supplements added to phase II compost at time of spawning. cMeans followed by the same letter are not significantly different according to Tukey’s HSD at P=0.05. dLambert’s T6M (36 g) was used as a standard comparison; All 18 g corn supplements also contained 18g Lambert’s T6M to obtain a 36g supplement by compost dry weight.

30" 100" 25" A" A A A A 80" A A" A A A AB" 20" B AB" B" 60" 15" %"BE" %"BE" 40" 10" 5" 20" 0" 0"

36CR" 36CB" 36CR" 36CB" 36T6M" 36CGF" 36T6M" 36CGF"

18CR+18T6M" 18CB+18T6M" 18CR+18T6M" 18CB+18T6M" 18CGF+18T6M" 18CGF+18T6M" Figure 3-8. Groupings from analysis of variance for supplement added at spawning for yield and biological efficiency (BE). Supplements: CGF = corn gluten feed, CR = Grit-O’Cobs® 80, CB =corn bran. T6M = Lambert commercial supplement. Columns

26 with the same letter are not significantly different according to Tukey’s HSD (Crop 3; MRC 1302).

3.3 Simple Correlation Coefficients

Significant (P=0.001) simple correlation coefficients (r) with r-values >0.4 for compost factors, mushroom yield, and BE are presented in Table 3-5. The highest r- value, as expected, was 0.939 for the comparison between BE at crop termination and yield at crop termination. A negative correlation (-0.751) was observed between ash at spawning and hemicellulose at spawning. We also observed a negative correlation (-0.667) between lignin content at spawning and hemicellulose at spawning. Only BE at crop termination was significantly correlated (-0.45) with ash content of compost at crop termination.

Table 3-5. Simple correlation coefficients (r) with r-values >0.4 of compost factors, mushroom yields and BE, ranked from high to low.

Variable By Variable ra BE at Tb Yield at T 0.939 Ash at Sc Hemicellulose at S -0.751 Nitrogen at S Nitrogen at T 0.710 Lignin at S Hemicellulose at S -0.667 Ash at S Lignin at S 0.641 Nitrogen at S Ash at T 0.630 Ash at S Cellulose at S -0.549 Ash at T Lignin at T 0.488 Spawn run rating at S Nitrogen at T 0.473 BE at T Ash at T -0.450 Cellulose at S Nitrogen at S -0.433 Lignin at S Nitrogen at T -0.413

27 Ash at S Trichoderma at T 0.401 ______a All values significant at P = 0.001. b T= crop termination (end of break 3). c S= at spawning.

3.4 Bulk Density

SMC addition to phase I compost significantly increased bulk density of phase II compost (Fig. 3-9). 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 not a significant difference between the 80LC and 80Ctl formula in relation to bulk density, suggesting no significant major structural differences between the 80Ctl and 80Lc formulations.

140 A A 120 B 100

3 80

kg/m 60

0 Ctl 80Ctl 80LC

28 3.5 Metagenomic Analysis

Analysis of bacterial populations of two phase II composts (Ctl, 80LC) revealed greater variability between crops than between compost types. Of the 15 known 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 (Fig. 3-10). Bacterial populations from the phylum Gemmatimonadetes were the only populations statistically different between 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 populations with significant differences in the composts, but still only accounted for about 2% of the total population in the Ctl compost (Fig. 3-11).

Compost type 29 Control 80% Lignocellulose

a b Crop 1 Proteobacteria 22.1 20.7 19.5 19.5 Actinobacteria

Chloroﬂexi Firmicutes 17.3 27.0 17.9 33.0 Deinococcus-Thermus Bacteroidetes

Planctomycetes Crop 2 8.5 5.9 Verrucomicrobia

TM7 c d

BRC1 19.6 27.9 23.4 26.1 Gemmatimonadetes Acidobacteria 6.5 15.4 Spirochaetes 12.1 24.3 17.4 Tenericutes Fusobacteria 7.9 1 8.2 Crop 3 Unclassiﬁed 2 e Unclassiﬁed Domain f 19.7 27.1 26.5 22.6 3.6 4.6 9.5 15.4 21.1 4.2 18.0 15.5

6.5

Figure 3-10. Percentage of bacteria detectable by phyla at spawning: a) Crop 1, Ctl b) Crop 1, 80LC c) Crop 2, Ctl d) Crop 2, 80LC e) Crop 3, Ctl and f) Crop 3, 80LC 1Unclassified Phylum 2Unclassified Domain (Not Bacteria)

2.5 A 2

1.5 B

1 Percent Population 0.5

0 Ctl 80LC

31 4. Discussion

We were successful in producing mushrooms from both formulations of compost containing SMC. Our results confirm that at least 20% SMC (without the casing layer) can effectively 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 N, organic content and minerals, reclaiming nutrients left over in SMC for compost preparation might be an economical prospect for growers.

Formulation appears to be an important aspect of utilizing SMC as an ingredient in compost. For example, the 80LC compost contained raw material ingredients that were selected to compliment the lower availability of hemicellulose and cellulose in

SMC (Sonnenberg and Blok 2011). We chose ingredients such as corn cobs, wheat straw, corn stover and cottonseed hulls that are higher, as a group, in these fiberous components than horse manure (major ingredient in the Ctl formulation). This may be one of the reasons that 80LC mushroom yields were higher on this formulation than the 80Ctl formulation. All composts were formulated to contain 1.45% N at build. Although SMC may have a a slower N release rate than other N-based raw materials, SMC as a nitrogen source does not appear to be a significant factor contributing to differences in mushroom yield in this study.

32 Another positive aspect of this study was finding an increase in bulk density of compost prepared with SMC. Bulk densities were nearly 20% higher in both compost types prepared with SMC relative to the Ctl compost suggesting that commercial growers could fill more substrate in the same volume of production space when SMC is used as an ingredient in compost. While at first glance, this may seem like an advantage, it is not known if increased bulk density would present problems with conducting a proper phase II conditioning and pasteurization process for commerial growers. Clearing ammonia from our experimental composts seemed to be similar to our control compost. However, excess moisture coinciding with reduced structure is more prone to anaerobic conditions. Pasteurization and conditioning of compost containing SMC may be less of a concern where bulk preparation of compost is practiced (i.e., use of phase II tunnels) compared to phase II conducted inside mushroom production houses. With bulk preparation of compost, forced air is used and can be controlled based on oxygen demand whereas with phase II conducted in production houses, less control is available. Less air provided to microbial populations in compost may result in anaerobic compost, uneven temperatures and inadequate pasteurization to kill pests. It remains to be determined if potential higher bulk densities could be considered an advantage for commercial growers.

Some commercial farms are now experimenting with using SMS in compost formulations, but this work has only just begun.

33 Supplementation of compost with delayed release supplements is known to increase mushroom yields (Carroll and Schisler 1976). Much progress in supplement development has been made since the early work of Carroll and Schisler (1976).

Supplements with various levels and types of protein, lipids and carbohydrates are now available commercially (Wach and Wheeler 1998). Recent work has shown that it is economically feasible to add delayed release supplement types and quantities based on analysis of compost. For example, MCSubstradd® is using near infrared reflectance spectroscopy (NIRS) to analyze fiberous and biomass fractions in phase

III compost (spawn run in tunnel) and then adding the type and amount of supplement needed to optimize economic yield (De Leeuw 2011). To date, however, supplements are not available to replace or compliment composts that may be lacking in optimum hemicellulose or cellulose contents. We sought, therefore, to add selective supplements, such as corn bran, Grit-O’Cobs®, and corn gluten feed, that might serve this purpose. We were not successful in this regard because none of the supplements we added were superior in stimulating yield compared to the commercial supplement Lambert®T6. It should be noted that T6 contains small pellets of newsprint and other waste paper materials (perhaps 15-20% of the supplement dry wt) that would supply extra cellulose to the growing mycelium. This may be one of the components that would be limited in compost that is prepared with SMC as an ingredient. More work is warranted in this area, i.e., where

34 supplements could be designed or mixed with supplemental materials to compliment composts that may be dificient in some fiberous components.

It is currently not known how much SMC may be added to compost raw materials before structural or nutritional problems may be encountered. The negative correlation between ash and cellulose content suggests multiple reuses of SMC may require significant changes to the formulation beyond what has been proposed in this study. Scale-up experiments for phase I in bunkers and phase II in tunnels should be conducted to observe temperature profiles of composts containing various quantities of SMC. This may shed light on activities of microbial populations in the compost and how they may be manipulated to produce high-yielding substrates.

Bacterial population analysis of control compost containing no SMC or compost formulated with 20% SMC revealed that the majority of component bacterial populations were not statistically different. 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 compliment SMC may be more important than particular profiles of microbial populations in the compost.

Knowledge of the bacteria that contribute to conditioning of substrate, however, may help us understand what makes the substrate selective. Firmicutes have been found to produce current using acetate as an electron donor in microbial fuel cells (

Wrighton et al., 2008). Firmicutes have also been shown to control N metabolism using a single transcription regulator within the CodY gene (controls amino acid

35 supply), using isoleucine as a sensor within the environment (Guedon et al., 2005).

Another study revealed populations of both Firmicutes and Proteobacteria (α, β, ε) increased in coal combustion wastewater undergoing continuous ammonium injection (3 months) (Vishnivetskaya et al., 2013). Proteobacteria were also found to dominate agricultural soils undergoing arsenic contamination (Das et al., 2013).

Actinomycete populations have been found in environments ranging from anaerobic digestors of olive oil residue (Rincon et al., 2013) to caves composed of calcium sulfate maintaining 55°C all year and 100% humidity (Quintana et al., 2013). In a study comparing bacterial community structure of a semi- arid haloalkaline soil with a normal soil, clones of Proteobacteria, Chloroflexi, Firmicutes, and Actinobacteria were found in both environments (Keshri et al., 2013). These findings suggest that phase II might not be the best point within the composting cycle to compare how

SMC may affect the microflora of the compost.

Although genus Truepera was significantly more abundant in the Ctl than the

80LC formula for the second and third crop, there was no difference in the population counts in the first crop. Further replicates of this research would better indicate if SMC might inhibit persistence of this genus. More work is needed in this area, and our work with quantification of bacterial populations may be useful as a beginning in this process.

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Appendix A

Significant Correlations (complete)

Spearman Variable by Variable Prob>|ρ| ρ Amt corn supplement at Amt base supplement -0.962 <.0001 2S at S BE at 1T Yield at T 0.9385 <.0001 Ash at S Hemicellulose at S -0.7514 <.0001 Nitrogen at S Nitrogen at T 0.7099 <.0001 Lignin at S Hemicellulose at S -0.6671 <.0001 Ash at S Lignin at S 0.6406 <.0001 Nitrogen at S Ash at T 0.6297 <.0001 Ash at S Cellulose at S -0.5489 <.0001 Ash at T Lignin at T 0.4882 <.0001 Spawn Run rating Nitrogen at T 0.4734 <.0001 BE at T Ash at T -0.4502 <.0001 Cellulose at Spawning at N beginning at S -0.4328 <.0001 S Lignin at S Nitrogen at T -0.4134 <.0001 Ash at S Trichoderma at T 0.401 <.0001 Yield at T Ash at T -0.3987 <.0001 Ash at S Lignin at T -0.39 <.0001 Lignin at S Hemicellulose at T -0.3886 <.0001 Lignin at S Trichoderma at T 0.3786 <.0001 Amt T6M at S BE at T 0.3655 <.0001 Lignin at S Ash at T -0.3648 <.0001 Amt T6M at S Nitrogen at T 0.3632 <.0001 Hemicellulose at S Hemi End at T 0.3624 <.0001 Spawn Run rating at S BE at T 0.3624 <.0001 Amt T6 at S Nitrogen at S 0.3604 <.0001 Amt corn supplement at Nitrogen at S -0.3604 <.0001 S Trichoderma at T Ash at T -0.3588 <.0001

42 Ash at S Hemicellulose at T -0.3582 <.0001 Nitrogen at S Cellulose at T -0.3573 <.0001 Hemicellulose at S Trichoderma at T -0.3516 <.0001 Ash at S Ash at T -0.3446 <.0001 Hemicellulose at S Lignin at T 0.3382 <.0001 Amt corn supplement at BE at T -0.3282 <.0001 S Nitrogen at S Trichoderma at T -0.3269 <.0001 Amt corn supplement at Nitrogen at T -0.3161 <.0001 S Spawn run rating Yield Bk1-3 at T 0.3079 <.0001 Spawn run rating Base Supplement at S 0.3077 <.0001 Amt T6M at S Yield Bk1-3 at T 0.3048 <.0001 Amt T6M at S Hemicellulose at S -0.2973 <.0001 Amt Corn Supplement at Hemicellulose at S 0.2973 <.0001 S Ash at T Hemicellulose at T -0.2925 <.0001 Amt Supplement at S Yield Bk1-3 at T -0.2854 <.0001 Spawn Run rating at S Amt Supp at S -0.2777 <.0001 Hemicellulose at S Ash at T 0.2767 0.0006 Nitrogen at T Cellulose at T -0.2756 <.0001 Nitrogen at S Lignin at T 0.2743 0.0007 Cellulose at S Hemi End at T 0.2715 0.0008 Nitrogen at T Hemicellulose at T 0.2575 0.0002 Cellulose at T Hemicellulose at S 0.2447 0.0027 BE at T Nitrogen at T 0.2427 0.0004 Yield at T Trichoderma at T 0.2415 <.0001 Cellulose at S Cellulose at T 0.2327 0.0044 Nitrogen at S Lignin at S -0.2269 <.0001 Lignin at S Cellulose at S -0.2266 <.0001 Ash at S Cellulose at T -0.2258 0.0058 Ash at T Cellulose at T -0.2201 0.0013 BE at T Trichoderma at T 0.2177 <.0001 Lignin at S Lignin at T -0.2165 0.0082 Spawn Run rating Ash at T -0.2134 0.002 Nitrogen at S Spawn Run rating 0.2102 <.0001 BE at T Hemi at T 0.2041 0.003

43 Nitrogen at S Yield at T -0.2019 <.0001 Trichoderma at T Lignin at T -0.1958 0.0044 Hemicellulose at S Yield at T -0.1946 <.0001 Cellulose at S Ash at T -0.1942 0.0172 Yield Bk1-3 at T Hemicellulose at T 0.1933 0.0049 Base Supplement at S Lignin at T -0.1932 0.005 Ash at S Nitrogen at T -0.1917 0.0196 Ash at S Yield at T 0.191 <.0001 Base Supplement at S Ash at S 0.1841 0.0001 Amt corn supplement at Ash at S -0.1841 0.0001 S Hemicellulose at S Nitrogen at T 0.1836 0.0255 Amt corn supplement at Hemicellulose at T 0.1804 0.0088 S BE at T Lignin at T -0.172 0.0125 Ash End at T Nitrogen at T -0.1664 0.0158 Amt Supp at S Lignin at T 0.1653 0.0165 Lignin at S Yield at T 0.1635 0.0006 Cellulose at S Yield at T 0.1614 0.0007 Amt T6M at S Hemicellulose at T -0.159 0.0212 Nitrogen at T Lignin at T -0.153 0.0267 Cellulose at S BE at T 0.1442 0.0024 Spawn Run rating at S Hemicellulose at T 0.1437 0.0398 Amt T6M at S Cellulose at S -0.1283 0.007 Amt corn supplement at Cellulose at S 0.1283 0.007 S Hemicellulose at S BE at T -0.1229 0.0098 Nitrogen at S BE at T -0.1203 <.0001 Amt base supplement at Lignin at S 0.12 0.0116 S Amt corn supplement at Lignin at S -0.12 0.0116 S Ash at S BE at T 0.1084 <.0001 Lignin at S BE at T 0.1043 <.0001 Base Supplement at S Trichoderma at T 0.099 0.0091 Nitrogen at S Hemicellulose at S 0.0951 0.046 1All values significant at P=0.001, 1T= at crop termination, 2S= at spawning

Appendix B

Metagenomic Data (raw)

TOTAL R ank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 no rank 13052 18330 12342 13294 14668 18207 Bacteria domain 13049 18310 12331 13280 14645 18184 Proteobacteria phylum 2549 5106 3348 2753 3829 4113 Alphaproteobacteria class 1230 2510 1599 1501 2035 1907 Rhizobiales order 1083 2099 1305 1346 1798 1614 Hyphomicrobiaceae family 374 463 228 374 437 332 Hyphomicrobium genus 29 41 22 15 12 24 Filomicrobium genus 10 13 12 7 11 18 Devosia genus 2 6 10 2 5 10 Rhodoplanes genus 0 1 1 0 0 0 Unclassified 333 402 183 350 409 280 Phyllobacteriaceae family 105 240 195 122 232 177 Hoeflea genus 5 3 4 3 4 6 Pseudaminobacter genus 5 4 6 2 7 3 Chelativorans genus 1 1 0 1 1 0 Aquamicrobium genus 0 1 0 0 0 0 Mesorhizobium genus 0 0 2 2 0 0 Aminobacter genus 0 0 0 0 1 0 Unclassified 94 231 183 114 219 168 Beijerinckiaceae family 34 195 96 89 91 112 Chelatococcus genus 32 186 88 84 89 105 Camelimonas genus 1 0 0 0 0 0 Unclassified 1 9 8 5 2 7 Methylobacteriaceae family 0 2 0 1 2 3 Microvirga genus 0 2 0 1 2 2 Methylobacterium genus 0 0 0 0 0 1 Rhizobiaceae family 0 1 3 0 3 2 Rhizobium genus 0 1 3 0 3 1 Sinorhizobium genus 0 0 0 0 0 1 Brucellaceae family 0 0 2 2 3 4 Ochrobactrum genus 0 0 1 1 0 3 Pseudochrobactrum genus 0 0 0 0 1 0 Unclassified 0 0 1 1 2 1 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Rhizobiales_incertae_sedis family 0 0 2 0 0 1 Vasilyevaea genus 0 0 1 0 0 0 Bauldia genus 0 0 0 0 0 1 Unclassified 0 0 1 0 0 0 Rhodobiaceae family 0 0 1 0 0 2 Parvibaculum genus 0 0 0 0 0 1 Unclassified 0 0 1 0 0 1 Bradyrhizobiaceae family 0 0 1 0 0 3 Balneimonas genus 0 0 1 0 0 3 Methylocystaceae family 0 0 0 0 0 1 Methylocystis genus 0 0 0 0 0 1 Unclassified 570 1198 777 758 1030 977 Rhodospiralles order 43 140 74 36 27 62 Rhodospirillaceae family 34 63 36 22 14 8 Tistlia genus 6 6 1 2 1 0 Inquilinus genus 0 3 5 0 0 1 Pelagibius genus 0 1 4 0 0 0 Dongia genus 0 0 1 0 0 1 Magnetospirillum genus 0 0 1 0 0 0 Rhodospirillaceae 28 53 24 20 13 6 Acetobacteraceae family 0 4 5 2 0 2 Roseomonas genus 0 1 0 0 0 1 Stella genus 0 0 1 0 0 0 Unclassified 0 3 4 2 0 1 Sphingomonadales order 9 24 25 12 33 20 Erythrobacteraceae family 8 20 15 7 26 12 Altererythrobacter genus 3 7 3 2 6 1 Porphyrobacter genus 0 0 1 1 2 0 Croceicoccus genus 0 0 1 0 1 2 Unclassified 5 13 10 4 17 9 Sphingomonadaceae family 0 2 4 2 1 1 Sphingomonas genus 0 1 0 1 0 0 Novosphingobium genus 0 0 1 0 0 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Sphingosini cella genus 0 0 0 0 0 1 Unclassified 0 1 3 1 1 0 Unclassified 9 73 33 12 13 52 Rhodobacterales order 5 13 30 3 11 11 Rhodobacteraceae family 5 13 30 3 11 11 Labrenzia genus 0 1 1 0 0 0 Agaricicola genus 0 0 1 0 0 0 Pannonibacter genus 0 0 1 0 0 0 Paracoccus genus 0 0 1 2 3 1 Ketogulonicigenium genus 0 0 1 0 0 1 Amaricoccus genus 0 0 0 1 0 3 Rhodobacter genus 0 0 0 0 0 1 Unclassified 5 12 25 0 8 5 Unclassified 1 2 6 3 6 7 Sneathiellales order 0 0 2 0 2 0 Sneathiellaceae family 0 0 2 0 2 0 Sneathiella genus 0 0 2 0 2 0 Rickettsiales order 0 0 1 0 0 0 Rickettsiaceae family 0 0 1 0 0 0 Orientia genus 0 0 1 0 0 0 Caulobacterales order 0 0 2 0 1 4 Caulobacteraceae family 0 0 2 0 1 4 Phenylobacterium genus 0 0 1 0 1 3 Brevundimonas genus 0 0 0 0 0 1 Unclassified 0 0 1 0 0 0 Alphaproteobacteria_incerta e_sedis order 0 0 0 0 1 0 Geminicoccus genus 0 0 0 0 1 0 Gammaproteobacteria class 847 1623 1007 716 1074 1188 Xanthomonadales order 445 985 560 403 618 704 Xanthomonadaceae family 261 535 330 204 276 306 Pseudoxanthomonas genus 92 171 108 96 140 118 Luteimonas genus 53 78 54 34 33 44 Lysobacter genus 17 24 6 5 5 6 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Pseudofulvimonas genus 8 61 19 0 0 19 Stenotrophomonas genus 0 0 0 1 0 0 Unclassified 91 201 143 68 98 119 Sinobacteraceae family 181 440 228 197 334 391 Steroidobacter genus 181 440 228 197 334 391 Unclassified 3 10 2 2 8 7 Pseudomonadales order 40 56 75 41 129 72 Pseudomonadaceae family 39 53 70 41 125 67 Serpens genus 37 39 47 37 87 58 Pseudomonas genus 1 3 3 0 3 2 Cellvibrio genus 0 1 7 0 13 0 Azomonas genus 0 0 0 0 1 0 Unclassified 1 10 13 4 21 7 Moraxellaceae family 0 3 5 0 4 4 Acinetobacter genus 0 3 4 0 3 1 Enhydrobacter genus 0 0 0 0 1 0 Unclassified 0 0 1 0 0 3 Unclassified 1 0 0 0 0 1 Chromatiales order 24 13 6 12 6 13 Ectothiorhodospiraceae family 10 7 5 2 1 2 Natronocella genus 0 0 0 1 0 0 Unclassified 10 7 5 1 1 2 Unclassified 14 6 1 10 5 11 Alteromonadales order 1 2 0 0 0 0 Idiomarinaceae family 1 2 0 0 0 0 Pseudidomarina genus 1 1 0 0 0 0 Idiomarina genus 0 1 0 0 0 0 Oceanospirillales order 0 6 2 0 2 1 Halomonadaceae family 0 1 0 0 1 0 Kushneria genus 0 1 0 0 1 0 Unclassified 0 5 2 0 1 1 Legionellales order 0 1 1 1 5 0 Legionellaceae family 0 1 1 1 5 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Legionella genus 0 1 1 1 5 0 Gammaproteobacteria_incert ae_sedis order 0 2 3 2 2 3 Porticoccus genus 0 0 1 1 0 1 Unclassified 0 2 2 1 2 2 Enterobacteriales order 0 0 1 0 1 0 Enterobacteriaceae family 0 0 1 0 1 0 Serratia genus 0 0 0 0 1 0 Unclassified 0 0 1 0 0 0 Methylococcales order 0 0 2 0 2 1 Methylococcaceae family 0 0 2 0 2 1 Methylocaldum genus 0 0 2 0 2 1 Aeromonadales order 0 0 0 1 0 0 Aeromonadaceae family 0 0 0 1 0 0 Aeromonas genus 0 0 0 1 0 0 Unclassified 337 558 357 256 309 394 Unclassified 90 234 160 104 162 196 Deltaproteobacteria class 281 406 264 277 259 419 Myxococcales order 254 287 194 236 206 312 suborde Cystobacterineae r 50 34 24 38 16 37 Cystobacteraceae family 39 18 14 22 8 24 Anaeromyxobacter genus 3 4 4 1 2 3 Melittangium genus 0 0 0 0 0 2 Unclassified 36 14 10 21 6 19 Unclassified 11 16 10 16 8 13 suborde Sorangiineae r 87 132 97 108 100 195 Polyangiaceae family 41 85 55 54 72 115 Sorangium genus 0 6 11 1 0 34 Unclassified 41 79 44 53 72 81 Unclassified 46 47 42 54 28 80 suborde Nannocystineae r 0 0 1 0 0 1 Nannocystaceae family 0 0 1 0 0 1 Nannocystis genus 0 0 1 0 0 1 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Unclassified 117 121 72 90 90 79 Bdellovibrionales order 1 40 19 1 21 41 Bdellovibrionaceae family 1 39 19 1 21 41 Bdellovibrio genus 1 14 6 1 10 12 Vampirovibrio genus 0 25 13 0 11 29 Bacteriovoracaceae family 0 1 0 0 0 0 Peredibacter genus 0 1 0 0 0 0 Desulfuromonadales order 0 0 0 0 0 1 Desulfuromonaceae family 0 0 0 0 0 1 Unclassified 0 0 0 0 0 1 Unclassified 26 79 51 40 32 65 Betaproteobacteria class 50 152 164 63 145 201 Burkholderiales order 32 114 126 44 90 117 Alcaligenaceae family 16 47 67 23 55 57 Kerstersia genus 2 0 2 2 1 3 Pusillimonas genus 1 3 5 1 8 1 Parapusillimonas genus 1 2 1 0 4 2 Pigmentiphaga genus 1 0 0 0 5 1 Bordetella genus 0 3 8 0 2 8 Unclassified 11 39 51 20 35 42 Comamonadaceae family 7 36 43 11 18 35 Schlegelella genus 7 35 40 11 16 29 Hylemonella genus 0 0 1 0 0 0 Hydrogenophaga genus 0 0 0 0 1 1 Comamonas genus 0 0 0 0 1 0 Unclassified 0 1 2 0 0 5 Burkholderiales_incertae_sed is family 0 0 0 0 0 1 Unclassified 0 0 0 0 0 1 Unclassified 9 31 16 10 17 24 Hydrogenophiales order 4 2 4 4 17 21 Hydrogenophilaceae family 4 2 4 4 17 21 Hydrogenophilus genus 4 2 3 4 14 21 Unclassified 0 0 1 0 3 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Rhodocyclales order 2 7 5 1 11 7 Rhodocyclaceae family 2 7 5 1 11 7 Shinella genus 1 3 2 0 2 5 Azoarcus genus 0 3 0 0 2 0 Methyloversatilis genus 0 0 2 0 0 0 Unclassified 1 1 1 1 7 2 Methylophilales order 0 1 0 0 1 1 Methylophilaceae family 0 1 0 0 1 1 Methylobacillus genus 0 0 0 0 0 1 Unclassified 0 1 0 0 1 0 Unclassified 12 28 29 14 26 55 Unclassified 141 415 314 196 316 398 Actinobacteria phylum 4305 4449 2605 3591 2256 1734 Actinobacteria class 4305 4449 2605 3591 2256 1734 Actinobacteridae subclass 3062 3112 2182 2254 1529 1323 Actinomycetales order 3062 3112 2181 2254 1529 1323 Corynebacterineae suborder 40 101 65 25 26 15 Mycobacteriaceae family 18 53 7 13 12 1 Mycobacterium genus 17 49 7 13 11 1 Nocardiaceae family 19 45 57 10 13 8 Rhodococcus genus 2 0 8 1 5 4 Gordonia genus 3 0 1 0 5 3 Micropolyspora genus 14 45 44 9 1 1 Millisia genus 0 0 0 0 2 0 Unclassified 1 4 0 0 1 0 Corynebacteriaceae family 3 1 0 0 0 2 Corynebacterium genus 3 1 0 0 0 2 Unclassified 0 0 4 0 0 0 Dietziaceae family 0 0 0 0 0 1 Dietzia genus 0 0 0 0 0 1 Unclassified 0 2 1 2 1 3 Micrococcineae suborder 303 518 255 283 375 219 Dermabacteraceae family 5 5 1 2 0 1 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Brachybacterium genus 4 4 0 2 0 1 Unclassified 1 1 1 0 0 0 Cellulomonadaceae family 78 145 82 57 77 77 Cellulomonas genus 15 28 15 5 16 8 Actinotalea genus 35 59 40 29 33 47 Unclassified 28 58 27 23 28 22 Microbacteriaceae family 10 26 22 13 44 25 Leucobacter genus 2 1 1 2 4 1 Microbacterium genus 1 9 7 5 13 7 Klugiella genus 0 0 0 0 1 0 Unclassified 7 16 14 6 26 17 family 41 58 19 55 38 18 Ornithinicoccus genus 31 42 16 46 25 10 Janibacter genus 0 0 0 0 0 1 Unclassified 10 16 3 9 13 7 Bogoriellaceae family 22 18 5 8 3 2 Georgenia genus 22 18 5 8 3 2 Promicromonosporaceae family 8 7 0 13 22 2 Cellulosimicrobium genus 7 7 0 13 21 2 Xylanimicrobium genus 1 0 0 0 0 0 Isoptericola genus 0 0 0 0 1 0 Demequinaceae family 31 78 27 24 39 9 Demequina genus 31 78 27 24 39 9 Micrococcaceae family 1 2 5 0 0 1 Yaniella genus 1 0 0 0 0 0 Nesterenkonia genus 0 1 1 0 0 0 Arthrobacter genus 0 1 3 0 0 1 Unclassified 0 0 1 0 0 0 Beutenbergiaceae family 0 1 2 1 0 1 Unclassified 0 1 2 1 0 1 Brevibacteriaceae family 0 1 0 0 0 0 Brevibacterium genus 0 1 0 0 0 0 Sanguibacteraceae family 0 0 3 0 3 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Sanguibacter genus 0 0 3 0 3 0 Jonesiaceae family 0 0 5 0 0 1 Jonesia genus 0 0 5 0 0 1 Unclassified 107 177 84 110 149 82 Streptosporangineae suborder 1661 1785 1241 1420 713 737 Nocardiopsaceae family 1204 961 773 1068 435 241 Thermobifida genus 1137 904 696 1013 403 215 Nocardiopsis genus 5 4 3 2 0 0 Murinocardiopsis genus 1 0 0 0 0 0 Marinactinospora genus 0 3 1 1 1 1 Unclassified 61 50 73 52 31 25 Thermomonosporaceae family 59 218 81 39 36 53 Thermomonospora genus 58 214 79 36 34 53 Actinomadura genus 1 3 0 0 0 0 Unclassified 0 1 2 3 2 0 Streptosporangiaceae family 263 481 300 211 177 382 Microbiospora genus 31 1 3 5 0 0 Thermopolyspora genus 175 455 266 186 165 371 Unclassified 57 25 31 20 12 11 Unclassified 135 125 87 102 65 61 Micromonosporineae suborder 531 103 77 165 86 36 Micromonosporaceae family 531 103 77 165 86 36 Unclassified 531 103 77 165 86 36 Pseudomocardineae suborder 45 119 191 31 60 65 Pseudonocardiaceae family 45 119 191 31 60 65 Prauserella genus 5 5 3 1 0 1 Saccharopolyspora genus 11 53 81 2 3 1 Thermobispora genus 5 2 12 23 46 43 Saccharomonospora genus 7 12 12 0 3 1 Thermocrispum genus 8 26 34 4 6 18 Pseudocardia genus 0 1 2 0 0 0 Unclassified 9 20 47 1 2 1 Glycomycineae suborder 5 2 0 0 0 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Glycomycetaceae family 5 2 0 0 0 0 Stackebrandtia genus 3 0 0 0 0 0 Glycomyces genus 2 2 0 0 0 0 Propionibacterineae suborder 8 2 6 2 7 4 Propionibacteriaceae family 2 0 0 0 0 1 Propionibacterium genus 1 0 0 0 0 0 Microlunatus genus 0 0 0 0 0 1 Unclassified 1 0 0 0 0 0 Nocardioidaceae family 6 2 6 2 6 3 Aeromicrobium genus 2 0 1 0 2 1 Nocardioides genus 2 0 3 1 0 0 Marmoricola genus 2 1 2 0 3 2 Thermasporomyces genus 0 0 0 1 0 0 Unclassified 0 1 0 0 1 0 Unclassified 0 0 0 0 1 0 Actinomycineae suborder 1 0 0 0 0 0 Actinomycetaceae family 1 0 0 0 0 0 Actinomyces genus 1 0 0 0 0 0 Streptomycineae suborder 0 5 5 1 14 28 Streptomycetaceae family 0 5 5 1 14 28 Streptomyces genus 0 5 3 0 12 27 Streptacidiphilus genus 0 0 1 0 0 0 Unclassified 0 0 1 1 2 1 Unclassified 468 477 341 327 248 219 Acidimicrobidae subclass 281 275 115 211 134 95 Acidimicrobiales order 281 275 115 211 134 95 Acidimicrobineae suborder 281 275 115 211 134 95 Iamiaceae family 77 59 19 60 40 18 Iamia genus 77 59 19 60 40 18 Acidimicrobineae_incertae_ sedis family 10 12 10 3 4 4 Aciditerrimonas genus 10 12 10 3 4 4 Acidimicrobiaceae family 7 24 4 12 3 10 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Ilumatobacter genus 6 23 4 12 3 10 Unclassified 1 1 0 0 0 0 Unclassified 187 180 82 136 87 63 Nitriliruptoridae subclass 19 22 6 40 11 13 Nitriliruptorales order 1 0 0 0 0 0 Nitriliruptoraceae family 1 0 0 0 0 0 Nitriliruptor genus 1 0 0 0 0 0 Euzebyales order 7 13 2 16 5 5 Euzebyaceae family 7 13 2 16 5 5 Euzebya genus 7 13 2 16 5 5 Unclassified 11 9 4 24 6 8 Rubrobacteridae subclass 26 55 12 22 25 18 Thermoleophilales order 3 10 1 2 3 0 Thermoleophilaceae family 3 10 1 2 3 0 Thermoleophilum genus 3 10 1 2 3 0 Solirubrobacterales order 3 10 3 6 3 3 Conexibacteraceae family 1 6 1 3 2 0 Conexibacter genus 1 6 1 3 2 0 Patulibacteraceae family 0 1 0 0 0 1 Patulibacter genus 0 1 0 0 0 1 Unclassified 2 3 2 3 1 2 Unclassified 20 35 8 14 19 15 Unclassified 0 0 1 0 0 0 Unclassified 917 985 290 1064 557 285 Chloroflexi phylum 775 1504 802 1126 1152 2814 Thermomicrobia class 539 550 232 594 842 570 Sphaerobacteridae subclass 532 532 227 584 820 564 Sphaerobacterales order 532 532 227 584 820 564 suborde Sphaerobacterineae r 532 532 227 584 820 564 Sphaerobacteraceae family 532 532 227 584 820 564 Sphaerobacter genus 532 532 227 584 820 564 Unclassified 7 18 5 10 22 6 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Caldilineae class 41 133 76 38 93 99 Caldilineales order 41 133 76 38 93 99 Caldilineaceae family 41 133 76 38 93 99 Caldilinea genus 41 133 76 38 93 99 Anaerolineae class 165 667 292 443 162 1759 Anaerolineales order 165 667 292 443 162 1759 Anaerolineaceae family 165 667 292 443 162 1759 Bellilinea genus 0 9 4 4 3 38 Unclassified 165 658 288 439 159 1721 Chloroflexi class 0 33 74 4 16 87 Chloroflexales order 0 32 70 3 14 83 Chloroflexaceae family 0 32 70 3 14 83 Chloroflexus genus 0 1 3 1 1 5 Heliothrix genus 0 13 28 0 6 27 Roseiflexus genus 0 1 0 0 0 0 Unclassified 0 17 39 2 7 51 Unclassified 0 1 4 1 2 4 Unclassified 30 121 128 47 39 299 Firmicutes phylum 2333 2223 1895 2294 2545 3282 Bacilli class 1522 1713 1431 1633 1857 1879 Bacillales order 1507 1699 1411 1614 1835 1852 Planococcaceae family 443 705 504 693 887 433 Ureibacillus genus 179 169 62 161 105 71 Planococcaceae_incertae_se dis genus 181 392 206 419 518 218 Rummeliibacillus genus 7 6 34 0 16 11 Solibacillus genus 2 14 10 0 2 8 Caryophanon genus 1 0 0 0 0 0 Sporosarcina genus 0 1 0 0 0 0 Bhargavaea genus 0 2 0 0 0 1 Kurthia genus 0 0 20 0 12 8 Planococcus genus 0 0 4 0 0 1 Planomicrobium genus 0 0 1 0 0 0 Psychrobacillus genus 0 0 0 2 1 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Unclassified 73 121 167 111 233 115 Thermoactinomycetaceae 2 family 77 91 97 55 52 22 Planifilum genus 77 91 97 55 52 22 Bacillaceae 2 family 58 27 37 35 9 8 Piscibacillus genus 4 1 0 0 0 0 Paucisalibacillus genus 1 1 0 0 0 0 Tuberibacillus genus 0 0 0 9 0 0 Gracilibacillus genus 0 0 0 0 0 1 Unclassified 53 25 37 26 9 7 Thermoactinomycetaceae 1 family 70 240 200 100 143 726 Thermoactinomyces genus 22 47 98 2 8 9 Thermofalvimicrobium genus 41 179 95 95 124 652 Laceyella genus 0 0 0 0 1 0 Unclassified 7 14 7 3 10 65 Bacillaceae 1 family 94 104 88 133 179 134 Aeribacillus genus 4 0 0 0 2 1 Bacillus genus 37 66 47 54 42 54 Geobacillus genus 19 3 16 43 97 42 Saccarococcus genus 2 1 0 1 2 1 Falsibacillus genus 0 1 0 2 3 2 Unclassified 32 33 25 33 33 34 Paenibacillaceae 1 family 63 39 39 40 30 55 Thermobacillus genus 39 15 25 23 13 22 Cohnella genus 3 0 0 0 1 4 Brevibacillus genus 1 0 2 0 0 0 Fontibacillus genus 0 2 1 1 0 1 Paenibacillus genus 0 1 2 2 1 4 Unclassified 20 21 9 14 15 24 Staphylococcaceae family 2 6 1 0 1 0 Staphylococcus genus 1 3 1 0 1 0 Jeotgalicoccus genus 0 3 0 0 0 0 Unclassified 1 0 0 0 0 0 Pasteuriaceae family 15 42 42 24 47 65 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Pasteuria genus 15 42 42 24 47 65 Paenibacillaceae 2 family 17 7 8 13 2 26 Ammoniphilus genus 16 7 4 12 1 18 Oxalophagus genus 0 0 2 0 0 1 Unclassified 1 0 2 1 1 7 Bacillales_incertae_sedis family 7 7 4 18 11 13 Caldalkalibacillus genus 7 5 2 12 3 5 Calditerricola genus 0 2 2 5 8 8 Unclassified 0 0 0 1 0 0 Unclassified 661 431 391 503 474 370 Lactobacillales order 2 3 6 0 5 2 Streptococcaceae family 1 2 0 0 4 0 Streptococcus genus 1 0 0 0 0 0 Lactococcus genus 0 2 0 0 4 0 Enterococcaceae family 1 0 3 0 1 1 Enterococcus genus 1 0 3 0 1 0 Unclassified 0 0 0 0 0 1 Lactobacillaceae family 0 1 0 0 0 0 Lactobacillus genus 0 1 0 0 0 0 Leuconostocaceae family 0 0 1 0 0 1 Leuconostoc genus 0 0 1 0 0 1 Carnobacteriaceae family 0 0 1 0 0 0 Desemzia genus 0 0 1 0 0 0 Unclassified 0 0 1 0 0 0 Unclassified 13 11 14 19 17 25 Clostridia class 279 295 303 278 383 1051 Clostridiales order 209 255 258 235 332 915 Clostridiales_Incertae Sedis XI family 39 19 23 34 43 49 Tepidimicrobium genus 38 16 20 26 36 35 Tissierella genus 1 0 0 2 1 3 Sedimentibacter genus 0 0 0 0 0 1 Unclassified 0 3 3 6 6 10 Ruminococcaceae family 33 60 34 38 72 303 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Sporobacter genus 1 0 0 1 1 0 Clostridium III genus 27 39 18 31 57 193 Acetivibrio genus 1 2 3 0 3 11 Clostriidum IV genus 0 0 2 0 0 0 Unclassified 4 19 11 6 11 99 Clostridiales_Incertae Sedis III family 8 2 2 4 3 2 Tepidanaerobacter genus 8 1 2 4 3 2 Unclassified 0 1 0 0 0 0 Clostridiaceae 4 family 2 0 3 0 1 3 Unclassified 2 0 3 0 1 3 Clostridiales_Incertae Sedis XVIII family 21 7 7 17 38 15 Symbiobacterium genus 21 7 7 17 38 15 Clostridiaceae 1 family 37 67 76 41 56 101 Clostridium sensu stricto genus 28 59 49 26 37 48 Anaerobacter genus 1 1 3 1 2 4 Sarcina genus 0 1 1 0 1 0 Proteiniclasticum genus 0 0 2 0 4 6 Fervidicella genus 0 0 0 0 0 1 Unclassified 8 6 21 14 12 42 Lachnospiraceae family 8 28 31 14 28 122 Cellulosilyticum genus 0 0 1 0 0 2 Parasporobacterium genus 0 0 1 1 0 0 Clostridium XIVa genus 0 0 0 0 0 1 Unclassified 8 28 29 13 28 119 Incertae Sedis IV family 4 0 2 5 1 0 Caldicoprobacter genus 4 0 2 5 1 0 Peptococcaceae 2 family 3 4 2 0 1 9 Desulfotomaculum genus 2 3 1 0 1 5 Unclassified 1 1 1 0 0 4 Eubacteriaceae family 3 1 0 1 0 1 Alkalibaculum genus 3 1 0 1 0 0 Garciella genus 0 0 0 0 0 1 Clostridiaceae 2 family 1 0 2 3 1 1 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Alkaliphilus genus 1 0 2 3 1 1 Syntrophomonadaceae family 0 2 0 1 1 4 Syntrophomas genus 0 2 0 1 0 1 Unclassified 0 0 0 0 1 3 Clostridiales_Incertae Sedis XVII family 0 1 0 2 1 0 Thermaerobacter genus 0 1 0 2 1 0 Gracilibacteraceae family 0 2 3 0 3 30 Lutispora genus 0 1 3 0 3 30 Unclassified 0 1 0 0 0 0 Peptococcaceae 1 family 0 2 0 0 0 6 Desulfonispora genus 0 2 0 0 0 1 Thermincola genus 0 0 0 0 0 2 Desulfitobacterium genus 0 0 0 0 0 2 Unclassified 0 0 0 0 0 1 Peptostreptococcaceae family 0 0 1 0 0 0 Clostridium XI genus genus 0 0 1 0 0 0 Clostridiales_incertae_sedis family 0 0 0 2 0 5 Proteiniborus genus 0 0 0 2 0 5 Clostridiales_Incertae Sedis XIII family 0 0 0 0 1 2 Anaerovorax genus 0 0 0 0 1 2 Heliobacteriaceae family 0 0 0 0 0 2 Unclassified 0 0 0 0 0 2 Unclassified 50 60 72 73 82 260 Natranaerobiales order 9 1 4 2 3 5 Natranaerobiaceae family 9 1 4 2 3 5 Dethiobacter genus 9 1 4 2 3 5 Halanaerobiales order 7 4 5 13 8 8 Halanaerobiaceae family 7 4 5 13 8 8 Halocella genus 1 0 0 0 0 0 Halothermothrix genus 0 0 0 1 0 0 Halanaerobiaceae 6 4 5 12 8 8 Thermoanerobacterales order 0 0 0 2 0 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Thermoanerobacteraceae family 0 0 0 2 0 0 Unclassified 0 0 0 2 0 0 Unclassified 54 35 36 26 40 123 Erysipelotrichia class 0 0 0 0 0 1 Erysipelotrichales order 0 0 0 0 0 1 Erysipelotrichaceae family 0 0 0 0 0 1 Coprobacillus genus 0 0 0 0 0 1 Negativicutes class 0 0 0 0 0 1 Selenomonadales order 0 0 0 0 0 1 Veillonellaceae family 0 0 0 0 0 1 Sporotalea genus 0 0 0 0 0 1 Deinococcus-Thermus phylum 173 228 103 248 142 87 Deinococci class 173 228 103 248 142 87 Deinococcales order 109 216 100 103 69 74 Trueperaceae family 109 216 99 101 68 74 Truepera genus 109 216 99 101 68 74 Unclassified 0 0 1 2 1 0 Thermales order 60 5 0 141 72 12 Thermaceae family 60 5 0 141 72 12 Thermus genus 56 5 0 134 66 11 Unclassified 4 0 0 7 6 1 Unclassified 4 7 3 4 1 1 Bacteroidetes phylum 230 550 573 197 954 756 Flavobacteria class 34 43 44 34 208 46 Flavobacteriales order 34 43 44 34 208 46 Flavobacteriaceae family 33 43 44 34 205 46 Flavobacterium genus 6 13 10 1 33 2 Empedobacter genus 1 0 0 0 2 3 Yeosuana genus 0 0 2 0 0 0 Ornithobacterium genus 0 0 0 0 5 1 Chryseobacterium genus 0 0 0 0 0 1 Unclassified 26 30 32 33 165 39 Unclassified 1 0 0 0 3 0 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Sphingobacteria class 143 362 326 126 607 326 Sphingobacteriales order 143 362 326 126 607 326 Rhodotermaceae family 85 111 87 95 162 113 Salisaeta genus 1 0 2 5 5 5 Rhodothermus genus 6 2 1 5 3 0 Unclassified 78 109 84 85 154 108 Chitinophagaceae family 36 180 164 18 253 151 Flavitalea genus 9 57 59 10 65 38 Filimonas genus 0 1 3 0 1 5 Gracilimonas genus 0 0 0 1 0 0 Unclassified 27 122 102 7 187 108 Sphingobacteriaceae family 9 12 17 2 94 9 Sphingobacterium genus 3 1 8 0 19 2 Parapedobacter genus 1 3 0 0 9 1 Pseudosphingobacterium genus 0 0 0 0 2 0 Pedobacter genus 0 0 0 0 0 1 Unclassified 5 8 9 2 64 5 Cytophagaceae family 4 24 8 1 22 19 Persicitalea genus 0 1 0 0 1 0 Sporocytophaga genus 0 0 0 1 0 6 Unclassified 4 23 8 0 21 13 Flammeovirgaceae family 0 1 1 0 1 5 Unclassified 0 1 1 0 1 5 Cyclobacteriaceae family 0 0 6 0 21 3 Aquiflexum genus 0 0 1 0 0 0 Unclassified 0 0 5 0 21 3 Unclassified 9 34 43 10 54 26 Bacteriodia class 45 42 57 32 68 203 Bacteroidales order 45 42 57 32 68 203 Bacteroidaceae family 41 3 1 22 11 18 Acetomicrobium genus 41 3 1 22 11 16 Bacteroides genus 0 0 0 0 0 2 Marinilabiaceae family 3 27 38 5 31 122 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Anaerophaga genus 2 17 26 2 18 93 Alkaliflexus genus 0 2 2 0 4 7 Unclassified 1 8 10 3 9 22 Porphyromonadaceae family 0 4 15 1 22 55 Petrimonas genus 0 0 6 1 15 13 Proteiniphilum genus 0 0 1 0 1 2 Parabacteroides genus 0 0 1 0 2 6 Tannerella genus 0 0 0 0 0 2 Unclassified 0 4 7 0 4 32 Prevotellaceae family 0 0 0 1 0 0 Prevotella genus 0 0 0 1 0 0 Unclassified 1 8 3 3 4 8 "Bacteroidetes"_incertae_sedi s class 1 49 88 0 2 95 Ohtaekwangia genus 1 49 87 0 2 95 Unclassified 0 0 1 0 0 0 Unclassified 532 215 161 383 305 350 Unclassified 7 54 58 5 69 86 Planctomycetes phylum 98 484 446 72 210 415 Phycisphaerae class 44 360 292 19 39 281 Phycisphaerales order 44 360 292 19 39 281 Phycisphaeraceae family 44 360 292 19 39 281 Phycisphaera genus 44 360 292 19 39 281 Planctomycetacia class 53 118 141 53 167 126 Planctomycetales order 53 118 141 53 167 126 Planctomycetaceae family 53 118 141 53 167 126 Planctomyces genus 23 34 27 20 82 52 Rhodopirellula genus 5 13 16 1 6 5 Blastopirellula genus 1 1 1 0 0 0 Singulisphaera genus 0 2 3 0 0 0 Unclassified 24 68 94 32 79 69 Unclassified 1 6 13 0 4 8 Verrucomicrobia phylum 4 30 29 6 40 26 Opitutae class 1 9 4 2 3 2 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Puniceicoccales order 0 2 1 1 0 1 Puniceicoccaceae family 0 2 1 1 0 1 Pelagicoccus genus 0 0 1 0 0 0 Unclassified 0 2 0 1 0 1 Unclassified 1 7 3 1 3 1 Verrucomicrobiae class 1 9 5 1 8 4 Verrucomicrobiales order 1 9 5 1 8 4 Verrucomicrobiaceae family 1 9 5 1 8 4 Verrucomicrobium genus 1 6 4 0 5 0 Unclassified 0 3 1 1 3 4 Subdivision3 class 2 5 10 2 15 3 Subdivision3_genera_incertae_ sedis genus 2 5 10 2 15 3 Spartobacteria class 0 5 7 0 5 12 Xiphinematobacter genus 0 1 1 0 0 0 Spartobacteria_genera_incerta e_sedis genus 0 1 0 0 2 4 Unclassified 0 3 6 0 3 8 Unclassified 0 2 3 1 9 5 TM7 phylum 1 4 15 1 4 6 TM7_genera_incertae_sedis genus 1 4 15 1 4 6 BRC1 phylum 3 44 24 10 13 16 BRC1_genera_incertae_sedis genus 3 44 24 10 13 16 Gemmatimonadetes phylum 3 5 7 1 0 1 Gemmatimonadetes class 3 5 7 1 0 1 Gemmatimonadales order 3 5 7 1 0 1 Gemmatimonadaceae family 3 5 7 1 0 1 Gemmatimonas genus 3 5 7 1 0 1 Acidobacteria phylum 31 90 36 48 65 75 Acidobacteria_Gp6 class 31 90 36 48 64 70 Gp6 genus 31 90 36 48 64 70 Gp16 class 0 0 0 0 1 0 Gp16 genus 0 0 0 0 1 0 Gp4 class 0 0 0 0 0 4 Gp4 genus 0 0 0 0 0 4 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).

TOTAL Rank Ctl 1 Ctl 2 Ctl 3 LC 1 LC 2 LC 3 Unclassified 0 0 0 0 0 1 Spirochaetes phylum 0 2 3 0 0 12 Spirochaetes class 0 2 3 0 0 12 Spirochaetales order 0 2 3 0 0 12 Leptospiraceae family 0 2 3 0 0 12 Leptonema genus 0 2 3 0 0 12 Tenericutes phylum 0 2 8 1 2 31 Mollicutes class 0 2 8 1 2 31 Haloplasmatales order 0 1 1 1 0 7 Haloplasmataceae family 0 1 1 1 0 7 Haloplasma genus 0 1 1 1 0 7 Acholeplasmatales order 0 1 7 0 2 24 Acholeplasmataceae family 0 1 7 0 2 24 Acholeplasma genus 0 1 7 0 2 24 Fusobacteria phylum 0 1 0 0 0 0 Fusobacteria class 0 1 0 0 0 0 Fusobacteriales order 0 1 0 0 0 0 Fusobacteriaceae family 0 1 0 0 0 0 Fusobacterium genus 0 1 0 0 0 0 Unclassified 2544 3588 2437 2932 3433 4816 Unclassified 3 20 11 14 23 23 Classification of amplifiable bacteria detected in A. bisporus substrate at spawning for 3 crops (1,2,3) with and without 20% SMC dwt (80LC, Ctl).