A Two-Phase Solid-State Fermentation Process for Mushroom (Agaricus bisporus) Production on Cereal Grains

M. A. Bechara, P. H. Heinemann, P. N. Walker, C. P. Romaine*

ABSTRACT. Grain-based substrates subjected to a two-stage solid-state fermentation (SSF) process for Agaricus bisporus mushroom production were tested as alternatives to the environmentally problematic compost-based substrate. Scytalidium thermophilum, the dominant thermophilic fungal species found in compost-based substrates, was used in the primary stage of the SSF process to pre-treat grain-based substrates, which was followed by inoculation with A. bisporus in the secondary stage. The objective of this study was to determine the effect of the two-stage SSF process on the substrate colonization period and mushroom productivity for A. bisporus and substrate bioefficiency using various grain-based substrates. To this end, three sterilized substrates composed of grain (rye, millet, and oat) and other minor ingredients were used as the basal nutrient substrates. The first experiment varied the incubation duration (0, 10, and 20 days) with S. thermophilum at 46C and grain type in the primary stage of the SSF process. The results indicated that incubation period, but not grain type, had a significant effect on mushroom yield and substrate bioefficiency (p < 0.05). Yield decreased significantly beyond the 10-day incubation period with S. thermophilum compared to that of the control treatment without S. thermophilum. Use of shorter primary stages with S. thermophilum (0, 2, 4, and 6 days) suggested that both incubation duration and the interaction of grain type and incubation duration were significant for mushroom yield and substrate bioefficiency (p < 0.05). The substrate colonization periods for A. bisporus using substrates pre-colonized by S. thermophilum were shorter (22 to 23 days) when compared to control treatments (44 to 50 days) without S. thermophilum pretreatment (p < 0.05). In conclusion, when using grainbased substrates for mushroom production, the addition of S. thermophilum in the primary stage of the SSF process shortened the time for substrate colonization by A. bisporus in the secondary stage. Further, the two-stage SSF process described herein increased mushroom yield when using an oat grain-based substrate. Keywords. Fungi, Scytalidium thermophilum, Solid-state fermentation, Thermophilic fermentation.

Submitted for review in February 2011 as manuscript number BET 9073; approved for publication by the Biological Engineering Editorial Board of ASABE in October 2011. The authors are Mark A. Bechara, ASABE Student Member, Operations Manager and Senior Research Scientist, Sylvan America, Kittanning, Pennsylvania, and former graduate student; Paul H. Heinemann, ASABE Member, Professor and Head, Paul N. Walker, ASABE Fellow, Professor Emeritus, and C. Peter Romaine, Professor, Department of Agricultural and Biological Engineering, Pennsylvania State University, University Park, Pennsylvania. Corresponding author: Paul H. Heinemann, Department of Agricultural and Biological Engineering, Pennsylvania State University, 250 Agricultural Engineering Building, University Park, PA 16802; phone: 814-865-2633; e-mail: hzh@psu.edu.
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roduction of the widely cultivated button mushroom (Agaricus bisporus) on partially composted substrates, a solid-state fermentation (SSF) process, is a mainstay in the industry. Although there are several variations in the substrate preparation process such as the long, Institut National de la Recherche Agronomique (INRA), and Anglo Dutch methods (Rai, 2004), the most widely used process in Pennsylvania was developed by Sinden and Hauser (1950) and is called the short method. The short method consists of two phases. In the first phase (phase I), raw ingredients (horse manure, chicken manure, hay, straw, gypsum, and others) are mixed, wetted, and placed in rows outdoors for uncontrolled self-heating, taking 7 to 14 days for completion. The materials are then transferred to temperature-controlled chambers in which the partially composted material is pasteurized to inactivate mushroom pathogens and then brought down to room temperature after being maintained at 45C to 50C for about 5 to 7 days. This concludes the second phase (phase II) of the substrate preparation step, after which the partially composted material, termed mushroom substrate, is ready for inoculation with mushroom fungus-colonized grain (spawning). Rye and millet are the most widely used grains for commercial mushroom spawn. The spawn is mixed with the mushroom substrate and incubated for 14 to 16 days, during which the time the mushroom fungus completely colonizes the composted nutrient substrate (spawn run). Although widely used, the traditional substrate preparation process has come under scrutiny due to the production of odors, nutrient-rich runoff, and leachate. Furthermore, the amount of spent mushroom substrate generated in some areas is too large for land disposal. Non-composted substrates have been shown to support mushroom production without using the environmentally problematic composted substrate, but mushroom yield has been consistently low (Till, 1962; Huhnke and Von Sengbush, 1968; Sanchez and Royse, 2001; Mamiro et al., 2007). Therefore, understanding some of the dynamics of the traditional mushroom substrate preparation process, especially the phase II portion, could help in the development of non-composted substrates with improved mushroom yield. During phase II, the dominant naturally occurring thermophilic species is a fungus, Scytalidium thermophilum (Straatsma et al., 1994). Artificially inoculating partially composted substrate with additional S. thermophilum biomass increased mushroom yield two-fold when compared to controls without inoculation (Straatsma et al., 1994). This observation was attributed to the role of thermophilic microflora in dominating the substrate at spawning and creating a selective niche for A. bisporus growth. Ross and Harris (1983) showed that heat and antimicrobial chemicals (alcohol and chloroform) destroy the selectivity of partially composted substrate by killing the thermophilic microflora. They went on to explain that the reduction in temperature of mushroom substrate after phase II and just before spawning produced static conditions (dormant) and not killing conditions in which the thermophilic microflora remained viable, ensuring the selectivity of the substrate for A. bisporus. In addition to forming a selective substrate, the thermophilic biomass could be a source of CO2, which has a stimulatory effect on A. bisporus growth. Wiegant et al., (1992) showed that a significant lag in mushroom fungus biomass formation was due to low CO2 concentrations in the sterile compost compared to non-sterile compost. However, the addition of S. thermophilum to the sterilized material either preincubated or at the time of spawning led to the same mycelium extension rate, with the latter characterized by a lag phase. A similar effect on mycelium extension rates was
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shown when CO2 concentrations were augmented from 0% to 0.5% (Wiegant et al., 1992) and up to 1% (San Antonio and Thomas, 1972). The explanation behind the stimulating effect of CO2 was described as an ecological effect, whereby the mushroom fungus senses the growth of other fungi and tries to colonize the substrate as rapidly as possible to ensure its survival (Wiegant et al., 1992). However, the more probable reason for CO2 stimulation is its role in anaplerotic reactions in which carbon fixation occurs for the formation of intermediate compounds in the tricarboxylic acid cycle and in the formation of purines, pyrimidines, and fatty acids (Maheshwari et al., 2000; Carlile et al., 2001). The major contribution of CO2 at cultivation temperatures of 24C was shown to be mainly due to the growth of the thermophilic fungi and less to the prokaryotic microflora (Wiegant et al., 1992). Wiegant et al. (1992) stated that although high CO2 concentrations stimulated extension rates, the growth-promoting effect of S. thermophilum either by secreting stimulus compounds or vital nutrients cannot be ruled out. It is noteworthy to mention that S. thermophilum produces large quantities of cellulases and xylanases (Zanoelo et al., 2004a, 2004b), and this could aid in substrate pre-conditioning. Bilay (2000) pre-incubated sterilized grain with Humicola insolens (S. thermophilum) and then inoculated the colonized grains with A. bisporus to form what was called experimental grain spawn (EGS). Bilay (2000) stated that the advantage of using EGS is in reducing the problem of mold contamination. San Antonio (1971) showed that A. bisporus mushroom production was possible on a substrate composed primarily of rye grain, but with a low yield. Bechara et al. (2006) showed that adequate mushroom production can be achieved by using a sterilized substrate composed of millet grain and various oilseeds, yielding up to 15.9 kg m-2 for a 2 cm layer of grain. However, this was still lower than the 30 kg m-2 obtained with a conventional compost-based substrate. Moreover, a 32-day spawn run was needed, which is considerably longer than the 14-day period typically used for composted substrates. Given the prolonged duration of spawn run and lower mushroom yield, an area worth pursuing is testing whether performance of sterilized cereal grain-based substrates would benefit from a thermophilic phase as encountered in traditional mushroom substrate preparation. The objective of this is study was to determine if a twostage SSF process starting with a thermophilic stage using a culture of S. thermophilum followed by inoculation with A. bisporus could effectively decrease spawn-run duration, and increase mushroom yield and substrate bioefficiency in cereal grainbased substrates.

Methods and Materials

Fungal Cultures Substrate Materials Rye grain spawn cultures of S. thermophilum (DC 295) and A. bisporus (MC 459) were obtained from the Mushroom Spawn Lab at Pennsylvania State University. Scytalidium thermophilum inoculum was stored at 37C, and A. bisporus was stored at 5C. The grain-based substrates were composed of 608 g of either oat, millet, or rye (moisture content = 60% wet basis), 40 g of cracked roasted soybean (Agway, Pleasant Gap, Pa.), 10 g of CaCO3 and 10 g of CaSO4 (both from Fisher Scientific, Hampton, N.H.), 15 g of vitamin and mineral supplement (DAC, Dover, Ohio), and either

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2000 or 500 mL of perlite (Therm-o-Rock, New Eagle Pa.), a water-holding material. Perlite was also added to the mushroom production trays (Bechara et al., 2008). Preparation of Grain Substrates Grains and the appropriate amount of water to achieve a final moisture content of 0.6 (kg kg-1, wet basis) were added to polypropylene containers (L = 0.37 m W = 0.25 m D = 0.14 m). The filled containers were covered with a double layer of aluminum foil, autoclaved for 45 min, and allowed to cool to room temperature. The grain substrate components were then mixed and added to autoclavable polypropylene bags (type 10, Unicorn Imp. and Mfg. Corp., Commerce, Tex.) containing either 2000 or 500 mL of perlite. The filled bags were then autoclaved for 3 h and allowed to cool in a laminar-flow hood. Once cooled, the bags were aseptically inoculated with 20 g of rye grain spawn (S. thermophilum and/or A. bisporus). Substrates inoculated with S. thermophilum were incubated at 46C for the treatment incubation duration and then inoculated with A. bisporus after cooling to room temperature. Substrates inoculated with either A. bisporus or a co-culture of A. bisporus and S. thermophilum (0 day treatment) were incubated at 22C. Once fully colonized, the mushroom substrates were transferred to mushroom production trays. Preparation of Mushroom Production Trays Polypropylene trays (L = 0.3 m, W = 0.16 m, D = 0.09 m) were used for mushroom production. Perlite was wetted, and each mushroom tray received 2000 mL of perlite. The trays were then autoclaved for 45 min and allowed to cool. Mushroom casing (lime-neutralized peat moss) was obtained in dried form from the Mushroom Research Center at Pennsylvania State University. The casing was wetted (7000 mL of casing + 5000 mL of tap water), supplemented with 10% (v/v) activated carbon (Fisher Scientific, Hampton, N.H.), and autoclaved for 2 h. The contents of each bag were added to the mushroom production trays, and treatments receiving delayed-release supplement, a class of nitrogenous compounds that mushroom growers use to increase mushroom yield, were amended with 40 g of Promycel Target (SpawnMate, Watsonville, Cal.). The sterilized casing (limed peat moss with activated carbon) was then added over the substrate (1200 g per tray). Mushroom production was carried out in a mushroom production chamber. Environmental Conditions in Mushroom Production Rooms Filled and cased mushroom production trays were placed in a tray environmental chamber (2.54 m 1.32 m). Temperature was maintained using a commercial air conditioning unit. The chamber contained three indoor greenhouses (Midwest Quality Glove, Inc., Chillicothe, Mo.), which consisted of metal structures with four shelves that could hold up to five trays, enclosed within a polyethylene cover. Slits measuring 9 cm were made in the polyethylene covers to improve ventilation. Two slits were made on the top of the chambers, and the rest were made on the front side. The temperature was maintained at 22C until mushroom mycelium appeared on the casing surface in 50% of trays. The temperature was then dropped to 16C and maintained until mushroom harvesting was completed.

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Table 1. Treatments with different high-temperature incubation periods without Promycel Target.[a] Primary-Stage Incubation at Secondary-Stage Promycel Treatment[b] Inoculation 46C (days) Inoculation Target (g) Control A. bisporus 0 -0 0 days A. bisporus + S. thermophilum 0 -0 10 days S. thermophilum 10 A. bisporus 0 20 days S. thermophilum 20 A. bisporus 0 [a] Each bag contained 2000 mL of perlite and 835 g of substrate. [b] For each incubation treatment, three types of grain (rye, millet, and oats) were used. Table 2. Treatments with shorter high-temperature incubation periods and supplemented with delayed-release Promycel Target.[a] Primary-Stage Incubation at Secondary-Stage Treatment[b] Inoculation 46C (days) Inoculation Control A. bisporus 0 -0 days A. bisporus + S. thermophilum 0 -2 days S. thermophilum 2 A. bisporus 4 days S. thermophilum 4 A. bisporus 6 days S. thermophilum 6 A. bisporus [a] Each bag contained 500 mL of perlite with 835 g of substrate. [b] For each incubation treatment, the three types of grain (rye, millet, and oats) were used.

Promycel Target (g) 40 40 40 40 40

Experimental Design and Analysis of Data Two experiments were undertaken. The treatments of the first experiment were made based on a randomized complete block design and tested two factors: incubation duration at 46C for 0, 10, and 20 days; and type of grain used (rye, millet, and oat). The treatments were compared to a control group without added S. thermophilum. Table 1 provides a summary of the first experimental setup. The second experiment was comprised of treatments executed in a completely randomized design and tested the same factors, but with shorter incubation durations. Table 2 summarizes the treatments for the second experiment. All treatments for both experiments were replicated three times, and mushrooms were harvested over a period of 26 days. The response variables used were mushroom yield, spawn-run duration, average mushroom size, and substrate bioefficiency. Spawn-run duration is defined as the number of days required to fully colonize the substrate (grains fully ramified with mycelium) with A. bisporus and ready for transfer to mushroom production trays. Average mushroom size was calculated by dividing the total mushroom yield (fresh weight) by the number of mushrooms picked, whereas substrate bioefficiency was calculated by dividing the total mushroom yield (fresh weight) by the oven-dry weight of the substrate components. Perlite was not counted as part of the substrate. The response variables were statistically analyzed using the General Linear Model ( = 0.05) with MINITAB statistical software (release 13.1, State College, Pa.). All pairwise comparisons of treatments were made using the Tukey method. Oxygen Uptake Rate Evaluation Oxygen uptake rates (OUR) of S. thermophilum and A. bisporus incubated separately at 16C, 24C, and 32C were measured using the Oxitop system (WTW, Giessen, Germany). The system is composed of a 1.14 L glass bottle that is partially filled with a sample, a bottle-sealing cap composed of a receptacle for a CO2 absorbent (typically NaOH), and a pressure sensor that transmits the enclosed bottle pressure
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reading to a controller. The OUR can be calculated based on the pressure drop in the apparatus. The pressure drop is assumed to be induced by the oxygen consumption of the living biomass. For this study, a 50 g sample of rye grain pre-colonized with either A. bisporus or S. thermophilum was added to the Oxitop system. The bottles and receptacles were sterilized with the exception of the pressure sensor head. Two grams of concentrated NaOH pellets were added to the CO2 absorbent receptacle, and the bottle cap was sealed with the pressure head. The treatment duration was 48 h. Dry matter content was measured using 10 g samples of the grain spawn subjected to 105C in an oven for 24 h. Pressure drop data were converted into OUR as shown in equation 1 (Sadaka et al., 2004):
mg O 2 OUR g DM day = min mg g hPa Pa slope 100 V gas 32 moleO 1000 g 1440 day min hPa 2 J 8.314 T W (1 MC ) mole K

(1)

where slope = slope of the pressure drop curve Vgas = volume of gas in Oxitop vessel (assumed total volume of flask, m3) T = temperature (K) W = weight of sample (g) MC = moisture content wet basis.

Results
Experiment with Longer Incubation Durations with S. thermophilum Table 3 provides a summary of results for the 0-, 10-, and 20-day primary stage SSF incubation durations. For the first trials with S. thermophilum added, the only significant effect on mushroom yield and substrate bioefficiency was incubation duration (p < 0.05) (table 3). Yields for the different grain-based substrates were not significantly different (p > 0.05), nor was the interaction effect of grain incubation duration (p > 0.05). Overall, yields for the 0- and 10-day durations were not significantly different from the control group. However, yield was significantly lower for the 20-day incubation duration. Additionally, some treatments of the 20-day incubation duration failed to grow; the grain spawn when added to the bags was inactive for weeks, and mushrooms were not produced when the treatments were transferred to trays. Although data are not reported, an observed decrease in spawn-run duration was detected in treatments inoculated with S. thermophilum compared to the control. Furthermore, average mushroom yield was lower for the 10-day incubation duration compared to the control and 0-day incubation duration. Therefore, it was decided to test shorter incubation durations. The highest yield was for the 0-day rye treatment, producing 19.4 kg m-2 with a corresponding biological efficiency (BE) of 249.3%. For average mushroom size, none of the factors or interaction effects were significant (p > 0.05).

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[a]

[b]

Table 3. Preliminary treatments involving treatment of grain-based substrate with S. thermophilum in the primary stage of the SSF process.[a] Yield Biological Efficiency Average Mushroom Treatment (kg m-2) (%) Size (g) Control - rye 16.6 8.1 a 213 104 a 16.2 2.4 a Control - millet 15.2 2.5 abc 195 33 abc 20.9 4.9 a Control - oats 15.4 1.5 abc 197 19 abc 18.7 1.7 a 0 days - rye 19.4 6.3 a 249 81 a 21.1 5.4 a 0 days - millet 16.2 6.5 ab 208 83 ab 18.6 1.0 a 0 days - oat 18.6 2.9 a 238 37 a 17.7 1.0 a 10 days - rye 9.3 4.1 abcd 119 53 abcd 15.5 0.6 a 10 days - millet 10.2 4.9 abcd 131 64 abcd 17.6 3.7 a 10 days - oat 8.5 4.5 abcd 109 58 abcd 15.3 1.7 a 20 days - rye[b] 2.8 2.9 cd 36 37 cd 15.8 16.4 a 20 days - millet[b] 3.4 5.4 cd 44 69 cd 32.4 17.4 a 20 days - oat[b] 1.5 1.5 d 19 19 d 4.1 7.0 a Values are means standard deviations. Treatments followed by the same letter are not significantly different (p < 0.05). Observed data are for 26 days of harvesting. Treatments have large standard deviations because one replicate failed to yield any mushrooms. Table 4. Time for colonization of various grain-based substrates by A. bisporus in the secondary phase of the SSF process as influenced by duration of the primary stage with S. thermophilum. Spawn-Run Duration[a] Total Duration[b] Treatment (days) (days) Control - rye 49 49 2 a Control - millet 44 44 8 ab Control - oats 50 50 4 a 0 days - rye 48 48 0 a 0 days - millet 28 28 2 bcd 0 days - oat 39 39 8 abcd 2 days - rye 32 30 5 bcd 2 days - millet 40 38 9 abcd 2 days - oat 37 35 12 abcd 4 days - rye 31 27 4 bcd 4 days - millet 28 24 2 d 4 days - oat 30 26 5 cd 6 days - rye 28 22 2 d 6 days - millet 29 23 2 d 6 days - oat 28 22 2 d Values are means standard deviations. Treatments followed by the same letter are not significantly different (p > 0.05). Total duration includes the days of incubation at 46C and the spawn-run period.

[a]

[b]

Experiment with Shorter Incubation Durations with S. thermophilum Table 4 summarizes the spawn run duration for all the treatments. Primary stage SSF incubation with S. thermophilum reduced the spawn-run duration from 4450 days for the control treatments to 22-23 days for the 6-day incubation duration: the longer the thermophilic incubation period, the shorter the spawn-run duration. The shortest spawn-run period was observed for treatments with the 6-day incubation period with S. thermophilum, whereas the longest period was observed for the control treatment without adding S. thermophilum.

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[a]

Table 5. Influence of duration of the primary-stage with S. thermophilum and grain type on A. bisporus mushroom yield and size and substrate bioefficiency.[a] Yield Bioefficiency Average Mushroom Treatment (kg m-2) (%) Size (g) Control - rye 13.9 4.9 b 179 63 b 27.7 12.5 ab Control - millet 21.3 0.6 a 273 8 a 53.5 8.5 ab Control - oat 11.7 3.0 b 151 39 b 25.0 15.5 b 0 days - rye 15.6 2.4 ab 200 31 ab 28.7 3.8 ab 0 days - millet 13.1 2.3 b 168 30 b 27.7 10.2 ab 0 days - oat 15.6 1.1 ab 200 14 ab 44.3 3.1 ab 2 days - rye 15.2 1.6 ab 195 20 ab 37.7 10.9 ab 2 days - millet 15.9 1.1 ab 205 14 ab 36 6 ab 2 days - oat 18.2 0.2 ab 233 3 ab 59.7 6.5 a 4 days - rye 13.1 1.2 b 168 16 b 31.3 9.7 ab 4 days - millet 14.2 2.6 ab 183 33 ab 45 25.2 ab 4 days - oat 15.8 4.6 ab 202 59 ab 35.7 17.6 ab 6 days - rye 13.5 1.6 b 173.4 20.2 b 32 4.6 ab 6 days - millet 15.0 3.3 b 192 43 b 42.7 3.8 ab 6 days - oat 11.2 2.0 b 144 26 b 21 8.7 b Values are means standard deviations. Treatments followed by the same letter are not significantly different (p < 0.05). Data reflect a 26-day harvest period.

Table 5 summarizes mushroom yield. The interaction effect (grain type incubation duration) and incubation duration were the only significant terms for mushroom yield (p < 0.05). Grain type as the main effect was not significant (p > 0.05). Yield for the oat-based substrate resembled a bell-shaped curve that peaked at the 2-day incubation duration. The rye substrate did not have a clear peak in yield, but the highest yield was for the 0-day incubation duration. Finally, for the millet substrate, the highest yield was observed for the control group and then decreased for all other incubation durations. The highest mushroom yield was observed for the millet control treatment, producing 21.3 kg m-2 (BE = 273%). BEs followed the same trend as that for yield. Grain type and incubation duration did not significantly influence average mushroom size (p > 0.05). However, the interaction of grain type incubation duration was significant (p < 0.05). While average mushroom size increased with incubation period for oat and rye, it decreased for millet. The highest observed average mushroom size was for the millet control treatment (53.5 g), and the lowest was for the 6-day oat treatment (21 g). Oxygen Uptake Rate Evaluation Figure 1 depicts the general trend of OUR for each fungal species. The OURs of A. bisporus and S. thermophilum were measured and the observed trend was anticipated, whereby a steady increase in OUR was observed for both fungal species until 32C. At 32C, the OUR of S. thermophilum continued to increase, whereas that of A. bisporus decreased because it approached the upper temperature limit of growth.

Discussion
A two-stage solid-state fermentation using co-inoculation with A. bisporus and S. thermophilum of non-composted grain-based substrate for A. bisporus mushroom production was shown to be useful in decreasing spawn-run duration for all grain-

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Figure 1. OUR measurements for A. bisporus and S. thermophilum.

based substrates, whereas yield and substrate bioefficiency improved for some grainbased substrates. This research represents the first report of testing three different grain types (rye, millet, and oat) for mushroom production with the addition of S. thermophilum as a pre-conditioning agent for these grain substrates. Earlier, our work showed that grain-based substrates (either commercial rye and millet spawn or non-commercial millet supplemented with oilseeds) supported high mushroom yields (16.9 kg m-2), but comparatively lower than the typical 30 kg m-2 obtained with conventional composted substrates (Bechara et al., 2005, 2006). However, substrate bioefficiencies for the grain-based substrates were markedly higher than for compost-based substrates (Bechara et al., 2006) because on a wet weight comparison, grain-based substrates have more dry matter (more nutrition) than compost based substrates. In effect, adopting a grain-based mushroom production system with the current conditions would require approximately 75 days from start to finish compared to 70 days for compost-based mushroom production. Spawn-run durations for the grainbased substrates, as shown in this study, are considerably longer (44 to 50 days; table 4) compared to composted substrate (14 days). However, using a grain system would reduce the volume of substrate needed due to higher bioefficiencies. Therefore, pre-incubating the grain-based substrate with S. thermophilum is a viable solution to reduce spawn-run duration and ultimately shorten the total mushroom production process for grain-based substrates. Other methods of reducing spawn-run duration are increasing the inoculum amount per unit mass of substrate and increasing the inoculation points by using smaller particles, such as millet grain spawn, as have been used in traditional compost-based mushroom production (Mamiro, 2007). In addition, there was no difference in yield between the three grains used herein. Hence, a commercial producer could use the lowest-cost grain for mushroom production. An important factor that was observed to influence growth of A. bisporus in substrate pre-incubated with S. thermophilum was the addition of perlite to the substrate. Agaricus bisporus failed to colonize the pre-incubated substrates when perlite was absent because condensate accumulated at the bottom of the bag, leading to a loss of structure and increased substrate stickiness. The amount of perlite was reduced in the
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second experiment (500 mL) because the 2000 mL amendment in the first experiment was excessive, making it difficult to pack the entire substrate in the mushroom production containers. This was not apparent for control treatments lacking S. thermophilum but was observed in the 0-, 2-, 4-, 6-, 10-, and 20-day treatments. Otherwise, S. thermophilum, although a thermophile, appeared to grow adequately at both 24C and 16C, indicating that this fungus actually remains viable, and the CO2 output from the thermophilic biomass at each temperature is a contributing factor for the reduction of spawn-run duration, as many other studies have indicated. The difference in OUR trends at 32C for A. bisporus and S. thermophilum was due to the fact that A. bisporus had reached its upper temperature limit for growth. Hence, adding S. thermophilum to the sterilized grain-based substrate without the high-temperature incubation period could prove to be as effective as the 2-, 4-, and 6-day elevatedtemperature incubation periods. Additionally, there are energy savings for the former compared to the latter. Additional research is needed to optimize grain-based substrate composition to further increase yield and bioefficiency.

Conclusions
Mushroom yield, substrate bioefficiency, and spawn-run duration in noncomposted grain-based substrates were enhanced to varying degrees with the addition of S. thermophilum as a primary stage SSF inoculant. Oat, millet, and rye grain-based substrates were all shown to support mushroom yields (17.9, 21.3, and 19.4 kg m-2, respectively) with high substrate bioefficiencies. One important observation is the decrease in spawn-run duration associated with a pre-treatment of the substrates with S. thermophilum (incubation treatments of 6 days or less) compared to controls without S. thermophilum, which served to significantly shorten the overall production cycle. Otherwise, the effect of S. thermophilum was dependent on the type of grain type. Oat-based substrates benefited the most from the addition of S. thermophilum by way of increased mushroom yield and substrate bioefficiency, whereas rye- and milletbased substrates were unaffected. In summary, the introduction of S. thermophilum in the primary stage of a two-stage SSF process using certain grain-based substrates shortened the spawn-run period and increased mushroom yield. Acknowledgements The authors would like to acknowledge the assistance of John Pecchia, general manager, and the staff of the Mushroom Research Center at Pennsylvania State University. Funding was provided by the College of Agricultural Sciences and the Department of Agricultural and Biological Engineering, Pennsylvania State University.

References
Bechara, M. A., P. Heinemann, P. N. Walker, and C. P. Romaine. 2005. Cultivation of Agaricus bisporus on a mixture of cereal grain spawn and delayed-release nutrient supplement. Mushroom News 53(8): 6-10. Bechara, M. A., P. H. Heineman, P. N. Walker, and C. P. Romaine. 2006. Evaluating noncomposted grain substrates for the production of Agaricus bisporus and Agaricus blazei mushrooms. ASABE Paper No. 067089. St. Joseph, Mich.: ASABE.

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Bechara, M. A., P. Heinemann, P. N. Walker, and C. P. Romaine. 2008. Enhanced production of Agaricus bisporus using non-composted substrate in the presence of delayed-release supplements and control agents for water release. Biol. Eng. Trans. 1(3): 245-253. Bilay, V. T. 2000. Growth of Agaricus bisporus on grain pre-colonized by Humicola insolens and growth of mushroom mycelium from this spawn on compost. In Proc. 15th Intl. Congress on the Science and Cultivation of Edible Fungi, 425-429. International Society for Mushroom Science. Carlile, M. J., S. C. Watkinson, and G. W. Gooday. 2001. The Fungi. 2nd ed. New York, N.Y.: Academic Press. Huhnke, W., and R. Von Sengbush. 1968. Mushroom cultivation on non-composted nutritive substrates. Mushroom Sci. 7: 405-409. Maheshwari, R., G. Bharadwaj, and M. K. Bhat. 2000. Thermophilic fungi: Their physiology and enzymes. Microbiol. and Molecular Biol. Reviews 64(3): 461-488. Mamiro, D. J. 2007. Non-composted and spent mushroom substrates for mushroom production of Agaricus bisporus. PhD diss. State College, Pa.: Penn State University, Department of Plant Pathology. Mamiro, D. P., D. J. Royse, and R. B. Beelman. 2007. Yield, size, and mushroom solids content of Agaricus bisporus produced on non-composted substrate and spent mushroom compost. World J. Microbiol. Biotech. 23(9): 1289-1296. Rai, R. D. 2004. Production of edible mushrooms In Fungal Biotechnology in Agricultural, Food, and Environmental Applications, 233-246. New York, N.Y.: Marcel Dekker. Ross, R. C., and P. J. Harris. 1983. An investigation into the selective nature of mushroom compost. Scientia Horticulturae 19(1-2): 55-64. Sadaka, S. S., T. L. Richard, T. D. Loecke, and M. Liebman. 2004. Determination of compost respiration rates using pressure sensors. ASAE Paper No. 047019. St. Joseph, Mich.: ASAE. San Antonio, J. P. 1971. A laboratory method to obtain fruit from cased grain spawn of the cultivated mushroom Agaricus bisporus. Mycologia 63: 16-21. San Antonio, J. P., and R. L. Thomas. 1972. Carbon dioxide stimulation of hyphal growth of the cultivated mushroom Agaricus bisporus (Lange) Sing. Mushroom Sci. 8: 623-629. Sanchez, J. E., and D. J. Royse. 2001. Adapting substrate formulas used for shiitake for production of brown Agaricus bisporus. Bioresource Tech. 77(1): 65-69. Sinden, J. W., and E. Hauser. 1950. The short method of mushroom composting. Mushroom Sci. 1: 52-59. Straatsma, G., T. W. Olinjnsma, J. P. G. Gerrits, J. P. M. Amsin, H. J. M. Op den Camp, and L. J. L. D. Van Griensven. 1994. Inoculation of Scytalidium thermophilum in button mushroom compost and its effect on yield. Applied and Environ. Microbiol. 60(9): 3049-3054. Till, O. 1962. Champignonkultur auf sterilisiertem nahrsubstrat und die wiederver wendung von abgetragenem compost. Mushroom Sci. 5: 127-133. Wiegant, W. M., J. Wery, E. T. Buittenhuis, and J. A. M. de Bont. 1992. Growth promoting effect of thermophilic fungi on the mycelium of the edible mushroom Agaricus bisporus. Applied and Environ. Microbiol. 58(8): 2654-2659. Zanoelo, F. F., M. L. T. M. Polizeli, H. F. Terenzi, and J. A. Jorge. 2004a. Purification and biochemical properties of a thermostable xylose-tolerant beta-D-xylosidase from Scytalidium thermophilum. J. Ind. Microbiol. Biotech. 31(4): 170-176. Zanoelo, F. F., M. L. T. M. Polizeli, H. F. Terenzi, and J. A. Jorge. 2004b. -glucosidase activity from the thermophilic fungus Scytalidiumn thermophilum is stimulated by glucose and xylose. FEMS Microbiology Letters 240(2): 137-143.

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