Open Access

Agaricus bisporus production on substrates pasteurized by self-heating

AMB Express20177:135

https://doi.org/10.1186/s13568-017-0438-6

Received: 6 May 2017

Accepted: 19 June 2017

Published: 24 June 2017

Abstract

The aim of this work was to determine if the self-heating pasteurization procedure is technically applicable to the cultivation of Agaricus bisporus. Firstly the substrates alone (corncob, Pangola grass and a mixture of both ingredients with wood shavings) were tested. Two supplementation trials were then undertaken using soybean, wheat bran, sheep manure, sesame seed, black bean and chia. Highest production values (BE = 176.3% and Y = 26.6 kg/m2) were obtained using a 9% supplement, with a formula consisting of 25% each of soybean, black bean, wheat bran and chia, added at spawning and at casing. These results were comparable to those obtained with the Phase II compost traditionally used for A. bisporus cultivation.

Keywords

Portobello Substrate preparation Button mushroom Mushroom cultivation

Introduction

Agaricus bisporus is the fourth mushroom species cultivated in the world, with 15% of global production (34 × 106 t edible mushrooms, Royse et al. 2016). The method usually used to cultivate the white button mushroom was described by Sinden and Hauser (1950) and improved through the years by a great number of research findings (Wuest 1982). The process involves two composting phases and is used worldwide because of excellent results. Phase I is a composting treatment requiring between 6 and 14 days, according to raw materials used. Phase I helps soften straw and other raw materials, break down soluble sugars and lower the C/N ratio. Biological (bacterial) and chemical (ammonia) activities increase during Phase I composting (Straatsma et al. 1995; Gerrits et al. 1995). This phase brings environmental challenges to growers because of odors and slurries (Mamiro et al. 2007; Beyer 2017). Phase II is a pasteurization process devoted to reduce competitor microbiota and give the substrate its biological selectivity. In Phase II, ammonia is reduced to levels that are non-toxic to A. bisporus (Laborde et al. 1993). Besides environmental problems, the Phase II technology for producing A. bisporus requires time, labor and investment (Miller et al. 1990). The loss of matter during composting is also an argument for the development of mushroom cultivation alternative methods.

Agaricus bisporus is considered a litter secondary decomposer, which means that bacteria and other fungi have to break down raw materials before the mushroom can grow, however Till (1962), demonstrated that A. bisporus could be cultivated on a non-composted substrate, like autoclaved sawdust. Since that time, several studies about cultivation (San Antonio 1971; Sanchez and Royse 2001; Bechara et al. 2005, etc.) and also on ligninolytic activity of this mushroom (Durrant et al. 1991; Wood and Leatham 1983) have placed A. bisporus as a basidiomycete able to degrade lignin and to grow on several raw, non-composted substrates.

Several alternative methods have been developed to grow A. bisporus. They consist in preparing a non-composted sterilized or pasteurized substrate at different temperatures, for avoiding the long composting traditional procedure (Till 1962; Mee 1978; San Antonio 1971; Sanchez and Royse 2001; Bechara et al. 2005, 2006; Coello-Castillo et al. 2009). However, none of them have been applied commercially.

A low input technology has been proposed to cultivate oyster mushrooms Pleurotus ostreatus (Villa-Cruz et al. 1999; Hernández et al. 2003; Barrios-Espinoza et al. 2009; Avendaño-Hernandez and Sánchez 2013). This technology is based upon the use of substrates pasteurized by self-heating. It consists on mixing raw materials with calcium hydroxide (2%) at controlled moisture levels and depositing the substrate in a wooden crate to allow heat to warm up the entire mass for pasteurization purposes. The entire process is accomplished within two days, and has been used successfully to cultivate strains of Pleurotus species like P. djamor, P. citrinopileatus and P. eryngii, and also other edible mushroom genera including Auricularia and Agrocybe (Morales and Sánchez 2017).

The aim of this work was to determine if the self-heating pasteurization procedure is technically applicable to A. bisporus cultivation, and if production yields are comparable to those obtained using the two-phase traditional method.

Materials and methods

Strains

For spawning, the commercial variety Portobello Heirloom of Agaricus bisporus was purchased from Amycel (San Miguel de Allende, Guanajuato, México). Inocula of one or five percent were used as indicated.

Substrates and supplements

Three cultivation substrates were tested using the following agricultural byproducts obtained from local markets: (1) Pangola grass (Digitaria decumbens) cut into 2–2.5 cm lengths, (2) corncob (Zea mays) cut into 1–2 cm lengths, and (3) a mixture of wood shavings of the primavera tree (Tabebuia rosea) (16.6%), with corncob (41.6%) and Pangola grass (41.6%). These substrates were combined with 2% hydrated lime Ca(OH)2 (Super Cal hidratada Grijalva), and the moisture was adjusted to 65% with tap water.

The following supplements were obtained locally: soybean (Glycine max), wheat bran, sheep manure, sesame seed (Sesamum indicum), chia (Salvia hispanica) and black bean (Phaseolus vulgaris). Supplements were ground (2 mm screen), autoclaved in high-density polyethylene bags at 1.05 kg/cm2 (121 °C) for 20 min and mixed into the substrate both at spawning and prior to casing (9% dry wt each time). Two trials of 13 and 16 treatments were conducted using single or combined supplements (Tables 1, 2).
Table 1

Treatments used in the first supplementation trial (four ingredients) for the cultivation of A. bisporus on self-heated pasteurized Pangola grass

Treatment

Supplement

Soybean

Sheep manure

Wheat bran

Sesame seed

1

0.125

0.125

0.625

0.125

2

0

1

0

0

3

0.333

0

0.333

0.333

4

0.333

0.333

0.333

0

5

0

0

0

1

6

0.25

0.25

0.25

0.25

7

0.125

0.125

0.125

0.625

8

0.125

0.625

0.125

0.125

9

0.625

0.125

0.125

0.125

10

0.333

0.333

0

0.333

11

1

0

0

0

12

0

0

1

0

13

0

0.333

0.333

0.333

Table 2

Treatments used in the second supplementation trial (five ingredients) for the cultivation of A. bisporus on self-heated pasteurized Pangola grass

Treatment

Supplement

Soybean

Sesame seed

Black beans

Wheat bran

Chia

1

0.1

0.6

0.1

0.1

0.1

2

0

1

0

0

0

3

0.1

0.1

0.1

0.1

0.6

4

0.25

0

0.25

0.25

0.25

5

0

0

0

1

0

6

0.25

0.25

0

0.25

0.25

7

0.25

0.25

0.25

0

0.25

8

0.1

0.1

0.6

0.1

0.1

9

0.6

0.1

0.1

0.1

0.1

10

0

0

0

0

1

11

0

0.25

0.25

0.25

0.25

12

1

0

0

0

0

13

0

0

1

0

0

14

0.2

0.2

0.2

0.2

0.2

15

0.25

0.25

0.25

0.25

0

16

0.1

0.1

0.1

0.6

0.1

Substrate preparation

Substrates were homogenized in a mixer MC-50 (Maquinaria Agropecuaria HML, Xalapa, Mex.), placed in a wooden crate measuring 1 m3, and then pasteurized by allowing the temperature to rise to 60–65 °C. After 30 h, it was removed, turned and placed back in the crate. The process was completed after 45 h by aerating the substrate to decrease the temperature (Sánchez et al. 2016; Morales and Sánchez 2017).

Two batches (20 kg each) of full Phase II mushroom compost, designated C1 and C2, were obtained from two mushroom companies located near Mexico City and Xalapa, Veracruz. The composts were prepared using the following ingredients: wheat straw, chicken manure, canola, urea an gypsum. Full Phase II mushroom compost two (C2) had the following composition: moisture content 69.2%, pH 7.6, Nitrogen 2.2%, C/N ratio 15–17, and ashes 25%.

Cultivation

Substrate and spawn (5% inoculation rate, unless indicated differently) were mixed manually in 1 kg portions and placed in transparent polyethylene bags. To allow gas exchange, the upper end of the plastic bag was fitted into a plastic cylinder (4 cm diameter) and the hole covered with a clean white paper towel. Incubation lasted 3 weeks at 24–26 °C and after the substrate was colonized by the mushroom mycelium, the bag was opened and a casing (peat: lime: water 1:1:3; no thermal treatment) overlay (4 cm deep) was applied. The substrate was further incubated at 18 °C and 90% relative humidity for three more weeks. Irrigation was applied daily. When used, supplements were mixed into the substrate at spawning and before applying the casing (9% each time). Mushrooms were harvested (three breaks) when the pileus was open and the veil broken. No attempts were made to control weed molds and diseases.

Treatments

To test the technical feasibility of A. bisporus cultivation on self-heated pasteurized substrates, three substrates without supplements were assayed initially. Two sets of supplementation treatments were then carried out, the first with four ingredients and 13 treatments, and the second with five ingredients and 16 treatments (Tables 1, 2). Supplements (on a dry weight basis) were added 9% at spawning and 9% at casing. Controls were two non-supplemented full phase composts (C1 and C2) using two spawning rates of 1 and 5% for C1, and only 5% for C2, as indicated.

Chemical analysis

To determine Carbon/Nitrogen (C/N) ratios, reducing sugar and lipid levels, 200 g samples were dried in a 65 °C oven for 5 days, ground into a fine powder and sent to the Bromatology Laboratory (Ecosur) for analysis. Carbon and nitrogen levels were determined using a Flash 2000 Analyzer (Thermo Fischer Scientific, http://www.thermoscientific.com/). Reducing sugars were measured using the 3,5 dinitrosalicylic acid (DNS) method (Miller 1959), and total lipids were determined according to Williams (1984).

Evaluated parameters

Biological efficiency (BE) values were computed using the following calculation: fresh mushroom weight divided by dry substrate weight and multiplied by 100. The production rate (PR) was calculated by dividing BE by the number of days needed to obtain three flushes. Mean size of mushrooms (MMS) was calculated by dividing the weight of mushrooms by the number of mushrooms harvested per bag. Yields were estimated by dividing the production of each bag by the horizontal area exposed to mushroom production and are expressed in kg/m2. The incidence of contamination was estimated visually: at the end of the spawn run, and after the first harvest, the percentage of contaminated area per each substrate bag was estimated. The result was expressed as an average.

Statistical analysis

A completely randomized design with five repetitions was used (Tables 3, 4, 5). To investigate the impact of the ingredients of the supplement mixtures on yield, a mixture design was used (Table 7). In each case, five repetitions were used and an analysis of variance and a mean separation was evaluated using Tukey’s test with a significance threshold of p < 0.05. Statistical analysis was carried out using JMP version 4 (SAS 2000).
Table 3

Production variables of Agaricus bisporus cultivated on three substrates pasteurized by self-heating

Substrate

Production (g/bag)

BE (%)

MMS (g)

PR (%)

Y (kg/m2)

Mixture

132.0 ± 56.0a

37.7 ± 16.0a

36.6 ± 5.1a

1.2 ± 0.5a

5.5 ± 2.3a

Corncob

65.0 ± 41.1b

18.5 ± 11.7b

35.0 ± 26.1a

0.6 ± 0.3b

2.7 ± 1.7b

Grass

181.2 ± 55.7a

51.7 ± 15.9a

31.25 ± 9.7a

1.7 ± 0.5a

7.6 ± 2.3a

p

0.000118

0.000118

0.9367

0.000118

0.000118

Same letter in the same column indicates no statistical difference between substrates (alpha = 0.05)

BE biological efficiency, MMS mean mushroom size (g), PR production rate (%), Y yield

Table 4

Production variables of A. bisporus cultivated on Pangola grass supplemented (9%) with 13 different mixtures at spawning and casing

Treatment

Supplement

Production variables

Soy bean

Sheep manure

Wheat bran

Sesame seed

Production (g/1 kg wet substrate)

BE (%)

MMS (g)

PR (%)

Y (kg/m2)

C11%

531.2 ± 202.6ab

151.7 ± 57.9ab

54.2 ± 20.6b

5.0 ± 1.9ab

22.4 ± 8.5ab

C15%

623.5 ± 56.7a

178.1 ± 16.2a

43.1 ± 10.8b

5.9 ± 0.5a

26.3 ± 2.3a

0

240.6 ± 58.6d

68.7 ± 16.7d

45.2 ± 12.0b

2.2 ± 0.5d

10.1 ± 2.4d

1

0.125

0.125

0.625

0.125

540.2 ± 106.8ab

154 ± 30ab

50.0 ± 12.4b

5.1 ± 1.0ab

22.7 ± 4.5ab

2

0

1

0

0

393.2 ± 151.8bcd

112.3 ± 43.3bcd

41.4 ± 9.8b

3.7 ± 1.4bcd

16.6 ± 6.4bcd

3

0.333

0

0.333

0.333

497.6 ± 47.9abc

142.1 ± 13.7ab

39.8 ± 11.1b

4.7 ± 0.4ab

20.9 ± 2.0abc

4

0.333

0.333

0.333

0

433.4 ± 57.2abc

123.8 ± 16.3abc

39.2 ± 7.0b

4.1 ± 0.5abc

18.2 ± 2.4abc

5

0

0

0

1

363.2 ± 31.8bcd

103.7 ± 9.0bcd

44.2 ± 7.1b

3.4 ± 0.3bcd

15.3 ± 1.3bcd

6

0.25

0.25

0.25

0.25

493.2 ± 57.0abc

140.9 16.3abc

50.0 ± 8.7b

4.6 ± 0.5ab

20.8 ± 2.4ab

7

0.125

0.125

0.125

0.625

401.6 ± 30.0bcd

114.7 ± 8.5bcd

42.8 ± 11.6b

3.8 ± 0.2bcd

16.9 ± 1.2bcd

8

0.125

0.625

0.125

0.125

384.4 ± 42.3bcd

109.8 ± 12.1bcd

48.0 ± 7.6b

3.6 ± 0.4bcd

12.2 ± 1.7bcd

9

0.625

0.125

0.125

0.125

391.6 ± 118bcd

111.8 ± 33.7bcd

46.0 ± 13.9b

3.7 ± 1.1bcd

16.5 ± 1.7bcd

10

0.333

0.333

0

0.333

394.6 ± 89.5bcd

112.7 ± 25.8bcd

35.2 ± 11.1b

3.7 ± 0.8bcd

16.6 ± 3.7bcd

11

1

0

0

0

273.2 ± 52.6 cd

78.1 ± 15.03 cd

41.5 ± 8.0b

2.6 ± 0.5 cd

11.5 ± 2.2 cd

12

0

0

1

0

323.2 ± 52.6bcd

92.1 ± 47.5bcd

41.0 ± 29.34b

3.07 ± 1.5bcd

13.6 ± 7.0bcd

13

0

0.333

0.333

0.333

407.6 ± 235.1abc

116.4 ± 67.1abc

77.2 ± 52.1a

3.8 ± 2.2abc

17.1 ± 9.9abc

p

0.0007

3.31e-07

4.524e-05

3.31e-07

3.31e-07

Same letters in the same column indicate no statistical difference between treatments (alpha = 0.05)

BE biological efficiency, MMS mean mushroom weight (g), PR production rate (%), Y yield

Table 5

Production variables of A. bisporus cultivated on Pangola grass and supplemented (9% dw) at spawning and casing with different mixtures of soybean, sesame seed, black bean, wheat bran and chia

Treatment

Supplement

Production variables

Soy bean

Sesame seed

Black beans

Wheat bran

Chía

Production (g/1 kg wet substrate)

BE (%)

MMS (g)

PR (%)

Y (kg/m2)

C25%

438.4 ± 105.8bc

125.3 ± 30.2bc

53.4 ± 12.8abc

4.1 ± 1.0bc

18.4 ± 4.4bc

0

155 ± 59.9d

44.2 ± 17.1d

38.0 ± 6.7bc

1.4 ± 0.5d

6.5 ± 2.5d

1

0.1

0.6

0.1

0.1

0.1

598 ± 90.0ab

170.9 ± 25.7ab

47.6 ± 12.7ab

5.6 ± 0.8ab

25.2 ± 2.5ab

2

0

1

0

0

0

450 ± 112.2abc

128.6 ± 32.0abc

43.4 ± 5.0ab

4.2 ± 1.2abc

18.9 ± 4.7abc

3

0.1

0.1

0.1

0.1

0.6

543.6 ± 138.1abc

155.3 ± 39.4abc

36.4 ± 8.1c

5.1 ± 1.3abc

22.9 ± 5.8abc

4

0.25

0

0.25

0.25

0.25

617.2 ± 128.7a

176.3 ± 36.7a

48.0 ± 10.8abc

5.8 ± 1.3a

26.0 ± 5.4a

5

0

0

0

1

0

538.4 ± 128.4abc

153.8 ± 27.1abc

44.0 ± 9.4abc

5.1 ± 1.2abc

22.7 ± 5.4abc

6

0.25

0.25

0

0.25

0.25

449.2 ± 95.0abc

128.3 ± 22.9abc

58.2 ± 9.2abc

4.2 ± 0.9abc

18.9 ± 4.0abc

7

0.25

0.25

0.25

0

0.25

475.6 ± 80.3abc

135.9 ± 24.0abc

43.0 ± 11.8abc

4.5 ± 0.8abc

20.0 ± 3.3abc

8

0.1

0.1

0.6

0.1

0.1

471.8 ± 84.1abc

134.8 ± 24.0abc

41.6 ± 11.0abc

4.4 ± 0.7abc

19.9 ± 3.5abc

9

0.6

0.1

0.1

0.1

0.1

423 ± 10.36bc

120.9 ± 2.9bc

53.0 ± 5.7abc

4.0 ± 0.8bc

17.8 ± 0.4bc

10

0

0

0

0

1

494.8 ± 88.7abc

141.4 ± 15.0abc

66.8 ± 5.7a

4.7 ± 0.8abc

20.8 ± 3.7abc

11

0

0.25

0.25

0.25

0.25

369 ± 52.7c

105.4 ± 15.0c

43.8 ± 3.0abc

3.5 ± 0.5c

15.5 ± 2.2c

12

1

0

0

0

0

370.2 ± 41.4c

105.8 ± 11.8c

51.8 ± 9.4abc

3.5 ± 0.3c

15.6 ± 1.7c

13

0

0

1

0

0

442.4 ± 53.9abc

126.4 ± 15.4abc

64.0 ± 31.3ab

4.2 ± 0.5abc

18.6 ± 2.7abc

14

0.2

0.2

0.2

0.2

0.2

451 ± 38.3abc

128.9 ± 10.9abc

45.4 ± 11.1abc

4.2 ± 0.3abc

19.0 ± 1.6abc

15

0.25

0.25

0.25

0.25

0

448.2 ± 34.3abc

128.1 ± 9.8abc

45.0 ± 4.4abc

4.2 ± 0.3abc

18.9 ± 1.4abc

16

0.1

0.1

0.1

0.6

0.1

451.2 ± 36.9abc

128.9 ± 10.5abc

48.8 ± 8.3abc

4.2 ± 0.3abc

19.0 ± 1.5abc

p

1.958e-09

1.958e-09

0.002284

1.958e-09

1.958e-09

Same letters in the same column indicate no statistical difference between treatments (alpha = 0.05)

BE biological efficiency, MMS mean mushroom weight (g), PR production rate (%), Y yield

Results

Cultivation on substrates without supplements

Table 3 shows the production variables of A. bisporus on three substrates pasteurized by self-heating. Biological efficiency values varied between 18.5 (corncob) and 51.7% (Pangola grass), Production Rates between 0.6 and 1.7%, and yields between 2.7 and 7.6 kg/m2. Statistical analysis indicated that the cultivation of the mushroom on the mixture or on Pangola grass alone gave similar results, and that both substrates were significantly different from corncob, whose results were lower (p = 0.000). The weight of individual mushrooms ranged between 31.2 and 36.6 grams, and no significant difference was found between all three substrates (p = 0.9367).

Cultivation on supplemented substrate

Table 4 shows the production variables of A. bisporus cultivated on Pangola grass supplemented with 13 different mixtures of soybean, sheep manure, wheat bran and sesame seed. Biological Efficiency values varied from 68.7% (T0, non supplemented Pangola grass) to 178.1% (C2, full compost with 5% spawning rate). Statistical analysis indicated significant differences among treatments (p = 0.000). Table 5 shows the production variables of A. bisporus in a second evaluation trial, cultivated on Pangola grass supplemented with 16 different mixtures of soybean, sesame seed, black bean, wheat bran and chia. Biological Efficiency values varied from 44.2 ± 17.1 (T0) to 176.3 ± 36.7 (T4, formulation with 25% each of four ingredients excluding sesame seed). Statistical analysis indicated significant differences among treatments, establishing four statistical groups. Group “a” with the highest BE values varying between 128.1 and 176.3%. T4 generated a BE value significantly higher than that obtained with control Phase II compost (125.3%; p = 0.000), and also produced larger mushrooms (p = 0.0022) and higher yields (p = 0.000) than the full compost. Yields varied between 6.5 kg/m2 (T0) and 26.0 kg/m2 (T4).

Effect of supplements

Table 6 shows the nutritional value of the substrates and supplements used in these experiments. Since the substrates used for cultivating A. bisporus were of low nutritional value, BE and yield values recorded with the substrates alone were also low. Therefore, in order to improve these production parameters, it was necessary to create better formulations through the application of supplements. In all cases, the supplemented treatments produced higher yields than the control (Pangola grass alone, T0) and the highest BE values recorded were comparable to (Table 4, p = 0.000), or even better than (Table 5, p = 0.000), those obtained with the full Phase II compost. A correlation analysis was performed between supplement content (nitrogen, C/N, lipids and reducing sugar) and production variables (BE, MMS, PR and yield). Correlation coefficients varied between −0.54 and 0.36 (data not shown), indicating there was no correlation between the variables tested.
Table 6

Chemical composition of substrates and supplements used in this investigation

Substrates

Carbon (g/100 g)

Nitrogen (g/100 g)

C/N

Fat (g/100 g)

Reducing sugars (g/100 g)

Corncob

51.92

0.42

123.6

34.92

Grass

49.24

0.82

60.0

1.3

17.2

Mixture

51.12

0.56

91.2

16.11

Supplements

 Sesame seed

50.68

3.29

15.4

47.97

13.7

 Soybean

42.46

5.61

7.6

22.52

30.1

 Sheep manure

38.2

2.24

17.1

1.0512

15.3

 Wheat bran

35.64

2.57

13.9

4.53

66.8

 Chia

47.84

3.37

14.2

33.49

18.65

 Black beans

35.99

2.95

12.2

1.65

85.14

Analyses of supplements using a mixture design (Table 7) indicated that the principal components of the substrate exerted a significant effect (p values between 0.000 and 0.001) but there were no significant differences among the interactions between those components (p values >0.05).
Table 7

Parameter estimates of the mixture design of two supplementation trials evaluated

Term

 

Estimate

Std error

t ratio

Prob> |t|

Trial 1

 Intercept

Zeroed

0

0

 Soybean (mixture) & RS

 

75.828024

12.3874

6.12

0.0017

 Sheep manure (mixture) & RS

 

110.41891

12.3874

8.91

0.0003

 Wheat bran (mixture) & RS

 

97.727195

12.3874

7.89

0.0005

 Sesame seed (mixture) & RS

 

101.22084

12.3874

8.17

0.0004

 Soybean (mixture)*sheep manure (mixture)

Biased

−139.0873

112.6716

−1.23

0.2719

 Soybean (mixture)*wheat bran (mixture)

Biased

285.2221

112.6716

2.53

0.0524

 Sheep manure (mixture)*wheat bran (mixture)

Biased

181.01433

121.169

1.49

0.1954

 Soybean (mixture)*sesame seed (mixture)

Biased

224.53256

121.169

1.85

0.1231

 Sheep manure (mixture)*sesame seed (mixture)

Zeroed

0

0

 Wheat bran (mixture)*sesame seed (mixture)

Zeroed

0

0

Trial 2

 Intercept

Zeroed

0

0

 Soybean (mixture) & RS

100.61448

24.08483

4.18

0.0058

 

 Sesame seed (mixture) & RS

140.73471

24.08483

5.84

0.0011

 

 Black beans (mixture) & RS

125.73009

24.08483

5.22

0.002

 

 Wheat bran (mixture) & RS

151.73587

24.08483

6.3

0.0007

 

 Chía (mixture) & RS

143.47691

24.08483

5.96

0.001

 

 Soybean (mixture)*sesame (mixture)

63.101291

384.2923

0.16

0.875

0.891

 Soybean (mixture)*black bean (mixture)

344.78915

384.2923

0.9

0.4042

0.1963

 Sesame seed (mixture)*black beans (mixture)

−242.7836

382.7513

−0.63

0.5493

0.3501

 Soybean (mixture)*wheat bran (mixture)

−183.1043

343.033

−0.53

0.6127

0.8353

 Sesame seed (mixture)*wheat bran (mixture)

Zeroed

0

0

 Black bean (mixture)*wheat bran (mixture)

Zeroed

0

0

 Soybean (mixture)*chía (mixture)

Biased

151.41592

343.033

0.44

0.6744

 Sesame seed (mixture)*chía (mixture)

Zeroed

0

0

 Black bean (mixture)*chía (mixture)

Zeroed

0

0

 Wheat bran (mixture)*chía (mixture)

Zeroed

0

0

Contamination

Most of the spawned bags (replicates) produced mushrooms, although some exhibited poor yields due to the presence of contaminants: when the mushroom was cultivated on corncob, Trichoderma sp. was present in three of 20 bags spawned with A. bisporus (15%). Also, after the second flush on corncob, the myxomycete, Didymium iridis, was detected. To our knowledge, this is the first time this myxomycete has been reported to be a contaminant of mushroom substrate. Among the 80 bags spawned for the first supplemented trial on Pangola grass (Table 4), both treatments T12 and T13 had one bag contaminated with the mold Trichoderma sp. by the end of the colonization period. These two bags did not produce mushrooms. One bag in each of T11 and T12 were also about 20% contaminated with the same mold by the end of the first harvest, which would explain the low mushroom production recorded with these two supplements (soybean and wheat bran alone). The general incidence of contamination by Trichoderma sp was 6.6% in Supplementation Trial 1, and the fact that contamination appeared only in some bags of the last treatments suggests it occurred during spawning. For the second Supplementation trial (Table 5), also using Pangola grass as the substrate, none of the 90 bags spawned were contaminated and all the bags produced mushrooms on a regular basis.

Discussion

The results obtained clearly demonstrate that it is possible to use the technique of pasteurization by self-heating to produce A. bisporus Portobello mushrooms. When the substrates were used alone, BE and yield values obtained were low compared to commercial data such as estimated average yields of button mushrooms in the USA (26.9 kg/m2 or 6.33 lb/sq foot, NASS 2016). These low yields may be due to the low nutritive value of the substrates used (nitrogen content between 0.42 and 0.82, Table 6). However, it is noteworthy that full substrate colonization was achieved in less than three weeks, suggesting a certain selectivity for mushroom growth. The selectivity observed on the traditional full mushroom compost for the growth of A. bisporus is based on the presence of thermophilic fungi developed during phase II pasteurization (Straatsma et al. 1989, 1994; Wiegant et al. 1992). However, in the case of self-heating pasteurization, the total length of the treatment and the thermophilic phase was less than 45 h, so the substrate was less degraded and thermophilic microorganisms had shorter time to colonize the raw substrate. The raw material was pasteurized scarcely in two days, a short time compared with the phase II composting method that requires 12–20 days (Zied et al. 2010).

In previous investigations, researchers have shown that Phase I is not a prerequisite for Phase II (Wiegant et al. 1992; Straatsma et al. 1994; Sánchez et al. 2008; Sánchez and Royse 2009; Coello-Castillo et al. 2009). Also, by using sterile substrates, it has been demonstrated that phase II is also not necessary to grow A. bisporus (San Antonio 1971; Sanchez and Royse 2001; Bechara et al. 2005). In this work, it was found that A. bisporus can be cultivated in several pasteurized substrate that have not received a phase I or Phase II process. The ability to colonize and then to fructify in a self pasteurized substrate could be linked to the ligninolytic capacity of the white button mushroom (Kabel et al. 2017; ten Have et al. 2003; Bonnen et al. 1994) but also to the microbiota associated to the mycosphere (Torres-Ruiz et al. 2016). Therefore, it would be interesting to undertake further studies on this system to understand better the microbial diversity and its relationship on the development of A. bisporus.

Sánchez et al. (2008) used pasteurized grass (6 h, 60 °C) for A. bisporus production and obtained 11% BE and 25 g mean mushroom weight. Differences may be due to strain difference, but also to a possible beneficial effect of the microbiota developed in the self-heated pasteurized substrate (Torres-Ruiz et al. 2016).

The data obtained through the supplementation trials (BE = 176.3%; Y = 26 kg/m2) are interesting since Schisler (1982), for example, stated that BE values between 80 and 90% are already attractive commercially. They also compare favourably with previously results (BE = 73.1% and Y = 12.7 kg/m2) obtained by cultivating brown varieties of A. bisporus on non-conventional, pasteurized substrates (Coello-Castillo et al. 2009; Sánchez et al. 2008). Furthermore, they exceed BE and yield values (99.5–108.2% and 25.8–28.6 kg/m2, respectively) obtained by Pardo Giménez et al. (2016) who cultivated a white hybrid of A. bisporus on unsupplemented and supplemented (with defatted pistachio meal) Phase III compost. Sánchez and Royse (2009) using a Scytalidium thermophilum-colonized and supplemented substrate obtained BE and yield values of 99.3% and 21.19 kg/m2, respectively.

With regard to supplementation, our data with soybean, a supplement used successfully on a commercial scale since first reported by Schisler and Sinden (1962), confirmed those earlier results. Sheep manure, a supplement reported to be beneficial for A. bisporus cultivation (Mee 1978; Sanchez and Royse 2001), increased yields when used at low concentrations. The manure was obtained in dry form and no information about conditions of drying were available. In order to preserve quality, the drying process may need to be carefully controlled. Sesame seed is a grain high in fat and nitrogen. This product contributed to increased mushroom production only when it was applied at low concentrations in the supplementation formula. Conversely, chia, with a similar nitrogen content to sesame seed and less fat, had a positive impact on mushroom production. Comparing the treatments where ingredients were applied individually, chia and wheat bran showed higher increase in BE than the other ingredients (BE = 141.4 and 153.8%, respectively, Table 4). Specific components of each supplement with regard to amino acids, fatty acids, etc. may provide an explanation for why some mixtures promoted fruiting whereas others did not.

Supplementation is a highly complex variable where several group of formulations are recommended including proteins, lipids and carbohydrates (Wheeler and Wach 2006). It may be necessary to investigate micronutrients and individual ingredients present in the supplements in order to detect positive correlations. In this context, it has been reported that a single amino acid (isoleucine) positively stimulates mushroom yields when administered into the mushroom compost after the second flush (Royse and Sánchez 2008).

Successful colonization of the substrate by the mushroom and the low level of contamination recorded indicate that the use of raw substrates pasteurized by self-heating is a viable alternative method for cultivating Portobello mushrooms. The use of adequate supplement formulations improves mushroom development and allows the grower to obtain better yields statistically comparable to those obtained using traditional substrates (Phase II and III composts).

In regard to scaling up of the process, the self-heating pasteurization technique should be further studied (Sánchez et al. 2016). If conditions are improved, the duration of treatment may be reduced. Also, optimizing aeration would facilitate removal of the mass and reduce heat loss during turning. The technique used in this work is suitable for small growers because a crate of 1 m3 can process about 220 kg of grass or 380 kg corncobs with 65% moisture. If processing a larger amount of substrate is required, it would be possible to increase the volume of the container. However, the question rises to what extent it would be feasible, referring to the traditional technique of two phases. The larger the mass, the greater the heat generated, but also the greater the effort required to obtain a uniform temperature throughout the whole substrate, and also the greater the difficulties to moisten the substrate homogeneously.

Abbreviations

C/N: 

carbon/nitrogen ratios

DNS: 

3,5 dinitrosalicilic acid

BE: 

biological efficiency

PR: 

production rate

MMS: 

mean size of mushrooms

Y: 

yield

Declarations

Authors’ contributions

SCC assisted in the experimental design, carried out the experiments and assisted in the interpretation of the data, JES designed the experiments, supervised the execution, analyzed the results and wrote the manuscript, and JVM coordinated the statistical analysis, and critically reviewed the manuscript. All authors read and approved the final manuscript.

Acknowledgements

The authors thank Dr. Gerardo Mata and Ing. Luis Aguilar, for providing the full Phase II mushroom compost samples, Lilia Moreno for her technical support, and René Andrade for identifying D. Iridis. We also thank Miguel A. López-Anaya and Guadalupe Pérez-Escobar for their help with the chemical analyses and Dr. J.A. Buswell for critically reviewing the manuscript and correcting linguistic errors.

Competing interests

The authors declare that they have no competing interests.

Availability of data and materials

The data supporting the findings of this study are included in the main manuscript file.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Funding

This research was supported financially by Fondos Mixtos Conacyt through the Project FOMIX-13149 “Design, construction, equipment and startup of a state center for innovation and technology transfer for the development of coffee growing in Chiapas, Mexico” and through the Project MT-11063 of Ecosur “Social and environmental innovation in coffee growing areas for reducing vulnerability”.

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Authors’ Affiliations

(1)
Instituto de Biociencias
(2)
El Colegio de la Frontera Sur.

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Copyright

© The Author(s) 2017