World J Microbiol Biotechnol (2011) 2715311339 DOT 10.1007/s11274-010-0582. ORIGINAL PAPER es Agaricus blazei production on non-composted substrates based on sunflower seed hulls and spent oyster mushroom substrate Gonzélez Matute « D. iglas - N. Curvetto Received: 20 July 2010/ Accepted: 30 September 2010 Published online: 13 October 2010 (© Speinger Science+Business Media B.V. 2010 Abstract Agaricus blazei Murrill is usually cultivated using the same biphasic composting method employed for A. bisporus. Because cultivation of A. blaze’ on traditional A. bisporus composts poses some disadvantages, non- composted substrates were studied for A. blacei cultivation. Mycelial growth rate and productive performance of A, blazei were evaluated on substrates containing sunflower seed hulls, Pleuromus spp. spent mushroom substrate, or their combination, in the absence or in the presence of different supplements (vermicompost, peat or brewery residues). Substrates were prepared by initially soaking them and then they were sterilized (1 atm for 120 min), In addition, each substrate’s degradation was measured after cultivation by obtaining the lignin, cellulose, hemicellu lose, organic matter, total fiber, ash, carbon and nitrogen contents before spawn-run and al the end of two Aushes of A. blacei. The cultivation of A. bluzei on non-composted substrates is possible and with a low rate of contamination when using the spent mushroom substrate as the main ‘component or combined 50:50 with sunflower seed hulls. In addition, the best yields were obtained on those sub- strates containing spent Pleurotus mushroom substrate with supplements and those mixtures with sunflower seed hulls , Gonailez Matute (E) -D. Flas N. Curvero Laboratory of Biotechnology of Edible and Medicinal Mushrooms, CERZ0S (CONICET), CC. 738, 3000 Bahia Blanca, Argentina emma: rmatated@eriba eda. ar . Gonzilez Matute-D. Figlas CComisin de Investigaciones Cienfcas de la Provincia de Bucnos Aires, Buenos Aires, Argentina N. Curveto Departamento de Agronomia, Universidad Nacional del Sur, $000 Bahia Bhinca, Argentina ‘and vermicompost. These yields were similar to those reported on composted substrates, Substrate changes in composition measured at the end of two flushes indicate that the lignin-hemicellulose fraction was preferentially uused and that the substrates exhibiting the best yield showed greater biodegradation of lignin-hemicellulose fraction than the others did Keywords Almond portobello - A. brasiliensis Productivity » Axenic substrate - Vermicompost Introduction Agaricus blazei Murrill ss. Heinemann is one of the most valued cultivated edible mushrooms in the world market, mainly because of its medicinal properties (Stijve and Amazonas 2001). Another names, A. brasiliensis , Wasser (Wasser et al. 2002, 2005) or A. subrufescens Peck (Kerrigan 2005, 2007), have been proposed for this mushroom, however this controversy sUill remains, that is the reason why its original nomenclature was used throughout this text. ‘The same methodology used for cultivation of the white button mushroom (Agaricus bisporus) has been normally used with variations in the initial C/N ratio (Ferreira da Eira 2003). However, preparation of this traditional compost has some disadvantages: a long processing time (15-40 days, depending on the system), many employees, a high cost of composting equipment (turning machines, front-end loaders and conveyors systems), and sometimes additional costs of forced aeration bunkers, and Phase IL tunnels. In addition, there is a loss of substrate dry matter due to microbial activity during the composting process, and odors and effluents may become a problem for the D Springer1332 World J Microbiol Biotechnol (2011) 27:1331-1339 environment and the surrounding community (Duns et al 2004), ‘Non-composted substrate (NCS) is an alternative pro- cedure for the preparation of the substrate for cultivation of| Agaricus bisporus. Till (1962) obtained yields comparable to Phase II compost after grinding, mixing, filling, steril- izing and spawning substrate consisted of a mixture of 69.0% wheat straw, 12.3% peat, 9.8% CaCOs, 4.1% soy- ‘bean flour and 4.1% cotton seed meal. Mee (1978) obtained “good quality”” mushrooms on a NCS mixture of cold manure, Sphagnum peat moss and gypsum. More recently, Sanchez. and Royse (2001) reported procedures to obtain ‘good yields of A. bisporus on complex NCS. Garcia etal (2005) used vermicompost (verm) as part of the formula (0-12%), Moreover, Mamiro and Royse (2008) examined the effect on mushroom production of combined NCS with spent mushroom compost obtaining the best yields from a 50/50 mixture of those materials ‘The only reference to NCS cultivation of A. blazei is sterilized millet grain alone or with addition of different oilseeds (niger, safflower, and soybean) at different rates QS oF 30% of total substrate composition) (Bechara etal. 2006). Their best yield was obtained (15.9 kg/m*) using 30% niger (Guicoria abyssinica). Many agroindustrial residues have been used as alter- native substrates for cultivating mushrooms (Chang and Quimio 1982). Sunflower seed hulls (SSH), an abundant oil industry waste in the Buenos Aires Provinee, Argentina, is difficult to degrade and not suitable for animal consump- tion due to its high lignin content (23.9%) (Conghos et al. 2006). However, mushrooms are able to alter the chemical structure of the lignin by condensation and oxidation reactions (Fiyama et al. 1994), SSH has been successfully substituted for the main substrate for cultivation of many edible mushrooms, ic. Pleurotus spp. (Curvetto et al 2004), Lentinula edades (Curvetto et al. 2002), Ganoderma lucidum (Gonailez Matute et al. 2002), Hericium erinaceus (gles et al. 2007) and Agaricus bisporus (Gonzalez Matute et al. 2010), Sunflower seed hulls as part of « non-composted sub- strate for the cultivation of A. blaze has not been studied. It has @ good particle size that enables appropriate gas ‘exchange. Moreover, its relatively high lipid content may stimulate mushroom growth (Wardle and Schisler 1969; Nwanze et al. 2005), ‘This paper reports the mycelial linear growth (MLG) rate of A. blazei on various formulations of SSH on tradi- tional Phase II compost and on NCS in the absence or presence of vermicompost, brewery residues (BR), Sphagnum peat moss or spent Pleurotus pulmonarius (Fr) Quel. mushroom substrate (SMS). Some of the substrate formulations were evaluated in mushroom cultivation trial ‘Changes in C, N, total fiber (TF), organic matter (OM), D Springer hemicellulose, cellulose, lignin, and ash contents were also determined for the NCS on SSH. Materials and methods Mushroom strain Agaricus blazei Murrill (PL strain) was obtained from Brasmicel, SP, Brazil. This is a commercial strain that is fast growing and that produces good marketable size mushrooms, Preparation and conservation of the mushroom spawn ‘The mycelial culture was maintained in glass tubes on 20 malt extrac, 2g yeast extract, I g peptone and 20 g agar, per liter of medium MYPA and covered with sterile quid Vaseline® at room temperature until use. Another nutrient medium was prepared using the filtrate resulting from boiling 500 g of Phase IL sunflower seed hulls-based compost (Table 1, contol) in 1 1 of water and adding 2 2 yeast extract, 20g dextrose and 20g agar, per liter (CDYA) (Gonzalez Matute 2009), The medium was adjusted to pH 6.5 with HCI and sterilized at 1 atm for 30 min, A blaze’ mycelium was inoculated on Peti dishes containing the CDYA medium and incubated in darkness st 25°C for 10-15 days, at which time it was ready tobe used for spawn preparation. Wheat grain (Triticum aestivum L.) (250 g) was placed into a1 I glass botle and 1.3% CaCO and 190 ml of water were added and allowed to stand ovemight at room tem perature. Resulting mixture was autoclaved at 1 atm for 15 h. After mycelium inoculation (16 em? of colonized CDYA), the spawn was incubated at 25°C in darkness for 30 days, with occasional inspections. and shakings 10 facilitate complete grain colonization, Mycelial linear growth tests In Trial 1, the rate of mycelial growth of A. blazei was compared between Phase Hl compost (control, sunflower— Helianthus annuus—seed hulls based, supplemented with Table 1 Substrate composition (dry bass perentages) of treatments assayed daring Trial 1 ‘Treatment SSH_WB Verm Peat (HINQ:S0, CaCO, Comot = 29040 SD 20 Nes mo uo - 40 10 20 NCS+verm 160 60 110 40 10 20 [9H sunfower seed hull, Sphagnum peat moss. Final nat bran, verm vernicompon, peat istre ofall teatments was 60‘World J Microbiol Biotetnol (2011) 272133 Wheat bran and Sphagnum peat moss. prepared in 2 smaii scale system (Gonzalez, Matute etal. 2010) with a duration ‘of 14 and 15 days for Phase I and Il, respectively) and 1wo formulations of NCS (same ingredients as control but in different. proportions in order to reach similar N initial levels, 1.7-1.9%, and C/N, 20-24), in the absence and in the presence of vermicompost (non treated, 67% organic matter, 1.75% N, 1.25% K, 2.25% P, CIN 10, and 45% moisture content) (Table 1). Glass tubes (20 em long and 15 mm id), 10 per treat- ‘ment, were filled with 10 g substrate and depending on the particle size, compressed to 9.em (control and NCS + verm) and to 11 em (NCS). Tubes were closed at both ‘ends with cotton plugs, wrapped with newspaper and autoclaved (1 atm for 120 min), Once the substrate’s temperature cooled to 30°C, one end of the treatment substrate was inoculated with a 20-day-old agar mycelial plug (15 mm) of 4, blaze’ under aseptic conditions. One tube per treatment was used to measure the pH and mois- ture content. The remaining tubes were placed in a Percival 50036 (Percival, Boone, 1A, USA) chamber in darkness, 25°C and 80% of RH. Mycelial linear growth (MLG) rate in each tube was determined on day 28 by taking the average of four mea- surements ofthe linear mycelial growth around the tube. In this case, the apparent mycelial growth in the assayed substrates, as it was sawn by nude eve, were quite different, thus justifying the use of this more simple method for the analysis of mycelia growth, instead of using the determi- nation of biomass mycelium which while being a better growth parameter, requires more time and care (ie. ergosterol measurement eae 5 ‘of non-composted substrates. eee SSH ‘evaluation of A. blazei mycelia een - 7 ‘wth the resulting © N and C7 SSHUSMS (50:50)° 18.75 cesar ae Sera a acne a0 resend icsi" sen sean Som (054) and. water (60.0%) SSHIBR (70:20) oe SS SMS spent mushroom substrat ‘erm vermicompost, peat Sphagnum peat moss, BR brewery residues * Ingredients percentages in rackets are expressed in 8 dry ‘weight basis SMsWverm (80:20) = SMiverm (60:40) ~ SMSipeat (80:20, ~ SMisipest (60:40) — SMS/BR (80:20) _ 3.8: Rio Grande. Thera dei Furs, Arputza. or = brewer's industry residue (BR SO Ce Name 2 moisture content: Malteria Pampa. Buenos Aes. Ape tina), were mixed into base material of either dned «FC 48 h) spent Pleurotus pulmonarius mushroom. substrate (SMS), which had produced two ushes of mushrooms. or unmilled sunflower seed hulls (SSH). A 50/50 combination of SSH and milled SMS (18 mesh) (SMSM) or unmilled SMS were also used as the base material (Table 2) Procedures to fill tubes (n = 10) with substrates, sterit- ization, inoculation and incubation were the same as Trial 1. In Trial 2, substrates were packed into the tubes to obtain a length between 8.5 and 10.0 cm, depending on the par- ticle size ofthe formula components. A tube per treatment ‘was sacrificed after sterilization for both pH measurement and moisture content determination. ‘After 28 days, MLG of each tube was measured as in Trial 1, Data for both trials were analyzed using the ANOVA procedure of Statistica 6.0 (StaSoft Inc, OK. USA). Tukey HSD was used to compare means at 1% level Cultivation ‘Some of the substrates evaluated in Trial 2 were included in the cultivation study. Selection of treatment combina- tions from Trial 2 focused on the addition of SMS, both types, unmilled or milled, and 20% of each supplement, t0 ‘SSH. Additionally, treatments included 30 or 40% of BR to SMS Vem Pet «BRON CIN ~ e = 0671 1875 : ao 06 78 18:75 (milled) — = 6 06 78 - wo ors - [SOc os a4 - = 1s = Cates) e _ Bo os - 5 = 10 090 - - = nas 104 z - = 1500 110 3750 . a 0868 30.00 mH a «@ 0958 2250 Boo 09 as 30.00 e mo 09 a8 250 = 150 Lo as 30.00 - - 7009 D Springer‘World J Microbiol Biotechnol (2011) 27:1331-1339 the SSH. and 20% of BR to the SMS (Table 3). Substrates ‘sere prepared by initially soaking them separately Sy smmersion for 15-20 h in water-caleulated proportion Immeaisly following the soaking. period, 0.75 ky sub- 1m each formulation was placed into a high-density pelyeth lene bag 135 x 25 em, 75 microns), closed with a sovten sug and an elastic band in the neck and sterilized {atm for > hv, Thiteen replicates were prepared for each sscurmert, One autoclaved bag per treatment was used for re content determination and pH me: suremen: iter cooling 10 28°C, the remainder (n= 12) sere inoculated with 65% (on wet weight basis) of 35 day-old wheat spawn of A. blaze, under aseptic ions under a laminar flow hood. Substrates were randomly placed in @ room under darkness, 28 = 2°C and 70-80% RH, for incubation. Once at least 80% (21-27 days) of the substrate was colonized, a G4 ke of casing material (334% not sterilized peat moss, CaCO: \technical grade) and 40% water) was placed 6n top ofthe substrate of each bag and closed again. After 1 days. the cotton plug was removed and plastic bag borders were rolled down leaving « 2-3 em margin from the casing surface. Bags were then placed in a fruiting environment, ic. a plastic glasshouse 25 +t 4°C, 80-90% RH. ventilation (4fitered air exchanges) and artical light (200-400 tux, 12 h photoperiod) ‘Mushrooms were harvested when the veil was stretched, but before lamellae were exposed, and weighed. Biological efficiency (BE; calculated as fresh weight of mushrooms/kg ‘dry substrate expressed as a percentage), productivity expressed as BE/days from inoculation), average tuumiber of mushrooms per bag (N°M) from the first flush, and total yield after two mushroom fushes were analyzed with Statistica 6.0 by a simple ANOVA. Means were compared by Unequal N Tukey HSD or Tukey HSD, according to data balance, ata significance level of 5%. ‘Table 3 Substrate formulations forthe cultivation of Substrate composition determination Substrate samples (= 3) were randomly taken before inoculation and at the end of the second mushroom flush (Caken below the casing layer), dried (50°C for 48 h) and homogenized. A portion of each dried sample was milled (18 mesh) in a centrifugal grinder mill (Butt mod. am-a8, (60177, Tonomex, Argentina), Van Soest system was used to determine the cellulose and hemicellulose content (Van ‘Soest 1963, 1967) of the substrates. Acid detergent lignin was determined by sequential detergent analysis (Van Soest and Robertson 1980). Total nitrogen was determined by the wet oxidation Kjeldahl method and total organic carbon by dry combustion with a Leco Carbon Analyzer. CRI2, Leco Comporation, St. Joseph, MI, USA) (Nelson and Sommers 1996). Organic matter content was calculated by the method of weight loss by ignition at low temperature (430°C) in a muffle furnace (Thermolyne Furnatrol 133, Sybron Corp., Dubuque, IA) after 24 h. Calculation based con dry matter (DM) was: OM (‘e) = DM% — ash‘. Ash content was determined by ignition in muffle at $50°C for 15 min Results and discussion Mycelial linear growth tests In Trial 1, substrate moisture content and pH were 62% and 6.1 for the control, 57% and 6.2 for the NCS and 58% and 6.2 for NCS + verm, respectively. Highly significant differences (P< 0.01) were observed within the MLG rate (Table 4). A. blaze? grew four times and almost two times faster in the control (0.21 emiday) than in the NCS (0.05 em/day) or NCS. + verm (0,13 em/day), respectively. Vermicompost had a and resulting %N and C/N ratio ‘Treatment ssi SMS Verm Peat BR aN oN Conte (55H) 37.50 B 2 e = 06 7 'SSHSMS (50:50)" 895 1875 . a = 06 % SSHISMSM (50:50) 895 18:7 (milled) - - . 06 8 'SSHiverm (80:20) 3000 2 1s 2 = 07 58 ‘SSHipeat (80:20) 30.00 2 7s : os ss SSHIBR (80:20) 30.00 = a = 750 09 50 SSSHIBR (70:20) 26.28 = e : has 10 “4 SSSHIBR (60:40) 250 . a _ 15.00 MM 40 SSMS/BR (80:20) 30.00 S e 750 09 oo All treatments have the addition of CaSO, (2.0%), CaCO, (0.3%) and water (60.0%). Components are expressed as, SSH sunlower seed hols 'SMS spent mushroom subsite, verme vermicompost, peat Sphagnum peat ross, BR brewery residues * Ingredients percentages in brackets are expressed in a dry weight basis €) SpringerWorld J Microbiol Biotechnol (2011) 27:1331-1339 1335 ‘Table 4 Mycoial linear growth (envy) of A. blaze! on composted (contol) and a non-composted substrate (NCS), both based on sun- ower seed hulls. withthe addition or not of 30% (dry bass) oF vvermicompost (ver) Treatment ‘may Contr (0.21 (0.036) a* Nes 0.085 (0.025) ¢ NCSiverm (70:30) 0.13 (0015) 6 ‘Standard deviations are in brackets. w= 9 * Means in same column with a different Jeter are significantly dit ferent at 19 level according to Unequal N Tukey HSD. significantly positive effect on mycelial growth in non- composted substrates. Mycelia appeared whiter and denser in NCS than in contro. Galli (2007) studying A. brasiliensis in a similar MLG test, with composted and NCS, based on ray grass (Lolium ‘multiflioram Lam.) or elephant grass (Pennisetum purpur- eum Schum.) straw, found a superior MLG rate in NCS than in composted ones. Based on Trial 1, a NCS-SSH based, with or without vermicompost is inferior to the same formulated substrate that is composted. Recalling that the mycelia in NCS were denser, A. blazei more easily utilizes composted substrate with more readily available source of nutrients than the NCS. In our study, the vermicompost inclusion to this NCS improved the performance of the A. blazei mycelia growth. Experimentation with vermicompost has demonstrated that it contains plant growth regulators, including plant growth hormones and humic acids, responsible for seed germins- tion, plant growth and erop yields (Atiyeh et al. 2002; Arancon et al. 2006), In Trial 2, moisture content and pH of the different substrates after the autoclave sterilization were simiar, approximately 60% and 6, respectively Highly significant differences were observed (P € 0.01) in the MLG rate, among the different treatments. Substrates containing a 1:1 mix of sunflower seed hulls and spent mushroom substrate, unmilled or milled, produced @ slight increase in the MLG rate although not significant. However, substitution of SSH by SMS increased the MLG of A. blaze’ significantly (Table 5). Addition of 20% supplements to the SSH in Trial 2 resulted in an increase in MLG (cm/day), compared to control | (SSH) for both peat and vermicompost; no di ferences between both treatments were observed. BR sup- plementation did not affect mycelial growth. A 40% vermicompost ratio had a tendency to increase MLG. In contrast, « peat ratio increase decreased significantly the MLG (Table 5). Inclusion of 20% BR to SMS did not significant increase ‘LG. In contrast incorporation of 20 and 40% peat to the Table § Agaricus blazel mycelial linear growth rate (emiéay) obtained in diferent non-composted substrates based on sunflower seed hulls (SSH) andoe spent Pleurotus mushroom substate (SMS). tunmilled or milled (SMSM), in the absence or the presence of if ferent supplements cates Treatment cmlday Control 18H) 0.08 (0.011) e** SSSHISMS (50:50) ° 0.09 012) SSSHISMSM (50:50) 0.09 (0012) € SSsHverm (80:20) 0.11 @012) 4 SsHverm (60:40) 0.13 0.007) bed SSHipet (80:20) 0.13 0.012) bed S8Hpeat (60:0) 0.08 (0.005) ¢ SSSHBR (80:20) 0.08 (0.007) ¢ SSHIBR (70:20) 0.08 (0.006) € SSSHIBR (60:40) 0.08 (0011) & Control 2 (SMS) 0.1 015) 4 SSMS/BR (80:20) 0.120.018) ed SMsiverm (80:20) 019 0010) 3 SMsiverm (60:40) 0.20 (0.014) a SMSipest 80:20) 0.14 (0.008) be SMSipeat (60:40) 0.14 (0.005) 6 Verm vermicompost BR brewery residue, peat Sphagnuon peat moss ‘Standard deviations are in brackets. = 9 * Ingredients percentages in brackets are expressed in a dry weight basis “© Means in same column with a diferent leter are sigificanly ferent at 1% level according to Tokey HSD SMS produced a significant increase (27%) in MLG ‘compared to control 2 (SMS). However, between both peat proportions there were no significant differences. The presence of vermicompost (20 and 40%) significantly increased (72-80%) the MLG when compared to SMS alone. In turn, between both vermicompost proportions there were no significant differences but, in general, they ‘were significantly superior to the ones found with peat supplementations (Table 5). ‘Mycelial linear growth best values obtained in Trial 2 are almost 10 times greater than the ones presented by Coello Castillo (2006). When she evaluated the A. subru {fescens MLG rate on non-composted pangola grass (Digi ‘aria decumbens) (2.0-2.5 cm length) that was either pre- colonized or not with three strains of S, thermophilum, she observed values of 0.02 em/day, for treatments with S. thermophilum, and 0.01 cmiday in the absence of S. thermophitum. Cultivation trial Moisture content and pH values of substrates after auto- claving were approximately 60% and between 5.5 and 5.9, springer1336 World J Microbiol Biotechnol (2011) 27:1331-1339 respectively. Twenty days after incubation, several bags were discarded because of contamination, attributed to problems with the bags or closures. Two treatments, SSH/ BR (80:20) and SSH/BR (60:40), were lost completely Galli (2007) noted a high percentage of contamination in the sterilized substrates, based on straw of ray grass (Lolium muttiflorum Lam.) or of grass elephant (Pennise- ‘tum purpureum Schum), in comparison to composted oF pasteurized ones for the cultivation of A. brasiliensis. Pell (1992) also observed a high level of contamination in axenic mediums of A. bisporus. In sterilized substrates, fast growing competitors take advantage over the cultivated ‘mushroom due 0 the high readiness of easily available nutrients In addition, microbiota responsible for impeding the development of contaminants is eliminated. ‘The healthy bags showed a white vigorous and rhizo- morphic mycelia, These were exposed to the fruiting atmosphere 12 days after casing. In the ease of control 1 (SSH treatment), six bags were cased. No contamination was observed in treatments contain- ing SMS, i. SSH/SMS (50-50) and SMS/BR (80:20), and in the SSHiverm (80:20) only three bags were discarded. Al these treatments were also those that faster colonized the substrate and first fructified First flush of mushrooms started 60 days from spawne ing. Not all the bags within a treatment produced a second flush, ic. from SSH/SMS (50:50) and SMS/BR (80:20) a second flush of mushrooms was harvested from eight bays (67 and 61% of total of bags. respectively), from SSH/verm (80:20) seven bags (78%), from SSHVBR (70:30) three bags, (100%), from SSH/peat (80:20) two (50%) and from con- trol (SSH) and SSH/SMSM (50:50) one bag (17 and 20%, respectively). Second flush for all this treatments. was, obtained between day 110 from spawning and day 170. Table 6 Biological cffcieney (BE, 9%) and productivity (®, % /BELdays from spawning) of A. blazei mushrooms cultivated on ‘on-componted substrates based on sunflower seed hulls (SSH) and All supplements improved the final yield parameters with the exception of productivity (P) in the SSH/peat (80:20) that did not change. Biological efficiency (BE) and P after the first lush and atthe end of two flushes were not significanly different between treatments and control. However, first lush P and total BE were close to reaching 5% significant differences, P = 0.053 and 0.051, respec- tively. The highest first flush P was observed in SMS/BR (80:20), which was 43% superior to the contol, followed by SSHYBR (70:30), which also had the best firs sh BE. ‘SSHVSMS (50:50) ranked third for both yield parameters. In the total accumulated yield, the best BE and P were ‘oblained with SSH/BR (70:30), 52 and 46% superior tothe control, respectively, and 30 and 12% superior 10 the second better treatment—SMS/BR (60:20), respectively However, ar bore contamination affected large part ofthe SSEVBR (70:30) during te incubation, thus fom the ‘mushroom grower point of view itis preferable the SMS/ BBR (80:20) choice, where no contamination was observed. Treatments with good yields and none or low contamina tion were also the SSH/SMS (50:50) and SSH/verm (80:20) (Table 6). Hence, these observations do emphasize the point that substrates containing SMS are able to control the aboverentioned high contamination in the SSH substrate formulation. Also, the presence of secondary metabolites, coming from either P. pulmonarius or Eisenia foetida (Californian red worm), and/or the degree of lignocellu- Josic material degradation in the first ease, could eventually be the explanation for the observed low contamination ratio, These metabolites can be inhibitors of the develop- ‘ment of contaminants in the substrate. On the other hand, a it was demonstrated through the mycelial lineal growth test, the A. blaze’ mycelial growth rate was higher in the substrates containing SMS. Consequently, the substrate Pieurotas spent smushroom substrate unmilled (SMS) or milled (SMSM), in the absence or presence of different proportions of supplements ‘Treatment 1st Musk ‘Accumulted BE and P, 2 ashes BE P BEA) BEG P (BEY) NCB CConteol (SSH) 146 7.18) 0.13 (0.062) 16.4 (4.85) 0:13 045) 6 'SSHVSMS (50:50)" 1840.8), 0.17 (0.087) 22.4 (735) 0.19 074) 2 SSSHUSMSM (50:50) 79.91) 0.06 (0.030) 11 @51) 0.09 (0.063), 5 'SSH/verm (80:20) nsash 0.15 (0072) 230 (1200) 0.17 (0.098) 9 ‘SSHipest (80:20) 135 (1.67), 0.11 027) 19.1 6.60) 0.12 028) 4 ‘SSHIBR (70:30) 197482) 0.19 (0.085) 343 (7208) 024 (0081) 3 SMS/BR (80:20) 18.3 (8.99), 0.23 0.145) 240 (10.19) 021 (0.138) n Verm vernicompost, BR brewery residues, peat Sphagnum peat moss. NCB represents the number of son-contamtinated bags from the Ctal inoculated (a = 12) + Ingredients percentages in brackets are expressed in dey weight basis ‘Standard deviations are in brackets D springerWorld J Microbio! Biotechnol (2011) 27:1331-1339 ‘Table 7 Composition of non-composted substrates based on sun- flower seed hls (SSH) or spent mushroom compost (SMS, oyster 1337 harvest (AD. Hemicellulose (HCL), cellulose (CL), lignin (L}, total fiber (TF, organic matter (OM), ash, carbon (C) and nitrogen (N) mushroom) before (BI) and afte A. blace inoculation and two flushes average values (a = 3) Treatment Phase “HCL cs cL TF On Aah € N Con (SSH) BL 7.31 (L684) 37-31 (1.082) 1763 (1.542) 62.25 2982) SEB (O6K2) 849 (0828) 43390921) 059 00KT) Conia (SSH) AL 2.76 (1.278) 3869 (1.191) 1091 (1.106) 51.76 2421) 78.20.8TR) 11.21 (1.253) 39890787) 097 0.102) SSHISMS (500) BI 5.03 (0873) 3845 (2083) 16.74 (1.385) 55.22 2562) 78.20,0724) 15.0 (0523) 4048 (0754) 0.63 (0035) SSHISMS ($050) Al 4.6) (0684) 35.28 (1122) 1248 (0974) 52362707) 7602 (0485) 1486 (0.448) 38.03 (0628) 083 (0056) 8Huverm (80-20) BI 9.67 (1328) 3607 (2572) 1848 1495) 6422 (2314) 7600 (0872) 1383 (0979) 3748 (0885) 069 (0012) S8Hiverm (80-20) AL 457 (0777) 3741 (1.610) 808 (0651) 50.054 1910) 6701 (L840) 2469 (1.608) 3484 (0836) 080 (0009) SSHBR (70%) BE 1074 (2322) 2933 (1.937) 1491 (338) 5498 2608) 7602,2.982) 8:72 (0483) 4242 (0984) 0:99 0098) SSHBR (7030) AL $.50(1A0S) 3051 (1.678) 1086 (0953) 4687 (2101) 7488 (5.205) 18.18 (0.290) 37.24.1931) 1.00 (0230) SMS/BR (90:20) BI 7.3 (1847) 2054 (2.082) 15.00 (1498) 52.88 2761) HO. (2088) 1292 (0.724) 39.20 0986) 0:90 (0.059) SMSIBR (80:20) AL 3.37 (1475) 27:26(2.908) 1048 (1.358) $1.07 (4057) 6858 (L391) 2185 (@250) 34.19.1109) 097 (0.089) ‘Standard deviations ae in brsckaws ‘was colonized in a shorter time, thus permitting less growth of competitors No significant differences in the mushroom average number per bag were found between treatments and con- ‘rol; however, probability of seeing these differences was, also very close to the limit (P = 0.053). At the end of a second flush, treatments SSH/SMS (50:50) and SSH/BR_ (70:30) showed the higher average number of mushrooms per bag (3 mushrooms) and those giving the lower number ‘of mushrooms belonged to control (SSH) and SSH/SMSM (50:50) treatments (I mushroom). Galli (2007) compared yields of A. brasiliensis from substrates composted or non-composted, based on straw of ray grass (Lolium multflorum Lam.) or elephant grass (Pennisetum purpureum Schura,), sterilized or pasteurized, ‘with or without compost conditioning and cased with soil ‘a mix of soil/charcoal, The highest BE he obtained was 48.9% (pasteurized ray grass straw with soil as casing material), followed by 34.4% (elephant grass compost pasteurized with soil as a casing material), and remaining, ten treatments giving values among 33-27.1%. In the present study, the best BE obtained after two flushes was similar to the second better yield reported by Galli, and the BBE average from the three next best treatments was similar to the third best one that the author mentioned. However, neither quantities of harvested flushes nor cultivation duration are reported by Galli to allow productivity comparisons. ‘The average yields reported in our study are 25% lower than the ones noted on composted. substrates (Andrade et al. 2007; Kopytowski Filho and Minhoni 2004), Considering both the BE and the resistance to contam- ination, the two better substrates were those containing whole SMS in their formulations, in a 1:1 mixture with SSH, or supplemented with 20% BR. In the production of white and brown varieties of A. bisporus, Mamiro and Royse (2008) reported an improvement on non-composted substrate of oak sawdust and millet with the addition of an equal part of SMS from traditional champignon cultivation. Substrate analysis done on the NCS removed at the end of to flushes of A. blacei showed a significant reduction in organic matter between weatments. There was a 7.1% seduction of OM for control substrate (SSH) with 2.8 and 1.5% for SSH/SMS (50:50) and SSE/BR (70:30, respec- Lively. The higher OM reduction was observed in SSH/ ‘erm (80:20) of SMS/BR (80:20), 12 and 154%, respec- tively (Table 7). This overall response was simi 1 those jbserved during the cultivation on composted substrates (Adams and Frostick 2008: Gonzilez Matute 2009). The Joss of the substrte OM during mushroom production is due to mineralization of the substrate, which is directly related to catabolic activites (Sampedo et sl. 2009). Total fiber (TF) decreased in all the substrates. The greatest loss in TF corresponded in the two substrate treatments with the higher OM reduction (22% in both eases), followed by control (17%), SSH/BR (70:30) (15%) and lasly the SSH/SMS (50:50) (5%) (Table 7) In genera, the higher ash increases were observed in those NCS where greater loss of OM and/or TF was ‘observed. The highest increase corresponded to SSH/BR (70:30) (108%), followed by SSHiverm (80:20), SMS/BR (80:20) and control with 78, 67 and 32%, respectively (Table 7) In all the substrate formulations hemicellulose and lig- nin contents decreased by the end of A. blazei eukivation. However, cellulose increased between 3.5 and 5.5%, except in SMS/BR (80:20) where it diminished by 114% (Table 7). Sharma (1991) argued that 4, bisporus prefer- entially uses hemicelluoses associated with lignin instead fof the fraction in association with cellulose, The greatest hemicellulose loss was observed in control substrate (SSH) (62%), followed by SMS/BR (80:20), SSH/verm (80:20) 2 springer1338 World J Microbiol Biotest! (2011) 27:1531-1339 and SSH/BR (70:30), with decrease of 54, $3 and 49%, respectively. In the SSH/SMS (50:50), hemicellulose reduction was only 8.5%. Major lignin disappearance was observed in the SSH/verm (80:20) and control, 56 and 41%, respectively. In the remaining substrate treatments, 'SMS/BR (80:20), SSH/BR (70:30) and SSH/SMS (50:50), lignin reduction was 30, 27 and 25%, respectively (Table 7). During mushroom cultivation, the carbon source in substrate medium is consumed, transformed and partly liberated as CO, or energy, while a fraction of the nitrogen source in the substrate medium is absorbed and assimilated by the mushroom and hence transformed. Together with the residual Nin the substrate this makes the total N increase, hence decreasing the substrate C/N relationship (Sampedro et al. 2009). This was also true forall the NCS evaluated at the end of two flushes, The major N increase ‘was observed in the control (39%), followed by SSH/SMS. (50:50) (24%), SSH/verm (80:20) (149), SMSIBR (80:20) (7%) and SSH/BR (70:30) (1%), Carbon content reduction was between 6 and 13% (Table 7), Interestingly, the NCS with the greatest biodegradation of its components, SSH/verm (80:20) and SMS/BR (80:20), were also the ones with higher productivity, ie. 30 and 61% higher in relation to control substrate medium (0.13%). In addition, they showed the lower degree of substrate contamination, Conclusions Non-composted substrate containing sunflower seed hulls, waste from the edible oil industry, oF spent oyster mush- room substrate, based on SSH, with the addition of 20% peat or verinicompos, increases the A. blaze growth rate ‘compared to the growth media containing the main com- Ponents alone, Moreover, A. blazeé mycelia grow faster in spent oyster mushroom substrate than in sunflower seed hulls Cuhivation of A. blacei on non-compostod substrates containing sunflower seed hulls with an equal part of P. pulmonarius SMS, oF 20% verricompost, or SMS with 20% of brewery residue, is feasible with yields similar to composted substrates, The choice of additives to the for- mula may affect the resistance to competitor fungi during the mycelium running or incubation. Substrate nutrient enrichment posse the contamination prevention on top of the cares to be considered for a successful cultivation and for that reason it is recommended that extreme measures should be adopted to assure complete substrate sterilization before use, together with « very clean procedure on all the mushroom cultivation steps. Its also emphasized the need of preserving a clean environment by controlling possible springs contamination vectors and by filtering the air to be used for ventilation of the mushroom houses, as it is usual for a good cultivation practice. This approach of producing A. blazet in non-composted substrates should be considered as an alternative to the traditional composting. process, allowing more people to undertake Iow cast mushroom production. In general, the lignin-hemicetlulose fraction was pref- crentially used as carbon source over the cellulose-linked lignin during the cultivation of A. blazei in the sunflower based non-composted substrates. Mushroom productivity is positively linked to the loss of the lignin-hemicellulose fraction, Thus, the selection of compost formulations ‘would favor those with the greater loss of this fraction. Acknowledgments This research was supported by the Consejo Nacional de Investigaciones Cientifcas y Técnicas (CONICET), the Comisiin de Investigaciones Cientfeas dela Provincia de Buenos Altes (CIC, La Plats, Argentina) and Universidad Nacional det Sur (CNS, Bahia Blanca, Argentina). We also thank Ricardo Devalis for his helpful technical assistance, References ‘Adams JDW, Frostick LE (2008) Investigating microbial wtvitis in ‘compost using mushroom (Agaricus bisporus) cultivation as an experimental system. Bioresour Techs! 99:1097-1 102 Andrade MCN, Kopytowski Filho J, Minhoni MTA, Coutinho LN. Figueiredo MB (2007) Productivity, biological efciency, and number of Agaricus lace! mushrooms erown in compest inthe presence of Trichoderma sp. and. Chaetomium olivacearum contaminants. 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Eira ‘AF (2002) Isa widely culated culinary medicinal Royal Sun Agaricis (the Himematsutake mushroom) indeed Agarcur braced Murs? tne } Med Mushrooms 4267-290 ‘Wasser SP. Didukh MY, Amazonas MAL, Nevo B, Stamets P. Eira ‘AF (2005) Is widely cokivated eolinary-medicinal royal sun ‘Agaricus (Champignon do Brazil or the himematsutake mush- oom) Agaricus Braslenss S. Wasser etl. indeed a ssnonyin of A ebeufecens peck? Int Med Mushooens7:507-311 springer