Journal of Horticultural Science & Biotechnology (2004) 79 (2) 234-240

Soaking of substrate in alkaline water as a pretreatment for thecultivation of Pleurotus ostreatus

By E. P. CONTRERAS, M. SOKOLOV, G. MEJIA and J. E. SANCHEZ'.£1 Colegio de la Frontera Sur. Apdo. Postal 36, Tapachula, Chiapas 30700, Mexico(e-mail:

esanchez@tap-ecosur.edu.mx) (Accepted 16 August 2003)

SUMMARYSubstrate for cultivating Pleurotus ostreatus was prepared by soaking it in water containing 0.5% lime with no furtherthermal treatment. Five substrates were used: grass (Digitaria decumbens), com cob, com straw and two mixtures ofcom straw or grass with coffee pulp. Immersion time varied between 0-48 h. Mushroom yield measured as biologicalefficiency varied between 37.3 and 126%. No fungal contamination was found, however certain group of bacteria likepseudomonads, bacilli and coliforms were detected. This technique seems to have potential; however good control ofthe moisture of the substrate must be maintained to limit proliferation of substrate bacteria.

suggested by Hernandez et al. (2003). These authorsadjusted the initial pH values of the substrate with limeto 8.4-8.5 and found that even without composting it waspossible to achieve values of Biological Efficiency (BE)around 80% in two harvests.

Few reports are available on the cost of pasteurizing akilogram of substrate for Pleurotus cultivation. However,some estimates can be drawn from the work of Verma(2002) on total production cost. Although there are noexact figures for the difference in the cost of pasteuriza-tion by heat v pasteurization by soaking, it is clear thatthe cost of soaking is much lower because it requires noenergy outlay. This fact has significance in rural commu-nities where simple, fleXible technology is required, andwhere there is a need to identify substrates which maycontribute to energy savings and increased productionefficiency.

The object of this study was to evaluate the technicalviability of alkaline water soaking as a substrate prepa-ration technique for P. ostreatus cultivation. The BEsobtained using this method with five alternative sub-strates (or mixtures) was also determined.

MATERIALS AND METHODSStrain and spawn

Pleurotus ostreatus strain ECS-O152 from the ECO-SUR mycological collection was used for this study. Thesubstrate was inoculated at a rate of 5% with spawn pre-pared on sorghum seeds (Quimio,2002).

Substrate preparation: About 10 kg of raw materials werebroken into 3-4 cm pieces using a mill hammer (Sankeand Hunkel, Model A-1052) and then soaked in a 75 Iplastic container containing 20 I tap water (temperatureof 25 :I: 2°C) with 0.5% added lime (commercial limefrom Calhidra Balun, Canan Inc, Mexico with 81.5-82%calcium hydroxide content, 0.75-0.9% calcium oxide, and45.2-44.7 calcium content). Soaking time varied from 0 to48 h depending on the experiment. (Soaking for 5 minwas considered "0" time.) After the soaking period, the

A voidance of substrate contamination is an impor-tant part of the cultivation of edible mushrooms.

Competitor bacteria and undesirable fungi in the sub-strate drain nutrients as well as limiting mushroomgrowth and fruiting, thus reducing yield and quality.

Various treatments are used for the preparation of thesubstrate used to cultivate the oyster mushroomPieurotus spp.- steam pasteurization, submersion in hotwater, composting, chemical sterilization, and lactic fer-mentation. (Ferri, 1985; Kurtzman, 1979; Leiley andJanssen, 1993; Muez and Pardo, 2002; Soto Velazco et ai.,1989; Vijai and Sohi, 1987; Zadrazil and Schneidereit,1972). Each of these methods has advantages and disad-vantages depending on the prevailing technological situ-ation. (Laborde, 1989; Pani and Mohanty, 1998;Rajararhnam and Bano, 1987; Royse and Schisler, 1987).

In the state of Chiapas, Mexico, growers of Pieurotuscommonly prepare the substrate by simply soaking ligno-cellulosic residues in alkaline water for various times. Thisis a low output, empirical technique used by very smallscale growers. A farm at Jomanichim, in the municipalityof Tenejapa, which has a capacity of two tonnes of mush-rooms per month and grows P. ostreatus on com straw,represents the largest and best known of these smalloperations. This farm has operated fairly continuously fortwo years, apparently without contamination problemsand with biological efficiencies (BE) averaging 40-50%(Sanchez, 2001). The above-mentioned soaking techniquehas replaced submersion in hot water over the last fewyears in Chiapas because of the contamination problemsobserved (Guzman et ai., 1993; Lopez et ai., 1996).

Philippoussis et ai. (2001) reported that soaking thesubstrate in water with 2% calcium carbonate to a pH of6-7 followed by sterilization was an efficient method forpreparation for cultivating Pieurotus spp. This methodhad the additional advantage of a shorter colonizationtime than with composting.

The use of a more alkaline base than calcium carbon-ate to avoid the need to sterilize or heat pasteurize was

* Author for correspondence.

E.

P. CONTRERAS, M. SOKOLOV, G. MEJ1A and J. E. SANCHEZ 235

TABLE IPercent substrate (dry weight) used for each of six P. ostreatus crops

Coffee

pulp%

Grass%

Com cob%

Com straw%Substrate

123456*

to 100 bacterial colonies per dish were counted.Bacterial groups studied were: total coliforms,

Pseudomonas spp and Bacillus spp. The specific mediaingredients used for each culture were:Total calif arms: Brilliant green bile broth (Bioxon,Beckton Dickenson de Mexico, Inc.) The final pH of themedium was adjusted to 6.9-7.1, and 2% agar added.Pseudomonas group: A combination of Media #1, #54 and#81

(DSM, 1993). Per litre, 5 g peptone of casein (Difco),5 g dextrose (Sigma), 0.1 g. yeast extract (Difco), 0.3gNI4CI (J.T. Baker), 0.3 g KIzPO4 (Reproquifin), 0.3 gCaClz x 2 HzO (Omnichem), 0.2 g (J.T. Baker), 0.1 gNaCI (Merck), and 0.1 g MgO x 6HzO (Omnichem). Themedium was supplemented with a trace elements solutionbased on the SL-10 trace element solution (DSM, 1993)as follows: 10 ml HO; 1.5 g Fe Clz x 4H20; 70 mg ZnClz;80 mg MnCIZ x 2 HZO; 6 mg H3BO3; 190 mg CoClz x 6HzO ; 2 mg CuClz x 2 HZO; 24 mg NiCIZ x 6Hz); 36 mgNa2MoO4x 2 HZO; 3mg NaZSeO3 x 5HZO; 4 mg NaZ W04x HZO. All salts were dissolved in 100 mL of filter steril-ized distilled water. One mL of the stock trace elementsolution was added to each litre of medium.

100100

10070 30

5030

5070

* -Control (Grass 70%-Coffee Pulp 30%, composted)

substrate was placed in plastic trays to_permit drainageand the moisture content was adjusted to approximately70%. Six substrates were used, as shown in Table I.A con-trol was prepared as per Hernandez et al. (2003). Aftersoaking and draining, 950 g of substrate, mixed with 50 gspawn, were placed in 2 kg capacity polyethylene bags.

Spawn run and fruiting.Spawn run was completed in darkness for 15 d at 26°C.

Fruiting was induced at 23°C, 70% moisture, with contin-uous ventilation to maintain low COz (less than 800ppm) in the air. Fruiting bodies were collected andweighed for three flushes.

Bacillus group: Medium #1 (DSM, 1993) supplementedwith -the same salt composition as for the Pseudomonasgroup. Samples were pasteurized for 10 minutes by heat-ing in a water bath (Madigan 1999) for quantification.

Coliforms were incubated at 37°C and Pseudomonasand Bacillus spp were incubated at 300 C for 24 h.Bacterial populations were determined by the spreadplate method (Baton et ai., 1995).

Parameters evaluatedSubstrate moiSture: After soaking and just before spawn-ing, 5 g of substrate were evaluated gravimetrically threetimes for moisture content (80°C for 48 h). pH: Tengrams of substrate was placed in 125 ml of distilled waterand pH was determined with a Model 719A S-OO3584potentiometer (Orion Res. Inc., Boston, MA, USA)Williams (1984).

Biological efficiency (BE)After harvesting, BE was calculated using the follow-

ing formula:BE = Biomass (fresh weight) x 100

Substrate weight (dry basis)

Temperature inside the formed logs; Temperature insidethe logs was measured every 24 h during the spawn-runperiod using a mercury thermometer.

SEM visualization of bacterial contaminantsFor Scanning Electron Microscopy (SEM) a radial

section of 3 mm wide was cut covering all different areasfrom the centre to the edge of the pileus. Sections wererinsed for 5 min with 0.1 M phosphate buffer, pH 7.2,(PBS) and placed in 3% glutaraldehyde in PBS,overnight (Dykstra, 1993). The sections were removed 24h later and rinsed twice in PBS (5 min each) then dehy-drated with serial ethanol solutions (30,50,70,95,100,100%) for 45 minutes. Samples were critical-point dried withCO2 and mounted over an aluminum stub with silverpaint. Finally samples were gold-palladium coveredusing a ion-sputter Denton Vacuum Desk-II and wereobserved under high vacuum conditions at 5 kV.

Bacterial population: After soaking, samples were takento determine the population of various bacteria. Fivegrams substrate were blended with 100 ml of 0.85%NaCI solution in a commercial 12 speed blender(Osterizer, USA). One ml of supernatant was removedand six dilutions were performed serially. From eachdilution tube, 0.1 ml was spread onto the agar surface ofa petri dish containing appropriate medium for each bac-terial group under study. Triplicate petri dishes were pre-pared for each dilution sample; only dishes containing 50

TABLE IIMoisture changes in five substrates after soaking in a 0.5% Ca(OH)2 solution for 0-48

Soakingtime (h)

Grass(Sl)

Com cob(S3)

Grass-Coffeepulp(S4)

Com straw(52)

Com cob-Coffee pulp(55)

012243648

48.0 j: 0.6

71.6 j: 0.6

72.5 j: 1.0

72.0 j: 1.2

73.7 j: 0.3

56.0:t 1.771.5 :t1.471.6:t 0.573.5:t 2.472.9:t 0.0

54.9:1: 1.274.0 :1:0.576.0:1: 0.076.0:1: 1.776.0 :I: 1.5

48.0 ~ 0.573.0~0.072.5 ~ 0.771.6 ~ 0.570. 7~ 1.1

Mean:!: SE of the mean.

51.0 i: 1.069.9 i: 1.770.5 i: 0.570.0 i: 0.570.5 i: 0.5

236 Substrate treatment of pleurotus culture

TABLE IIIEvolution of pH in five substrates after immersion during 0-48 hours in 0.5% Ca(OHh solution. Mean %SE of the mean

Soakingtime (h)

Grass(51)

Com straw(52)

Com cOb(S3)

Grass-Coffee pulp(54)

Com cob-Coffee pulp(85)

0122436

48

12.1 :I: 0.20

11.5 :I: 0.11

11.2 :1:0.05

9.9 :I: 0..07

9.6 :I: 0.09

11.4 % 0.05

11.0 % 0.01

11.4 % 0.07

10.1 % 0.15

9.7%0.25

11.2 :I:

10.2 :I:

11.0 :I:

10.8:1:10.0:1:

11.6 :!: 0.06

11.0 :!: 0.05

10.5 :!: 0.06

9.5 :!: 0.05

8.9:!: 0.05

11.1 z 0.0610.1 :t 0.0410.5 :t 0.049.4 z 0.008.4 :t 0.01

logs contained carpophores with a shiny and wet appear-ance, as if they had recently been watered. (Care had infact been taken not to water at the final stages of devel-opment.) Their appearance was different from that ofnormal irrigated mushrooms; the liquid on the pileumwas slightly viscous and sticky to the touch, similar to abacterial film.. This film was probably the result ofPseudomonas bacteria. (Gea, 2002) See Figures 2 to 5.

Analysis of bacterial population was therefore per-formed on the substrate at the time of spawning, as seenin Figures 6 a-e. The bacterial population of each sub-strate at spawning was noted. In the grass substrate, thecorn straw, and the mixture of grass and pulp, the bacte-rial population remained at low levels from 0-36 h ofsoaking for all three bacteria under study(Pseudomonas, bacilli, ~nd coliforms). In these threesubstrates a high level of bacteria, principallyPseudomonads, (3x106- 107 cell ml-l) was observed aftersoaking in the alkaline solution for 48 h. In the treat-ments containing corn cobs, there was more contamina-tion, mainly from Pseudomonads, although bacilli andcoliforms were also observed.

Experimental design and statistical analysisThe experiment consisted of five t~atments with five

replicates per treatment arranged in randomized blocks,with blocks set in July, August, and September. An analy-sis of variance was performed on BE, and means wereseparated using Tukey's test with a = 0.05 (Steel andTome, 1988). For temperature, moisture and microbialpopulation, a repeated measures analysis was performed(Scheiner and Gurevitch, 1993). In all cases the ANOV Aprocedure (SAS Institute, 1999) was used.

RESULTSTemperature, moisture, and pH

The temperature of the liquid used for soakingremained at 28-29°C during the entire operation.Temperature at the centre of the logs during the incuba-tion also varied very little (25-28°C) for all treatments,with no statistical differences.

Table II shows changes in substrate humidity for eachtreatment, after soaking in lime and air draining.Moisture generally varied from 48 % (com cobs; comcobs+coffee pulp at 0 hours soaking) to 76% (com strawat 24-48 hours soaking.) Statistical analysis showed sig-nificant differences (P=O.OOOl) for time and the interac-tion of time with substrates. With minimum immersionstime (0), moisture varied between 48 and 56% depend-ing on the substrate, and for substrates soaked for 12 h ormore hours, mo~sture after rinsing and draining washigher, at 70- 76%. The pH of the substrate after soakingand draining and just before spawning is indicated inTable III. Observed values varied between 8.4 and 12.1.A statistical analysis indicated significant differences(P=O.OOl) for time and the interaction of time with sub-strates. Substrates for "0" soaking (immersion for 5 minfollowed by rinsing) had the highest pH values (11.1-12.1). These values decreased for all substrates withsoaking time and their value varied with the substrate,from 10 (com cob) to 8.4 (com cob + coffee pulp). ThepH observed for the control (grass + composted pulp)was 8.7.

Biological efficiencyTable IV presents the BE obtained for each substrate

spawned with P. ostreatus after 48 h of soaking. The effi-ciencies obtained vary from 82.9% (com straw) to 126%(grass). Statistical analysis resulted in the identificationof three significantly different groups: Group A, the grasssubstrate, with the highest BE (126%); Group B, whichincluded com straw; grass + coffee pulp; composted sub-strate), (BEs from 82.9 to 90%), and Group C (comcobs), with the lowest BE (60.9%)..

Table V presents the BEs obtained when P. ostreatuswas spawned in grass previously treated by soaking inalkaline water for different times. BEs obtained variedfrom 32% (immersion for 5 min and then rinsed) to 95%

TABLE IVBiological efficiency for ECS-O152 Pleurotus ostreatus cultivated on

five substrates and immersed in 0.5% lime for 48 h

Treatment Substrate Biological efficiency (%)*

1245

6

3

GrassCom straw

Grass coffee pulpCom cob coffee pulp

CompostedCom cob

126.0 a82.9 b84.0b87.2 b9O.0b60.9 c

Colonization and contamination levelsThe growth of P. ostreatus during incubation was

homogeneous for all treatments. It also appeared similarto the control and to other reports regarding pasteurizedsubstrates on which this mushroom has been cultivated.(Villa Cruz et al., 1999; Morales et al., 1995). The sub-strate was fully colonized in 14-15 d and no fungal con-tamination was observed (Figure 1).

At harvesting, although most carpophores appearednormal, soft, and dry to the touch, some of the harvested

* The same letter in the same column means no statistic~l difference at

the level a=O.5. C. V.=16.8%

0.040.060.010.010.05

E. P. CONTRERAS, M. SOKOLOV, G. MEJIA and J. E. SANcHEZ 237

TABLE V

Biological efficiency of Pleurotus ostreatus ECS-O152 cultivated onDigitaria decumbens grass, previously soaked in water with lime for

times indicated

Immersion time (h) Biological efficiency (%)

0

12

24

3648

37.3 b93.7 a

95.1 a93.2 a

92.5 a

* The same letter in the same column means no statistical difference atthe a=O.5IeveL C.V.=16.1 %

FIG. 3Surface of the pileus of a contaminated carpophore seen in the SEM.

FIG.!The appearance of healthy carpophores grown on unpasteurized D.decumbens grass, prepared by soaking in water with 0.5% Ca(OH)2. FIG. 4

Healthy carpophores of P. ostreatus.

FIG. 2P. ostreatus carpophores with evidence of bacterial contamination.

FIG.SSurface of the pileus of a healthy carpophores seen in the SEM.

Substrate treatment of pleurotus culture

(24 h soaking). Analysis resulted in the identification oftwo statistically different groups at the 0.5% level:Group A (control, and all soaking treatments from 12 to48 h) had higher BEs, and Group B (immersion fqr 15min) had a lower BE.

vests. The results, with BEs ranging from 37% to 126%BE, include data obtained with a control prepared bycomposting, and were comparable with results reportedwith other substrates prepared by various pasteurizationmethods. (Bermudez et al., 1994; Block et al., 1958;Gonzalez and Arzeta, 1994).

From this fact it may be concluded that adjusting pHto alkaline levels is a good means of inhibiting thegrowth of competitor fungi and bacteria without seri-ously affecting the growth of P. ostreatus. This confirmsreports by Stolzer and Grabbe (1999) and Hernandez etal. (2002), who suggested the use of alkaline pHs to cul-tivate this mushroom. In our experiment, the commondeuteromycete contaminants Trichoderma, Monilia,Penicillium, and Aspergillus, were not observed. Thepileus of some of the carpophores displayed someabnormal moist and slimy shininess, presumably fromPseudomonas bacteria, which cause yellow blotch onPleurotus spp. (Stamets and Chiltons, 1983; Fermor,1987; Gea, 2002), but the pronounced deformation of

DISCUSSIONThe technical feasibility of preparing substrate for the

cultivation of P. ostreatus by soaking in a 0.5% alkalinesolution without heat treatment was investigated. BEsvarying from 37 to 126% were obtained for various sub-strates and mixtures of substrates tested in three har-

a} Grass

d) Grass 70%- Coffeepulp 30 %

b) Corn straw

/'

18OE~

1.4OE~1.2OE~

:) 1.1IOE~8.00e.os

j e.ooeoOS'.ooeoos200eoOS ----~oooe.oo ..., , .,-

0 12 ~ Ie

TIME (HOURS)

..~1XE~1 ""~:: V~-

2' :JI

TIME (HOURS)

e

,

e) Com cob 500/... Coffeepulp 50%

l.--

c) Com cob

FIG. 6Population of three bacterial groups (pseudomonads, bacilli and coliforms) in five substrates as a function of soaking time in 0.5% linIe and water atroom temperature: a) Grass (Digitaria decumbens) b) Corn straw, c) Corn cob, d) Grass+coffee pulp, e) Corn cob + coffee pulp.

[ffiHJ Pseudomonas spp. D Bacillus spp. ~~ Coliforms

f~.~1V~+04 8~+04 7~+04 ::) ~~+04

u.: 5~+04

U '~+04".

E. P. CONTRERAS, M. SOKOLOV, G. MEJiA and J. E. SANCHEZ 239

toredior each substrate. The grass, straw, and straw + cof-fee pulp in 0.5% lime did not display bacterial growth inthe first 36 h of soaking, although after 48 h, bacterialgrowth did occur. The com cobs and coffee pulp + comcobs, however, displayed bacterial contamination from12 h of soaking onwards. The size of the com cob parti-cles was probably a factor in this, since it is a den.se mate-rial and even when broken into 3 cm pieces was not wellpenetrated by the lime, resulting in greater bacterialgrowth. Taking the BE and contamination results intoconsideration, the recommended soaking time for strawin 0.5% lime is 12-36 h.

The preparation of substrates by soaking in alkalinesolution is a technique which this study indicates haspotential. However good control of the moisture of thesubstrate must be maintained to limit proliferation ofsubstrate bacteria.

The authors would like to thank Consejo Nacional deCiencia y Tecnologia (CONACyT) for the financial sup-port given to this work (Proyecto No. 34199-B), to JavierValle for his help in the statistical analysis of the data andto Maria G. Nieto for her invaluable help in the SEM.

the carpophores reported by Bessette et al. (1985) wasnot observed. Since Pseudomonas bacteria proliferatein moist areas, and are disseminated primarily fromwater and secondarily through spawn and other materi-als used for mushroom cultivation (Stamets, 1983), highmoisture along with a high initial bacterial populationon the substrate explain the appearance of the symp-toms described on the affected mushrooms.

The substrate which produced the best BE and leastcontamination was the Digitaria decumbens grass. Thissubstrate has already been reported as suitable for thecultivation of P. ostreatus; however in that report it waspasteurized and composted (Hemandez et al., 2003).Other substrates tested were less efficient; however, it isprobable that with better moisture control, higher BEscould be obtained. Methods employing water soakingare known to present difficulties in controlling the initialmoisture of the substrate, since humidity varies betweenlots and even within a single lot. This causes differencesin fruiting and a higher risk of contamination (Lopezet aI, 1996). .

The effect of soaking in alkaline solution on the bac-terial population at the end of the soaking was moni-

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