Soaking of substrate in alkaline water as a pretreatment for

Journal of Horticultural Science& Biotechnology(2004)79 (2) 234-240
Soaking of substrate in alkaline water as a pretreatment for the
cultivation 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)
(Accepted16 August 2003)
SUMMARY
Substratefor cultivating Pleurotusostreatuswasprepared by soaking it in water containing0.5% lime with no further
thermal treatment.Five substrateswere used:grass(Digitaria decumbens),com cob, com strawand two mixtures of
com straw or grasswith coffee pulp. Immersiontime varied between0-48 h. Mushroomyield measuredas biological
efficiency varied between37.3and 126%.No fungal contaminationwas found, however certain group of bacteria like
pseudomonads,bacilli and coliforms were detected.This techniqueseemsto have potential; howevergood control of
the moisture of the substratemust be maintainedto limit proliferation of substratebacteria.
A
voidance of substrate contamination is an important part of the cultivation of edible mushrooms.
Competitor bacteria and undesirable fungi in the substrate drain nutrients as well as limiting mushroom
growth and fruiting, thus reducing yield and quality.
Various treatmentsare used for the preparationof the
substrate used to cultivate the oyster mushroom
Pieurotusspp.- steam pasteurization,submersionin hot
water, composting,chemicalsterilization, and lactic fermentation. (Ferri, 1985; Kurtzman, 1979; Leiley and
Janssen,1993;Muez and Pardo,2002;SotoVelazco et ai.,
1989; Vijai and Sohi, 1987; Zadrazil and Schneidereit,
1972).Each of thesemethodshas advantagesand disadvantagesdependingon the prevailing technologicalsituation. (Laborde, 1989; Pani and Mohanty, 1998;
Rajararhnamand Bano, 1987;Royseand Schisler,1987).
In the state of Chiapas,Mexico, growersof Pieurotus
commonlypreparethe substrateby simplysoakinglignocellulosicresiduesin alkaline water for varioustimes.This
is a low output, empirical technique used by very small
scalegrowers.A farm at Jomanichim,in the municipality
of Tenejapa,which hasa capacityof two tonnesof mushrooms per month and grows P. ostreatuson com straw,
represents the largest and best known of these small
operations.This farm hasoperatedfairly continuouslyfor
two years, apparently without contamination problems
and with biological efficiencies (BE) averaging40-50%
(Sanchez,2001).The above-mentionedsoakingtechnique
has replaced submersionin hot water over the last few
years in Chiapasbecauseof the contaminationproblems
observed(Guzmanet ai.,1993;Lopez et ai.,1996).
Philippoussis et ai. (2001) reported that soaking the
substratein water with 2% calciumcarbonateto a pH of
6-7followed by sterilization was an efficient method for
preparation for cultivating Pieurotus spp.This method
had the additional advantageof a shorter colonization
time than with composting.
The use of a more alkaline base than calciumcarbonate to avoid the need to sterilize or heat pasteurizewas
*Author for correspondence.
suggestedby Hernandez et al. (2003). These authors
adjustedthe initial pH values of the substratewith lime
to 8.4-8.5and found that evenwithout compostingit was
possibleto achievevalues of Biological Efficiency (BE)
around 80% in two harvests.
Fewreports are available on the costof pasteurizinga
kilogram of substratefor Pleurotuscultivation. However,
some estimatescan be drawn from the work of Verma
(2002) on total production cost. Although there are no
exactfigures for the difference in the costof pasteurization by heat v pasteurizationby soaking,it is clear that
the costof soakingis much lower becauseit requires no
energyoutlay.This fact has significancein rural communities where simple,fleXible technologyis required, and
where there is a need to identify substrateswhich may
contribute to energy savingsand increased production
efficiency.
The object of this study was to evaluatethe technical
viability of alkaline water soaking as a substratepreparation technique for P. ostreatuscultivation. The BEs
obtained using this method with five alternative substrates(or mixtures) wasalso determined.
MATERIALS AND METHODS
Strain and spawn
Pleurotus ostreatusstrain ECS-O152from the ECOSUR mycologicalcollection was used for this study.The
substratewasinoculated at a rate of 5% with spawnprepared on sorghumseeds(Quimio,2002).
Substrate
preparation:About 10kg of raw materialswere
broken into 3-4 cm piecesusing a mill hammer (Sanke
and Hunkel, Model A-1052) and then soaked in a 75 I
plastic containercontaining 20 I tap water (temperature
of 25 :I: 2°C) with 0.5% added lime (commercial lime
from Calhidra Balun, CananInc, Mexico with 81.5-82%
calciumhydroxide content,0.75-0.9%calcium oxide,and
45.2-44.7calciumcontent).Soakingtime varied from 0 to
48 h dependingon the experiment.(Soaking for 5 min
was considered"0" time.) After the soaking period, the
E.
P. CONTRERAS,
M. SOKOLOV,
G. MEJ1Aand J. E. SANCHEZ
TABLEI
Percentsubstrate(dry weight)usedfor each ofsix P. ostreatuscrops
Coffee
Grass
Substrate
1
%
Com straw
%
Com cob
%
pulp
%
100
100
2
3
4
5
6*
100
30
70
50
50
30
70
* -Control (Grass70%-CoffeePulp 30%, composted)
substratewas placed in plastic trays to_permit drainage
and the moisture contentwas adjustedto approximately
70%. Six substrateswere used,asshownin Table I.A control was prepared as per Hernandez et al. (2003).After
soakingand draining,950g of substrate,mixed with 50 g
spawn,were placed in 2 kg capacitypolyethylenebags.
Spawn run and fruiting.
Spawnrun wascompletedin darknessfor 15d at 26°C.
Fruiting wasinduced at 23°C,70% moisture,with continuous ventilation to maintain low COz (less than 800
ppm) in the air. Fruiting bodies were collected and
weighed for three flushes.
235
to 100 bacterial colonies per dish were counted.
Bacterial groups studied were: total coliforms,
Pseudomonas spp and Bacillus spp. The specific media
ingredients used for each culture were:
Total calif arms: Brilliant green bile broth (Bioxon,
Beckton Dickenson de Mexico, Inc.) The final pH of the
medium 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), 0.3g
NI4CI (J.T. Baker), 0.3 g KIzPO4 (Reproquifin), 0.3 g
CaClz x 2 HzO (Omnichem), 0.2 g (J.T. Baker), 0.1 g
NaCI (Merck), and 0.1 g MgO x 6HzO (Omnichem). The
medium was supplemented with a trace elements solution
based 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 6
HzO ; 2 mg CuClz x 2 HZO; 24 mg NiCIZ x 6Hz); 36 mg
Na2MoO4x 2 HZO; 3mg NaZSeO3 x 5HZO; 4 mg NaZW04
x HZO. All salts were dissolved in 100 mL of filter sterilized distilled water. One mL of the stock trace element
solution was added to each litre of medium.
Bacillus group: Medium #1 (DSM, 1993)supplemented
with -thesame salt compositionas for the Pseudomonas
group.Sampleswere pasteurizedfor 10 minutes by heating in a water bath (Madigan 1999)for quantification.
Coliforms were incubated at 37°C and Pseudomonas
and Bacillus spp were incubated at 300 C for 24 h.
Bacterial populations were determined by the spread
plate method (Baton et ai.,1995).
Parametersevaluated
SubstratemoiSture:After soakingand just before spawning,5 g of substratewere evaluatedgravimetricallythree
times for moisture content (80°C for 48 h). pH: Ten
Biological efficiency (BE)
gramsof substratewasplaced in 125ml of distilled water
After harvesting,BE was calculated using the followand pH was determined with a Model 719A S-OO3584 ing formula:
potentiometer (Orion Res. Inc., Boston, MA, USA)
BE = Biomass (fresh weight) x 100
Williams (1984).
Substrateweight (dry basis)
Temperatureinside the formed logs;Temperatureinside
the logs was measuredevery 24 h during the spawn-run
period usinga mercurythermometer.
Bacterialpopulation: After soaking,sampleswere taken
to determine the population of various bacteria. Five
grams substrate were blended with 100 ml of 0.85%
NaCI solution in a commercial 12 speed blender
(Osterizer,USA). One ml of supernatantwas removed
and six dilutions were performed serially. From each
dilution tube,0.1 ml wasspreadonto the agarsurfaceof
a petri dish containingappropriate medium for eachbacterial group under study.Triplicate petri disheswere prepared for eachdilution sample;only dishescontaining50
SEM visualizationof bacterialcontaminants
For Scanning Electron Microscopy (SEM) a radial
sectionof 3 mm wide was cut covering all different areas
from the centre to the edge of the pileus. Sectionswere
rinsed for 5 min with 0.1 M phosphate buffer, pH 7.2,
(PBS) and placed in 3% glutaraldehyde in PBS,
overnight (Dykstra, 1993).The sectionswere removed 24
h later and rinsed twice in PBS (5 min each) then dehydrated with serial ethanolsolutions(30,50,70,95,100,100
%) for 45 minutes.Sampleswere critical-point dried with
CO2 and mounted over an aluminum stub with silver
paint. Finally samples were gold-palladium covered
using a ion-sputter Denton Vacuum Desk-II and were
observedunder high vacuumconditions at 5 kV.
TABLE II
Moisture changes in five substrates after soaking in a 0.5% Ca(OH)2 solution for 0-48
Grass
time (h)
(Sl)
Com straw
(52)
0
56.0:t 1.7
71.5:t1.4
71.6:t 0.5
73.5:t 2.4
72.9:t 0.0
54.9:1: 1.2
74.0 :1:0.5
76.0:1:0.0
76.0:1: 1.7
76.0 :I: 1.5
Soaking
12
24
36
48
Mean:!: SE of the mean.
Com cob
(S3)
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
Grass-Coffee
pulp
(S4)
51.0i: 1.0
69.9i: 1.7
70.5 i: 0.5
70.0i: 0.5
70.5i: 0.5
Com cob-Coffee pulp
(55)
48.0~ 0.5
73.0~0.0
72.5~ 0.7
71.6~ 0.5
70.7~ 1.1
236
Substratetreatmentof pleurotus culture
TABLEIII
Evolution of pH in five substratesafter immersionduring 0-48hours in 0.5% Ca(OHh solution.Mean %SE ofthe mean
Soaking
time (h)
0
12
2436
48
Grass
Com straw
(52)
Com cOb
(S3)
Grass-Coffeepulp
(54)
Com cob-Coffeepulp
(85)
12.1 :I: 0.20
11.4 % 0.05
11.2 :I: 0.04
11.6 :!: 0.06
11.5 :I: 0.11
11.0 %0.01
10.2 :I: 0.06
11.0 :!: 0.05
10.5 :!: 0.06
11.1z 0.06
10.1:t 0.04
10.5:t 0.04
9.4z 0.00
8.4:t 0.01
(51)
11.2 :1:0.05
11.4 %0.07
11.0 :I: 0.01
9.9 :I: 0..07
10.1 %0.15
10.8:1:0.01
10.0:1:
9.6 :I: 0.09
9.7%0.25
Experimentaldesignand statisticalanalysis
The experimentconsistedof five t~atments with five
replicates per treatment arranged in randomizedblocks,
with blocks set in July,August,and September.An analysis of variance was performed on BE, and meanswere
separated using Tukey's test with a = 0.05 (Steel and
Tome, 1988). For temperature,moisture and microbial
population, a repeatedmeasuresanalysiswasperformed
(Scheinerand Gurevitch,1993).In all casesthe ANOV A
procedure (SAS Institute, 1999)wasused.
RESULTS
Temperature,
moisture,and pH
The temperature of the liquid used for soaking
remained at 28-29°C during the entire operation.
Temperatureat the centre of the logs during the incubation also varied very little (25-28°C) for all treatments,
with no statisticaldifferences.
Table II showschangesin substratehumidity for each
treatment, after soaking in lime and air draining.
Moisture generally varied from 48% (com cobs; com
cobs+coffeepulp at 0 hours soaking)to 76% (com straw
at 24-48hours soaking.) Statisticalanalysisshowedsignificant differences (P=O.OOOl)
for time and the interaction of time with substrates.With minimum immersions
time (0), moisture varied between 48 and 56% depending on the substrate,and for substratessoakedfor 12 h or
more hours, mo~sture after rinsing and draining was
higher, at 70-76%.The pH of the substrateafter soaking
and draining and just before spawningis indicated in
Table III. Observedvaluesvaried between8.4 and 12.1.
A statistical analysis indicated significant differences
(P=O.OOl)for time and the interaction of time with substrates.Substratesfor "0" soaking (immersion for 5 min
followed by rinsing) had the highest pH values (11.112.1). These values decreased for all substrates with
soaking time and their value varied with the substrate,
from 10 (com cob) to 8.4 (com cob + coffee pulp). The
pH observed for the control (grass + compostedpulp)
was8.7.
Colonizationand contaminationlevels
The growth of P. ostreatus during incubation was
homogeneousfor all treatments.It also appearedsimilar
to the control and to other reports regardingpasteurized
substrateson which this mushroomhas beencultivated.
(Villa Cruz et al., 1999;Morales et al., 1995).The substrate was fully colonized in 14-15d and no fungal contamination wasobserved(Figure 1).
At harvesting,although most carpophoresappeared
normal, soft,and dry to the touch,someof the harvested
0.05
9.5 :!: 0.05
8.9:!: 0.05
logs containedcarpophoreswith a shiny and wet appearance,as if they had recently beenwatered. (Care had in
fact been taken not to water at the final stagesof development.) Their appearancewas different from that of
normal irrigated mushrooms;the liquid on the pileum
was slightly viscousand sticky to the touch, similar to a
bacterial film.. This film was probably the result of
Pseudomonasbacteria.(Gea, 2002)SeeFigures2 to 5.
Analysis of bacterial population was therefore performed on the substrateat the time of spawning,as seen
in Figures 6 a-e.The bacterial population of eachsubstrate at spawningwas noted. In the grasssubstrate,the
corn straw,and the mixture of grassand pulp, the bacterial population remained at low levels from 0-36 h of
soaking for all three bacteria under study
(Pseudomonas,bacilli, ~nd coliforms). In these three
substrates a high level of bacteria, principally
Pseudomonads,(3x106-107cell ml-l) was observed after
soaking in the alkaline solution for 48 h. In the treatments containing corn cobs,there was more contamination, mainly from Pseudomonads,although bacilli and
coliforms were also observed.
Biological efficiency
Table IV presentsthe BE obtained for eachsubstrate
spawnedwith P. ostreatusafter 48 h of soaking.The efficienciesobtained vary from 82.9% (com straw) to 126%
(grass).Statisticalanalysisresulted in the identification
of three significantlydifferent groups:Group A, the grass
substrate,with the highest BE (126%); Group B, which
included com straw;grass+ coffee pulp; compostedsubstrate), (BEs from 82.9 to 90%), and Group C (com
cobs),with the lowestBE (60.9%)..
Table V presentsthe BEs obtained when P.ostreatus
was spawned in grass previously treated by soaking in
alkaline water for different times. BEs obtained varied
from 32% (immersionfor 5 min and then rinsed) to 95%
TABLE IV
Biological efficiencyfor ECS-O152Pleurotusostreatuscultivatedon
five substrates
and immersedin 0.5% lime for 48h
Treatment
1
2
4
5
6
3
Substrate
Biological efficiency(%)*
Grass
Com straw
Grasscoffee pulp
Com cob coffeepulp
126.0a
82.9b
84.0b
87.2 b
9O.0b
60.9c
Composted
Com cob
* The sameletter in the samecolumn meansno statistic~ldifferenceat
the levela=O.5.C.V.=16.8%
E. P. CONTRERAS,
M. SOKOLOV,
G. MEJIA and J. E. SANcHEZ
237
TABLE V
Biological efficiencyof Pleurotus ostreatus ECS-O152cultivated on
Digitaria decumbensgrass, previouslysoaked in water with lime for
timesindicated
Immersion time (h)
Biological efficiency(%)
0
37.3 b
12
93.7 a
24
95.1 a
36
93.2 a
48
92.5a
* The sameletter in the samecolumnmeansno statisticaldifferenceat
the a=O.5IeveLC.V.=16.1%
FIG.3
Surfaceof the pileus of a contaminatedcarpophoreseenin the SEM.
FIG.!
The appearance of healthy carpophores grown on unpasteurizedD
.decumbensgrass,preparedby soakingin water with 0.5% Ca(OH)2.
FIG.4
Healthy carpophores of P. ostreatus.
FIG.2
P. ostreatus carpophores with evidence of bacterial contamination.
FIG.S
Surfaceof the pileus of a healthy carpophoresseenin the SEM.
Substratetreatmentof pleurotus culture
(24 h soaking).Analysis resulted in the identification of
two statistically different groups at the 0.5% level:
Group A (control, and all soakingtreatments from 12 to
48 h) had higher BEs, and Group B (immersion fqr 15
min) had a lower BE.
DISCUSSION
The technical feasibility of preparingsubstratefor the
cultivation of P. ostreatusby soaking in a 0.5% alkaline
solution without heat treatment was investigated.BEs
varying from 37 to 126%were obtained for various substrates and mixtures of substratestested in three har-
vests.The results,with BEs ranging from 37% to 126%
BE, include data obtained with a control prepared by
composting,and were comparablewith results reported
with other substratesprepared by various pasteurization
methods. (Bermudez et al., 1994; Block et al., 1958;
Gonzalezand Arzeta, 1994).
From this fact it may be concluded that adjusting pH
to alkaline levels is a good means of inhibiting the
growth of competitor fungi and bacteria without seriously affecting the growth of P. ostreatus.This confirms
reports by Stolzer and Grabbe (1999)and Hernandezet
al. (2002),who suggestedthe use of alkaline pHs to cultivate this mushroom. In our experiment, the common
deuteromycete contaminants Trichoderma, Monilia,
Penicillium, and Aspergillus, were not observed. The
pileus of some of the carpophores displayed some
abnormal moist and slimy shininess,presumably from
Pseudomonasbacteria, which cause yellow blotch on
Pleurotus spp. (Stamets and Chiltons, 1983; Fermor,
1987; Gea, 2002), but the pronounced deformation of
a} Grass
d) Grass70%- Coffee
pulp 30 %
b) Corn straw
/'
18OE~
1.4OE~
f~.~
::)
8~+04
~~+04
V~+04
7~+04
u.:
5~+04
1.2OE~
:)
1
1.1IOE~
8.00e.os
j e.ooeoOS
'.ooeoos
200eoOS
--
U '~+04
".
~1XE~1
oooe.oo
""
~:: V~-
--~
...,
0
2'
:JI
,
12
~
.,Ie
..
TIME (HOURS)
e
TIME (HOURS)
,
e) Com cob 500/...Coffee
pulp 50%
l.--
c) Comcob
FIG. 6
Populationof three bacterial groups(pseudomonads,
bacilli and coliforms)in five substratesasa function of soakingtime in 0.5% linIe and water at
room temperature: a) Grass (Digitaria decumbens)b) Corn straw, c) Corn cob, d) Grass+coffee pulp, e) Corn cob + coffee pulp.
[ffiHJ
Pseudomonas
spp. D
Bacillusspp.
~~
Coliforms
E. P. CONTRERAS,
M. SOKOLOV,
G. MEJiA and J. E. SANCHEZ
the carpophores reported by Bessette et al. (1985)was
not observed. Since Pseudomonasbacteria proliferate
in moist areas, and are disseminated primarily from
water and secondarily through spawnand other materials used for mushroom cultivation (Stamets,1983),high
moisture along with a high initial bacterial population
on the substrate explain the appearanceof the symptoms described on the affected mushrooms.
The substrate which produced the best BE and least
contamination was the Digitaria decumbensgrass.This
substrate has already been reported as suitable for the
cultivation of P. ostreatus;however in that report it was
pasteurized and composted (Hemandez et al., 2003).
Other substratestested were lessefficient; however,it is
probable that with better moisture control, higher BEs
could be obtained. Methods employing water soaking
are known to presentdifficulties in controlling the initial
moisture of the substrate,since humidity varies between
lots and even within a single lot. This causesdifferences
in fruiting and a higher risk of contamination (Lopez
et aI, 1996).
.
The effect of soaking in alkaline solution on the bacterial population at the end of the soaking was moni-
239
toredior eachsubstrate.The grass,straw,and straw+ coffee pulp in 0.5% lime did not displaybacterial growth in
the first 36 h of soaking, although after 48 h, bacterial
growth did occur.The com cobs and coffee pulp + com
cobs,however, displayed bacterial contamination from
12 h of soaking onwards.The size of the com cob particleswas probably a factor in this,sinceit is a den.sematerial and evenwhen broken into 3 cm pieceswas not well
penetrated by the lime, resulting in greater bacterial
growth. Taking the BE and contamination results into
consideration,the recommendedsoakingtime for straw
in 0.5% lime is 12-36h.
The preparation of substratesby soaking in alkaline
solution is a technique which this study indicates has
potential. However good control of the moisture of the
substrate must be maintained to limit proliferation of
substratebacteria.
The authorswould like to thank ConsejoNacional de
Ciencia y Tecnologia(CONACyT) for the financial support givento this work (ProyectoNo. 34199-B),to Javier
Valle for his help in the statisticalanalysisof the data and
to Maria G. Nieto for her invaluable help in the SEM.
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