Mushroom Biology and Mushroom Products. Sánchez et al. (eds). 2002
UAEM. ISBN 968-878-105-3
SUBMERGED FERMENTATION OF LIGNOCELLULOSIC WASTES UNDER
MODERATE TEMPERATURE CONDITIONS FOR OYSTER MUSHROOM GROWING
SUBSTRATES
1
J. Pardo Núñez1 and C. García Mendoza2
Centro de Investigación, Experimentación y Servicios del Champiñón (CIES), Quintanar del Rey,
Cuenca (Spain).<jpardo.cies@dipucuenca.es>
2
Centro de Investigaciones Biológicas (CIB), CSIC, Madrid, Spain. <cgm@cib.csic.es>
ABSTRACT
Several waste materials derived from agriculture and agroindustrial products (cereal straw, vine
shoots and grapevine stems, dried olive mill waste, kenaf, cotton and flax wastes) have been utilized
as substrates for oyster mushroom cultivation. Ten pairs (1:1 w/w) of these materials were
immersed in water causing an spontaneous semi-anaerobic weak lactic acid fermentation produced
by the material and environmental microorganisms. The study of this microflora showed the
presence of several species of Bacillus, Microccocus, Streptomyces, Geotrichum and Mucor. When
the fermentation terminated the substrates were drained, pressed, spawned and put into polyethylene
bags. The strain used was Pleurotus ostreatus H9. All the conventional stages of the growth cycle
were carried out in environmentally controlled growing rooms. The total period of cultivation
ranged between 53-64 days. Seven of the ten substrates showed three production flushes whereas
the other three substrates exhibited only two flushes. The yield (biological efficiency) of the studied
substrates varied from 106.2 to 50.8 %.
INTRODUCTION
In commercial oyster mushroom (Pleurotus spp) production the preparation of the growing
substrate is considered to be the most critical point in the process. Much research and experience
throughout the world, particularly during the last two or three decades, has resulted in many
methods of treatment for substrates, such as soaking in water, thermal sterilization and semisterilization, dipping in hot water, steam pasteurisation, aerobic thermophilic fermentation,
chemical sterilization, submerged anaerobic or semi-anaerobic mesophilic fermentation, etc.
(Stamets 1993, Muez 1994, Oei 1996, Jandaik 1997).
Taking into account differences in safety risks and depending on the producers’ abilities, some of
these methods are more suitable than others. For this study, submerged semi-anaerobic mesophilic
fermentation was used. Principles of this method are mentioned in Lelley (1985), Lelley and Jansen
(1993) and Stamets (1993). The procedure is based on utilization of the natural resident microflora
from the bulk substrate, and the essence of this technology is to immerse the bulk material in water
for four to ten days in order to induce a weak lactic acid fermentation. The aim of this procedure is
to remove various easily soluble nutrients by means of the activity of natural microflora. This
microflora can then be consumed further on by the mushroom mycelium and the metabolites
generated in the process can kill internal competitors and protect the substrate from other external
competitors.
271
MATERIALS AND METHODS
Selected substrates
Several residual lignocellulosic materials derived from agriculture and agroindustrial products were
selected: cereal straw, vine shoots, grapevine stems, dried olive mill waste, kenaf, cotton and flax
wastes. Each material was characterized physio-chemically following the global technique of
Weende (Quarmby and Allen 1989). Various combinations of two materials were assigned, in
which one or both of the components were physically bulky (straw, cotton waste, kenaf and flax
waste), and the other was fine-structured or milled material (vine shoot and grapevine sawdust and
dried olive mill waste). The selected ten pairs of materials combined in a 1: 1 proportion by weight
were: barley straw plus cotton waste, kenaf plus vine shoot sawdust, kenaf plus dried olive mill
waste, cotton waste plus dried olive mill waste, barley straw plus vine shoot sawdust, kenaf plus
flax waste, kenaf plus grapevine stems, barley straw plus olive dried waste, barley straw plus kenaf
and barley straw plus grapevine stems.
Fermentation process
Each mixture of two materials was submerged in water at 25-26C in a proportion of 1:20-25
(material/water), maintaining the external temperature at 25C. Sucrose at different concentrations
and times was added as a fermentation stimulator (1% of the substrate weight distributed two times:
2/3 at the beginning of the experiment and 1/3 at the fourth day of fermentation). The different
processes were maintained for six to seven days and supernatant samples were taken for their
microbiological evaluation at 24, 72 and 144-168 h, measuring pH and temperature daily. The
process was finished when the pH values became stable or a slight increase was observed.
Microflora isolation and identification
Bacteria and actinomycetes were isolated on actidione-augmented nutrient agar plates. Yeasts and
moulds were isolated on aureomycin and terramycin-augmented Czapek agar plates. For
identification of the microflora the following keys and publications were consulted: Holt et al. 1994
and Barnett and Hunter 1972.
Mushroom cultivation
At the end of the fermentation processes the culture broth was discarded, the materials were
drained, pressed to a moisture content between 72.8-81.6 % (in a manual press), spawned, and then
put into polyethylene bags. The strain used was P. ostreatus H9 (Jacq. ex Fr.) Kumm. (3% fresh
weight substrate), and all the conventional stages of the growth cycle were carried out in
environmentally controlled growing rooms (incubation: 26C; fruiting: 15-17C; 85-90% relative
humidity). Three bags were filled with each substrate and the fresh weight for all the bags were
standardised at 4 Kg. The total period of cultivation ranged from 53 to 64 days.
RESULTS
The odors generated during the fermentation processes soon disappeared from the substrates when
the mycelial colonization began to take place. Table 1 shows the composition of the selected initial
materials. Changes in the pHs of the substrates during the fermentation are shown in Table 2. At
the end of the processes, pH values did not vary widely (5.8-6.0) and were close to the optimum
value for the oyster fungus development (5.8-6.0). Microflora identified in the distinct substrates
272
were: several species of bacteria (Bacillus and Microccocus), actinomycetes (Streptomyces) and
molds (Geotrichum and Mucor), with practically only minor quantitative differences between them
(Table 3). Later, all the substrates were fully colonized by the mycelium and no incidence of pests
and diseases was observed. Similar results are recorded by Lelley and Jansen (1993) and Stamets
(1993). The total period of mushroom cultivation lasted from 53 to 64 days. Seven of the ten
studied substrates showed three production flushes whereas the other three substrates exhibited only
two flushes (kenaf plus grapevine stems, barley straw plus kenaf, and barley straw plus grapevine
stems). After development of P. ostreatus H9 on the substrates, the best biological efficiency was
106.2% (barley straw plus cotton waste) ; the lowest biological efficiency was 50.8% (barley straw
plus grapevine stems) (Table 4).
DISCUSSION
Wood degrading mushrooms such as P. ostreatus are usually cultivated on sawdust or straw, but
new alternative substrates are emerging. The production of mushrooms using waste materials is a
boon for the restoration of the damaged ecosystem of planet earth. All the substrates used in this
study are derived from agriculture and agroindustrial products, in an attempt to adapt oyster
mushroom cultivation to recycling these wastes. Pleurotus spp are edible fungi that colonize
relatively microorganism-free substrates or substrates with low microorganism populations.
Substrates used for cultivating oyster mushrooms support diverse communities of bacteria and
fungi, depending on the type of fermentation techniques employed. The addition of easily utilized
carbohydrates as fermentation starters increases the presence of molds, which are a detriment to
further Pleurotus growth (with the exception of Mucor which exhibits the weakest effect). Bacteria,
on the other hand, rapidly multiply and quickly consume the readily utilizable carbohydrates. This
creates unfavourable conditions for the molds and also produces antibiotics and polysaccharides
which inhibit the growth of competitive microorganisms. However, actinomycetes are partly
stimulative because they are good synthesizers of certain vitamins that stimulate the growth of
mushroom mycelia.
This method does not result in consistent high yields compared to other more expensive heat
treatment techniques. However, if we take the figure 50 % as a minimum for acceptable biological
efficiency (Patra and Pani 1995), the biological efficiency of all the substrates tested can be
considered acceptable. Our results confirm that this method, depending mainly on cleanliness and
the aerobic state of the substrates during colonization (Stamets 1993), can be a suitable, simple and
cheap alternative for oyster mushroom production in small and medium-sized operations. It is also
an appropriate technology for developing countries.
REFERENCES
Barnett, H. L. and B. B. Hunter. 1972. Illustrated genera of imperfect fungi. Burgess. Minneapolis.
Holt, J. G., N. R. Krieg, P. H. A. Sneath, J. T. Staley, and S. T. Williams. 1994. Bergey's Manual of
Determinative Bacteriology. Williams and Wilkins. Baltimore.
Jandaik, C. L. 1997. History and development of Pleurotus cultivation in the world and future prospects. In:
Proceed. Indian Mushroom Conf. Mushroom Society of India, Solan India. 181-192.
Lelley, J. 1985. Pilze aus dem eigenem Garten. BLV Verlagsgellschaft. München.
Lelley, J., A. Janssen. 1993. Productivity improvement of oyster mushroom substrate with a controlled release
nutrient. Mush. News 41: 6-13.
Muez, M. A. 1994. Bases para el cultivo de Pleurotus. In: I Jornadas Técnicas del Champiñón y otros hongos
comestibles. Patronato de Promoción Económica. Diputación Provincial de Cuenca. 129-141.
Oei, P.1996. Mushroom cultivation. Tool Publications. Leiden.
Patra, A. K. and B. K. Pani. 1995. Evaluation of banana leaf as a new alternative substrate to paddy straw for
oyster mushroom cultivation. J. Phytol. Res. 8: 145-148.
273
Quarmby, C. and S. E. Allen. 1989. Organic constituents. In: S. E. Allen (ed). Chemical Analysis of
Ecological Materials. Blackwell Scientific Publication. 160-200.
Stamets, P. 1993. Growing Gourmet and Medicinal Mushroom. Ten Speed Press.
274
Table1. Composition of the all raw materials utilized as substrates.
Moisture
Total
Crude
Crude
N-free
Fat
Ash
content (%)
nitrogen
protein
fiber
extract
14.0
0.48
3.00
50.07
0.79
2.69
43.45
Raw materials
Barley straw
Organic
matter
97.31
117.6
C/N
Cotton waste
9.0
1.29
8.06
52.80
1.47
13.10
24.67
86.90
39.1
Kenaf
9.4
0.75
4.69
44.06
0.50
5.74
45.01
94.26
72.90
Dried olive mill waste
8.3
1.73
10.81
31.47
4.20
14.08
39.44
85.92
28.8
Vine shoot sawdust
11.5
0.80
5.00
52.65
3.07
4.82
34.46
95.18
69.0
Flax waste
Grapevine stem
sawdust
13.0
0.65
4.06
64.01
0.77
4.25
26.90
95.75
85.4
12.4
1.16
7.25
45.33
1.01
9.64
36.67
90.36
45.8
All the values except moisture content and C/N are expressed as % of the total dry matter
Days
Table 2. Evolution of pH along the fermentation process of the mixed materials.
Mixtures
K+OMD CW+OMD
BS+CW K+VSS
BS+VSS K+FW
K+GSS BS+OMDW
W
W
BS+K
BS+GSS
0 Mixtures
immersed (*)
1
-
-
-
-
-
-
-
-
-
-
5.97
5.77
6.07
5.98
6.02
5.88
5.72
6.19
5.96
6.09
2
5.84
6.03
5.21
5.48
6.17
6.11
6.03
5.32
6.00
5.97
3
4
(*)
5
6.15
6.18
5.42
5.65
5.85
5.57
6.23
5.43
5.66
6.27
6.15
5.55
5.85
6.06
5.56
5.44
6.46
5.66
5.66
6.32
5.87
5.17
5.78
5.98
5.50
5.37
6.12
5.73
5.46
6.18
6
5.77
5.18
5.68
5.82
5.56
5.40
5.90
5.92
5.51
5.87
7
5.89
-
5.55
-
5.58
5.42
5.56
-
5.58
5.85
(*) Sucrose addition
BS = barley straw; CW = cotton waste; K = kenaf; VSS = vine shoot sawdust; OMDV = dried olive mill waste; FW = flax waste; GSS = grapevine stem
sawdust
275
Table 3. Percentage of microorganisms isolated from different Pleurotus spp growing substrates.
Substrates
Bacillus spp Microccocus spp
Stretomyces Geotrichum Mucor spp
spp
spp
spp
Barley straw + Cotton
50
22
0
13
15
waste
Kenaf + Vine shoot
53
19
0
12
16
sawdust
Kenaf + Olive mill dried
46
16
11
12
15
waste
Cotton waste + Olive mill
51
20
0
13
16
dried waste
Barley straw + Vine shoot
58
23
0
19
0
sawdust
Kenaf + Flax waste
49
22
0
14
15
Kenaf + Grapevine stem
59
22
0
0
19
Barley straw + Olive mill
dried waste
Barley straw + Kenaf
44
18
16
10
12
52
23
0
14
11
Barley straw + Grapevine
stem
56
16
10
0
18
276
Table 4. Evaluation of different substrates for Biological Efficiency of Pleurotus ostreatus H9.
Fresh weight of
Moisture
Total yield per
Dry substrate per
Time taken for
Substrates (1:1 w/w) substrate per bag
content
bag
bag (g)
1th flush (days)
(g)
(%)
(g)(*)
Barley straw
4000
81.0
760
29
807 ab
Cotton waste
Kenaf
4000
78.7
852
23
848 a
Vine shoot sawdust
Kenaf
Dried olive mill
4000
77.0
920
27
779 ab
waste
Cotton waste
Dried olive mill
4000
72.8
1088
29
880 a
waste
Barley straw
4000
75.7
972
28
701 bc
Vine shoot sawdust
Kenaf
4000
75.8
888
27
585 cd
Flax waste
Kenaf
Grapevine stem
4000
78.6
856
31
544 d
sawdust
Barley straw
Dried olive mill
4000
76.3
948
28
584 cd
waste
Barley straw
4000
81.6
736
35
398 e
Kenaf
Barley straw
4000
76.4
944
32
480 de
Grapevine stem
(*) Values followed by different letters are significantly different ( p  0,05, Tukey´s test)
Yield values are the average of three replications. B.E. = g of fresh mushroom  100 / g of substrate dry matter
277
Biological
Efficiency
(B.E.) (%)
106.2
99.5
84.7
80.9
72.1
65.9
63.6
61.6
54.1
50.8