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UNIVERSITI PUTRA MALAYSIA
PRODUCTION BIOTECHNOLOGY WITH CONTROLLED ENVIRONMENTAL REGIMES, SUBSTRATES AND ENZYME
MARKERS FOR THE SHIITAKE MUSHROOM, LENTINULA EDODES
GANISAN KRISHNEN
FP 2001 17
PRODUCTION BIOTECHNOLOGY WITH CONTROLLED
ENVIRONMENTAL REGIMES, SUBSTRATES AND ENZYME MARKERS
FOR THE SHIITAKE MUSHROOM, LENTINULA EDODES
By
GANISAN KRISHNEN
Thesis Submitted in Fulfilment of the Requirement for the Degree of
Master of Science in the Faculty of Agriculture Universiti Putra Malaysia
June 2001
Abstract of thesis presented to the Senate ofUniversiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
PRODUCTION BIOTECHNOLOGY WITH CONTROLLED
ENVIRONMENTAL REGIMES, SUBSTRATES AND ENZYME MARKERS FOR THE SIDITAKE MUSHROOM, LENTINULA EDODES
By
GANISAN KRISHNEN
June 2001
Chairman: Tan Yee How, Ph.D.
Faculty : Agriculture
Mycelial growth of L. edodes was optimum at 25°C in six of the seven
strains of L. edodes tested. Mycelial biomass production was greater on sawdust than
grain with 0.53 to 1.60 mg glucosamine per 200 g substrate derived from sawdust
compared to 0.02 to 0.2 mg glucosamine per 200 g from grain. In fructification, a
fluctuating temperature set of 15°C (8 h)/25°C (16 h), with continuous lighting at 25-
35 lux, humidity of 80-95% and aeration of 1.3 - 2.5 mls produced the highest yield
or Biological Efficiency (RE.) in 11 of the 13 strains tested at three temperature sets.
Strains L38 (7.1-32.7% B.E.) and L300 (7.2-35.5% RE.) were the best yield
producers at all three temperature sets. Mycelial density during spawn-run was found
to be uncorrelated with yield in the 13 strains tested.
In the present study, specific activities of three lignocellulolytic enzymes,
laccase, carboxyrnethylcellulase (CMC'ase) and xylanase indicated that the second
week and 17th week· was the correct time to initiate fruiting on liquid and rubber
sawdust medium respectively. In liquid medium, laccase specific activity reached its
highest specific activity of 136.69 Umin-l (mg proteinrl on the second week of
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incubation before recording a sharp decline prior to primordia initiation on the third
week and recorded its lowest activity of 2.88 Umin-1 (mg proteiny} during the
development of mature fruit body which occurred at the fourth week of incubation.
In contrast, xylanase showed a decline in specific activity during the first two weeks
of incubation but increased prior to primordia initiation on the third week and until
the development of mature fruit body the fourth week where xylanase specific
activity was at its highest at 39.6 Umin-l (mg proteinyl. The drastic decline in
laccase specific activity and increase in xylanase specific activity after the second
week could be used as morphogenetic markers for primordia initiation. In solid
medium the sudden increase in CMCase specific activity on the 17ili week from
0.11 Umin-l (mg proteinyl to 0.60 Umin-l (mg proteinyl on 18ili week corresponded
to the onset and development of primordia.
C-serum supplementation in Leatham's mediUm and rubber sawdust had no
promotional effect on fruiting. Supplementation of C-serum from Hevea latex on a
Leatham's medium increased mycelial biomass production in the early stages of
growth. Mycelial biomass production in supplemented medium ranged between 17.8
- 249.5 mg while in control, the mats weighed at 7.0 - 173.8 mg over the 13 weeks
of incubation. Supplemented medium fruited at fourth week while the control only
on the sixth week of incubation. Meanwhile, C-serum supplementation on Hevea
wood sawdust significantly reduced yield from 24.4% B.E. to 8.3% B.E. even
though there was a slight increase in biomass of mycelia. Supplementation of C
serum in PDB and Acacia sawdust medium stimulated both mycelial growth and
fruiting. Supplemented PDB produced a yield of 2.8 - 49.8 mg while the control
failed to produce any fruit bodies. To date, no fruiting of shiitake on PDB as a
III
medium has ever been reported. In Acacia sawdust, C-serum supplementation
induced fruiting (18.1 % B.E.) while the control failed to produce any fruit bodies.
Three strains of L. edodes, L38, L272, L2161 grown on rubber wood
sawdust, palm pressed fibres (PPF) and five combinations of grass substrates
showed that rubber wood sawdust was better for mycelial growth compared to PPF
and grass substrates. Strain L38 required 36.0 days to colonize the rubber sawdust
medium while both L272 and L2 161 needed 37.0 days respectively. This growth was
significantly faster compared than one on PPF substrates where strain L38, L272 and
L2161 took 42.3, 4 1.0 and 38.5 days to colonize the substrate respectively. All three
strains recorded significantly slower colonization in all the grass substrates
compared to rubber sawdust and PPF. In fructification, only rubber wood sawdust
and grass treatment C (lmperata cylindrica) produced fruiting in strains L38 and
L2 161 with no significant differences in fruit body yield between both strains on
both substrates. Strain L38 recorded 21 .8% B.E. and 23.3% B.E. on rubber sawdust
and grass treatment C respectively while strain L 2161 recorded 1 1 .9% and 12.8%
B.E for the rubber sawdust and grass substrates respectively.
Knowledge of an optimal temperature - humidity - light - aeration regime for
a particular strain, employing morphogenetic enzyme markers and using productive
substrate and supplements all contribute to the development of a precision shiitake
cultivation technology.
IV
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains.
BIOTEKNOLOGI PENGHASILAN CENDA WAN DENGAN PERSEKITARAN TERKA WAL, SUBSTRA T DAN PENANDA ENZIM
UNTUK CENDA WAN SHllTAKE, LENT/NULA EDODES Oleh
GANISAN KRISHNEN
Pengerusi: Tan Vee How, Ph.D.
Fakulti : Pertanian
Jun 2001
Pertumbuhan miselium adalah optimum pada 25°C bagi enam dari tujuh strain
Lentinula edodes (L. edodes) yang diuji. Habuk kayu menghasilkan lebih banyak
miselia daripada bijirin di mana 0.53-1.60 mg dan 0.02-0.2 mg glukosamin dihasilkan
masing-masing bagi setiap 200 g medium habuk kayu dan bijirin. Set suhu
15°C(8j)/25°C(16j), cahaya berterusan (25-35 lux), kelembapan sebanyak 80-95%
dan kelajuan pengudaraan 1.3-2.5 mls menghasilkan Keefisienan Biologi (K.B) yang
tertinggi bagi 11 dari 13 strain pada tiga set suhu yang diuji. Strain L38 (7.1-32.7%
KB.) dan L300 (7.2-35.5% KB.) merupakan penghasil KB. yang terbaik bagi
kesemua set suhu yang diuji. Kepadatan miselia yang dihasilkan semasa inkubasi
adalah tidak berkolerasi dengan KB. bagi kesemua strain yang diuji.
Aktiviti spesifik dari tiga enzim lignoselulolitik iaitu laccase, CMC'ase dan
xilanase menunjukkan bahawa minggu kedua bagi medium cecair dan minggu
ketujuhbelas bagi media habuk kayu adalah waktu yang sesuai untuk memulakan
pembuahan cendawan. Aktiviti spesifik bagi laccase dan xilanase pada minggu kedua
adalah 136.69 Umin"l (mg protein)"l dan"7.35 Umin"l (mg protein)"l masing- masing.
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aktiviti spesifik laccase dan kenaikan aktiviti spesiftk xilanase selepas minggu
kedua sebelum penghasilan primordia pada minggu ketiga pengeraman, ini boleh
digunakan sebagai penanda morfogenetik bagi penghasilan primordium. Bagi
medium habuk kayu, hanya CMC' ase didapati sesuai digunakan sebagai penanda
morfogenetik. Peningkatan mendadak aktiviti spesiftk CMC'ase pada minggu
ketujuhbelas (0. 1 1 Umin1 (mg proteinyl) sehingga minggu ke1apanbelas (0.60
Umin-1 (mg proteinr1) adalah bersarnaan penghasilan primordium.
Penambahan C-serum pada medium Leatham dan habuk kayu getah tidak
menunjukkan kesan yang menggalakkan dari segi pembuahan cendawan.
Penambahan serum dari lateks getah ini hanya menunjukkan peningkatan biomas
miselia pada peringkat awal pertumbuhan. Miselia yang dihasilkan dengan
penambahan serum adalah sebanyak 17.8-249.5 mg berbanding dengan kawalan
sebanyak 7.0-173.8 mg sepanjang 13 minggu pengeraman. Media dengan
penambahan serum menghasilkan cendawan pada minggu keempat manakala
kawalan pula pada minggu keenam pengeraman. Manakala Habuk kayu getah
dengan penambahan serum mengurangkan K.B. dari 24.4% (kawalan) kepada 8.3%
(media ditambah serum). Bagi media Potato Dextrose Broth (PDB) dan habuk kayu
Acacia dengan penambahan C-serum menggalakkan penghasilan miselium dan hasil
cendawan. Media PDB dengan penambahahan serum menghasilkan cendawan
sebanyak 2.8 - 49.8 mg manakala kawalan pula gagal menghasilkan cendawan.
Sehingga kini tiada laporan tentang penghasilan cendawan pada PDB. Penarnbahan
serum ini pada habuk kayu Acacia menggalakan penghasilan cendawan ( 1 8.1%
K.B.) manakala kawalannya gagal sarna sekali untuk berbuat demikian.
V1
Tiga strain L. edodes, L38, L272, dan L2161 yang dikultur pada habuk kayu
getah, serabut kelapa sawit (PPF) dan lima kombinasi rumput menunjukkan bahawa
hanya habuk kayu getah sahaja menghasilkan pertumbuhan miselia terbaik
dibandingkan dengan PPF dan substrat rumput. Strain L38 memerlukan 36.0 hari
untuk mengkoloni habuk kayu getah manakala kedua-dua strain L272 dan L216 1
memerlukan 37.0 hari untuk tujuan yang sarna. Pertumbuhan ini adalah cepat jika
dibandingkan dengan PPF, di mana strain L38, L272 dan L2 161 masing-masing
mengambil 42.3, 4 1 .0 dan 38.5 untuk mengkoloni substrat. Kesemua strain
menunjukkan pertumbuhan yang lambat pada kesemua kombinasi rumput yang
diuji. Untuk penghasilan cendawan, hanya strain L38 dan L2161 berjaya
menghasilkan cendawan pada media habuk kayu getah dan rumput kombinasi C
(lmperata cylindrica) tanpa sebarang perbezaan signifikan. Strain L38 menghasilkan
K.B. yang bernilai 2 1.8% dan 23.3% masing-masing bagi media habuk kayu getah
dan rumput kombinasi C, manakala strain L2 161 pula menghasilkan K.B. yang
bernilai 1 1 .9% dan 12.8% bagi habuk kayu getah dan rumput kombinasi C.
Pengetahuan tentang suhu-kelembapan-cahaya-pengudaraan yang optima
terhadap strain yang tertentu, penggunaan penanda morfogenetik dan substrat yang
produktif serta penambahan serum menyumbang ke arah penghasilan teknologi
penanaman cendawan yang canggih.
VlI
ACKNOWLEDGEMENTS
I would like to take this opportunity to express my gratitude to my
supervisors Associate Prof. Dr. Tan Vee How, Associate Prof. Dr. Mohd. Noor
Abdul Wahab and Dr. Zainal Abidin Mior Ahmad for their invaluable advice,
guidance, support and encouragement throughout my project.
Special thanks are extended to Mr. V. Ganesan for his invaluable help in
preparation of this thesis and Dr. Jugah Kadir for his advice and encouragement.
Heartfelt thanks are offered to those helpful staff, Mr. Govindan, Mr.
Hamdan, Mr. Lim, Mr. Mohd. Khir from Department of Plant Protection and
Mr. Karim, Mr. Onn and Miss Ruhaidah from Department of Biochemistry and
Microbiology.
I wish to express my special thanks to my parents, sisters and brother and
other family members who have given so much of their love and encouragement.
Finally, I would like to thank all my friends who have either directly or
indirectly helped in this project.
viii
I certify that an Examination Committee met on 6th June 2001 to conduct the final examination of Ganisan Krishnen, on his Master of Science thesis entitled "Production Biotechnology with Controlled Environmental Regimes, Substrates and Enzyme Markers for the Shiitake Mushroom, Lentinula edodes" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:
KAMARUZAMAN SIJAM, Ph.D. Associate Professor Department of Plant Protection Faculty of Agriculture Universiti Putra Malaysia (Chairman)
TAN YEE HOW, Ph.D. Associate Professor Faculty of Agriculture Universiti Putra Malaysia ( Member)
ZAINAL ABIDIN MIOR AHMAD, Ph.D. Faculty of Agriculture Universiti Putra Malaysia ( Member)
MOHD. NOOR WAHAB, Ph.D. Faculty of Science and Environmental Studies Universiti Putra Malaysia ( Member)
........ .L�C1&..I ... MOHAYIDIN, Ph.D. Professor eputy Dean of Graduate School Universiti Putra Malaysia
Date: 2 8JUN 2001.
ix
This thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Master of Science.
x
AINI IDERIS, Ph.D. ProfessorlDean of Graduate School Universiti Putra Malysia
Date: 1 2 Jill 2001
DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that this thesis has not been previously or concurrently submitted for any other degree at UPM or any other institutions.
Signed
Date:
xi
TABLE OF CONTENTS
Page
A"BSTRACT ................................................................... ................................ 11 ABSTRAK ..................................................................................................... v AC.KNOWLEl>GEMENT . .... . .... . .. ............ .................... ............................... VlIl APfROV AL SHEETS ... ......... . . .. .. ................ .. ............... .......... ....... .............. IX 'DECLARATION FORM. . .. . . ............. ....................... .................................... XI LIST OF FIG'U"RES ..... .... . .. ..... .. .... .......... . .. ... .. ...... . .......... . .. ......................... XVI LIST OF TABLES .. . ... . ........ . . . . .... . . . . ... . .. . .. . . . . ... . . .. . . . . . . .. . . . ...... . ... . .. ..... . . . . . .. . . . .. XVI 11 LIST OF PLATES ... ... . .. . . ..... .. . ....... .. . ....... ... ..... . . .. ... . ...... ... . ... . . ... . . . ..... . ...... .. . XXXlll
CHAPTER
1.0 GENERAL INTRODUCTION ....... .. .. . . ... . ... .... . . .. ..... ... ....... .. 1
2.0 LITERATURE REVIEW .. . .. .. . . . . . . . .. . . .. . . . ... . ... ... . . .. . . ........ ... . .. 8 2.1. Biological Nature of Lentinula edodes .......... ......... ...... .... 8 2.2. Nutritional Requirement for the L. edodes Growth ... . .... . 10 2.3. Environmental Factors Influencing the Mycelial Growth
and Fruiting of L. edodes . . . .. . . ....... . ....... . . ... .. ................... 12 2.3.1. Temperature ...... . ............... . . ................................ 12 2.3.2. Light ........... ... . ..... ........... .. . ............ .......... . ............ 13 2.3.3. Humidity ... . .. . . .. . . ... . . . . . . . . . .. . .... . .. . . . . . . . . . . .. . ....... . ... . ... 14 2.3.4. Aeration . . ...... . .......... . . .... . .. . . .. . ... ............. ... ....... .... 1 5
2.4. Biomass and Glucosamine Estimation . ... . ... . .. . . ..... . . . .. . . ... 16 2.5. Lignocellulolytic Enzymes as a Morphogenetic Marker.. 17 2.6. Hevea Latex as a Growth Inducer for Shiitake .. . ........... . 2 1 2.7. Bioconversion of Lignocellulose into Edible Mushroom 23
2.7. 1 . Nutrient Content of Lignocellulosic Materials .... . 24
3.0 ANAMORPIDC ( MYCELIAL) AND TELIOMORPIDC ( FRUITING ) STUDIES ... .. . .. . . . .. . . .. . .... . ... .... . ..... ........... . . .. .... 26 3. 1 . Introduction .. . ....... .. ... . . . .. . . . . . .. . . . ... .... . . . . . . . . . .. . .. . . ... ............. 26
3.1.1. Anamorphic studies . . ...... ........... . .... .. ...... . . ... . ..... 27 3. 1 .1.1. Measurement of Mycelial Biomass on
Sawdust and Grain Substrates ............... 28 3. 1 .2. Teliomorphic/Fruiting Studies . ........................... 29
3.2. Materials and Methods ....... .... . ..... ............ ............... . ...... 30 3.2. 1 . Anamorphic (Mycelial) Studies .......................... 30
3.2. 1 . 1 . Mycelial Growth of L. edodes Strains on Potato Dextrose Agar ( PDA ) at Different Temperatures .. . ..... .............. . .. . 30
3.2.1 .2. Measurement of Mycelial Biomass on Sawdust and Grains Substrates ... . . .......... 3 1
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Page
3.2.1.2.1. Chitin Assay .. ......... ............ .... 33 3.2.2. Fruit Body Production at Three Different Sets of
Temperature .......... ... ............................................. 38 3.2.2.1. Strains and Spawn ........ ............................ 38 3.2.2.2. Production Bags .. ...................................... 39 3.2.2.3. Spawn Run .... ... ..................................... .... 40 3.2.2.4. Fruiting .......................... . .................... . ..... 40 3.2.2.5. Parameters .............. . ..... ............................ 42 3.2.2.6. Experimental Design and Statistical
Analysis . ......... ............. . .................... . ....... 44 3.3. Results ............... . ...... . ... ........ ........ .. . . .... ... . .......... ............ . . 45
3.3.1. Mycelial Growth of L. edodes Strains on Potato Dextrose Agar ( PDA ) at Different Temperatures. 45
3.3.2. Measurement of Mycelial Biomass on Sawdust and Grains Substrates ......... . ....... . .... . ................. . . . ........ 51
3.3.3. Shiitake Fructification at Different Sets of Temperature ................................. . . ...... ......... . . . . .... 55
3.3.4. Correlation Test Between Mycelial Biomass Production and Biological Efficiency .. ......... ........ 70
3.4. Discussion ..................... . . . ..................... ........................... 73 3.4.1. Mycelial Growth of L. edodes Strains on Potato
Dextrose Agar ( PDA ) at Different Temperatures. 73 3.4.2. Measurement of Mycelial Biomass on Sawdust and
Grains Substrates ........ .......... .... ............................. 74 3.4.3. Shiitake Fructification at Different Sets of
Temperature ........ ............ .............. ........ ................ 76 3.4.4. Correlation Test Between Mycelial Biomass
Production and Biological Efficiency .......... ......... 83 3.5. Conclusion ....................................................................... 85
4.0 EXTRACELLULAR LIGNOCELLULOL YTIC ENZYME PRODUCTION DURING MORPHOGENESIS IN LENTlNULA EDODES ........ ......................... ......... .......... ..... 86 4.1. Introduction ................................................ . .. ........... . ...... 86 4.2. Materials and Methods ................. . ............... .................... 88
4.2.1. Strain ... ........................................ . ......... ..... . .......... 88 4.2.2. Medium, Culture and Fruiting Conditions ........... 88
4.2.2.1. Liquid Medium ........ ............................... 88 4.2.2.2. Solid Medium .................... ........ . ............ 89
4.2.3. Preparation of Crude Enzyme Extracts .................. 90 4.2.3.1. Liquid Medium ............ ........................... 90 4.2.3.2. Solid Medium ........... ... ... ........................ 91
4.2.4. Enzymes Activity Determination .............. ........ ..... 93 4.2.4.1. Laccase Enzyme Assay ........................... 93
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4.2.4.2. CMC'ase Enzyme Assay........................ 94 4.2.4.3. Xylanase Enzyme Assay ........................ 95
4.2.5. Mycelial Biomass Measurement in the Sawdust Medium ................................................................ 96
4.3. Results .... ....... .... ... ... .. .. ... ... .. .. ... ..... .. ... ................ ............. 97 4.3.1. LignoceUulolytic Enzyme Specific Activity on
Liquid and Solid Me dium . ....... ... ......... ................ 97 4.3.2. Mycelial Biomass Measurement in the Sawdust
Medium .. ... . .... . . . .. . .. .. .. .... ..... ... ... .... .. . ... .... ....... .. .... 101 4.4. Discussion ... .. .. .. ... .. ..... .. ....... .. .. ..... .... .. .. ... ....... ..... ...... ... .. 103
4.5. Conclusion . .. ........ ....... .. .... .. ...... .... ... .... ..... . .. .. ... ... .. ........ . 110
5 .. 0 EFFECT OF SUPPLEMENTATION OF C-SERUM FROM RUBBER (HEVEA) LATEX ON GROWTH AND FRUITING IN LENTINULA EDODES . ... ..... ...... .. .. .... ... .. ... 111 5.1. Introduction . . ... . .. ..... .. .. ...... .............. .. . ... .... .. ............ .. ... ... 111
5.2. Materials and Methods ... .. . ..... ....... .... ...... ..... ..... ...... ........ 113
5.2.1. C-Serum Preparation . .. ... ... ...... . ... ..... ... .. ........... .... 113 5.2.2. The Effect of Latex on Mycelial and Fruit Body
Biomass in Liquid and Solid Media ..... . .... ..... ....... 113 5.2.2.1. Strain ... .. .. .. ... .... .... .. ..... .. .. .. .... .... .. ...... ... .... . 113 5.2.2.2. Liquid Medium . ... .. .. ...... . ........ ..... ...... ..... .. 115
5.2.2.2.1. Substrate Composition ..... .. ...... 115
5.2.2.2.2. Cultural Condition and Biomass Measurements ... ... .. ........ .. ... ...... 116
5.2.2.3. Solid Medium .. ... ...... .... .. .. .. .. .. .. ... . ... ...... . . . 116
5.2.2.3.1. Substrate Composition .. .. .... . .... 117
5.2.2.3.2. Substrate Preparation . ... .. .... ...... 117 5.2.2.3.3. Spawn Run . ... .. ... ..... ... .... .. ......... 118 5.2.2.3.4. Mycelial Biomass Measurement. 1 1 8
5.2.2.3.5. Fruiting ......... .. .. ........ .... ...... ...... ] 19 5.2.3. Mineral Determination on Rubber (Hevea) and
Acacia Sawdust .................................................... 12 0 5.3. Results .............................................................................. 121
5.3.1. The Effect of Latex on Mycelial Biomass in L iqu id Media ......... .. ... ... ......... ...... .... ... ... ... .. ... . ... ... ... ...... ... 121
5.3.2. The Effect of Latex on Mycelial and Fruit Body Biomass in Solid Media ..... .. .. .. ....... .. ... .... ... ... .. ... .. 13 1
5.3.3. Minerals (Cations) Determination on Rubber ( Hevea ) and Acacia Sawdust . .... . ,. ..... ........ ... ....... 139
5.4. Discussion .. ......... .......... ...... ... .... .... .... ... ..... ........ ....... ....... 141 5.5. Conclusion . .. .... .. ....... .. .. ..... ..... .... . .... ... .. .. .... . ... .. .. .. ... ... .... . 148
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Page
6.0 CULTIVATION OF THE SHIITAKE MUSHROOM ON AGRICULTURAL LIGNOCELLULOSIC WASTES OTHER THAN SAWDUST ...... . . . . . . ......... . . . . . ....................... 149 6.1. Introduction ...................................................................... 149 6.2. Materials and Methods . . . ............... .............. ..................... 151
6.2.1. Spawn and Production Bags Preparation . . .. .... ...... 151 6.2.2. Strain ......................................................... .. ........... 151 6.2.3. Media .... ...... ......... ....................... . .. .. .... .................. 151 6.2.4. Spawn Run ..... .. ....... ...... ......................... ........ ........ 154 6.2.5. Fruiting .... . . . . .. .. .. . . . . .. .. .... ........ .......... . . . . . . . . . . . . . . . . . . .. . . 154 6.2.6. Experimental Design and Statistical Analysis ....... 155
6.3. Results ..... .......................................................... ............... 156 6.4. Discussion ................. . .... .. . . ....................................... . . . .. . . . 168 6.5. Coclusion . ...................... . . . ......... ..... . . . . . .... ................ ........ .. 171
7.0 GENERAL DISCUSSION .......... ... ............ ............. ............ ... 172
REFERENCES ... ....................... ........... . ........................ ........... . . .. ... . ............. 183
APPENDIX A B C D
E
F G H
Preparation of Media/Solution ................................................ .
Preparation of Standard Curves ............................................... .
Standard Curve ........................................................................ . Enzyme Specific Activity Calculation .................................... .
Glucosamine Content (Biomass) Calculation .......................... .
Biological Efficiency (B. E) Calculation .................................. .
The Correlation Coefficient .................................................... .
Analysis of Variance ( ANOV A ) ........................................... .
194 196 199 203 206 207 208 209
VITA ...................................................... . . . . . ............... .......... ............... . ........... 247
xv
LIST OF FIGURES
Figure Page
3.1 Flow Chart of Chitin Assay ( Ride & Drysdale, 1972 ) ........ ......... 34
3.2 Correlation Between Time Needed for Seven Strains of L. edodes in PDA to Reach the Edge of Petri Dish and Visual Scoring of Mycelial Density at 20°C. The Correlation Coefficient ( r) Value =0.466 ............................................... ............................................ 49
3.3 Correlation Between Time Needed for Seven Strains of L. edodes in PDA to Reach the Edge of Petri Dish and Visual Scoring of Mycelial Density at 25°C. The Correlation Coefficient ( r ) Value =0.495 ........................................................................................... 49
3.4 Correlation Between Time Needed for Seven Strains of L. edodes in PDA to Reach the Edge of Petri Dish and Visual Scoring of Mycelial Density at 30°C. The Correlation Coefficient ( r ) Value = 0.233 ........................................................................................... 50
3.5 Correlation Between Glucosamine content ( mg) for 13 Strains of L. edodes in Sawdust Medium and Visual Scoring of Mycelial Density at 25°C. The Correlation Coefficient ( r) Value = 0.289 .. 54
3.6 Correlation Between Glucosamine Content ( mg ) for 13 Strains of L. edodes in Grains Medium and Visual Scoring of Mycelial Density at 25°C. The Correlation Coefficient ( r ) Value = 0.128 .. 54
3.7 Correlation Between Biological Efficiency and Glucosamine content ( mg ) for 13 Strains of L. edodes at Temperature Set of 15/25°C. The Correlation Coefficient ( r) Value = 0.185 ............ 71
3.8 Correlation Between Biological Efficiency and Glucosamine content ( mg ) for 13 Strains of L. edodes at Temperature Set of 20/25°C. The Correlation Coefficient (r) Value = 0.075 ............ 71
3.9 Correlation Between Biological Efficiency and Glucosamine content ( mg) for 13 Strains of 1. edodes at Temperature Set of Constant 25°C. The Correlation Coefficient ( r ) Value = 0.350 .. 72
4.1 Flow Chart of Enzyme Extraction ................................................. 92
4.2 Lignocellulolytic Enzyme Specific Activities of Lentinula edodes Grown on Chemically Defined Liquid Medium During Anamorphic and Teliomorphic Phases for 10 Weeks ................... 98
XVI
4.3 Lignocellulolytic Enzyme Specific Activities of Lentinula edodes Grown on Rubber Sawdust Medium During Anamorphic and Teliomorphic Phases for 18 Weeks .............................................. .
4.4 Mycelial Biomass (Glucosamine) Production on Rubber Sawdust Medium During Anamorphic and Teliomorphic Phases for 18 Weeks ....................................... ......... ............................. . . . . .......... .
XVll
p
LIST OF TABLES
Table
3.1 Days Needed to Reach the Edge of Petri Dishes by Seven Strains
Page
of L. edodes at Three Incubation Temperatures . .... .............. , ........ 46
3.2 Visual Scoring of Mycelial Density of Seven Strains of Full Grown L. edodes on PDA at Three Incubation Temperatures ..... 48
3.3 Glucosamine (Mycelial Biomass) Content (in mg) of 1 3 Strains of L. edodes (3 Months) Grown on 200 g of Sawdust and Grains Media at 2S ± 1°C ........................... .............................................. 52
3.4 Visual Scoring of Mycelial Density of 13 Strains of L. edodes on Sawdust and Grains Media After Three Months Incubation at 25 ±1°C .................... ............. .... . . ..... .......... . . . ............................ ..... 53
3.5 Biological Efficiency of 13 strains of L. edodes Fruited at Three Different Sets of Fructification Temperature . ......... ............... ....... 56
3.6 Yield in First and Second Flushes, Total Yield, Biological Efficiency and Ranking for 13 Strains of L. edodes Fruited at Temperature Set l 5/250C ............................ ........... ............ ........... 60
3.7 Yield in First and Second Flushes, Total Yield, Biological Efficiency and Ranking for 13 Strains of L. edodes Fruited at Temperature Set 20/2 5°C . ......................................... ....... ........... ,. 61
3.8 Yield in First and Second Flushes, Total Yield, Biological Efficiency and Ranking for 13 Strains of L. edodes Fruited at Constant 25°C ............. ................................ ..... ,...... ......... ........... . . . 62
3.9 Time (Days) Needed for the First Primodium to Appear for 13 Strains of L. edodes After Transfer to Fruiting Room at Three Sets of Temperature .................................. ...... ................ ... ......... . . ... ..... 63
3. 10 Percentage of Primodia That Developed to Fruit Bodies for 13 strains of L. edodes at Three Sets of Fruiting Temperature . . . . ...... 66
3.l1 Water Content (0/0) of Fruit Bodies of 13 Strains of L. edodes at Three Temperature Sets . .............. ........... ..... ...... ... ..... .. . . . . . . . . .. . ...... 68
3. 12 Contamination Rate During Fruiting of 13 Strains of L .edodes at Three Different Sets of Fruiting temperature .... ............. ..... .......... 69
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5.1 �Mycelial Biomass Production of L. edodes Strain L200 on
Page
Chemically Defined Leatham's Medium at 26 ± 1°C ................... 122
5.2 -Mycelial Biomass -Production of 1. edodes Strain L200 on Potato Dextrose Broth (PDB) Medium at 26 ± 1°C ................................ 124
5.3 Eruit -Body Biomass Production by 1. edodes Strain L200 on Chemically Defined Leatham's Medium at 26 ± 1°C ....... ............ 127
5.4 Fruit "Body Biomass Production by 1. edodes Strain L200 on Complex Potato Dextrose Broth (PDB) Medium at 26 ± 1°C ...... 130
5.5 -Mycelial Biomass Production by 1. edodes Strain L200 on Rubber (Hevea) Sawdust Media at 26 ± 1°C ........... ............... .... .... ......... .. 132
5.6 Mycelial Biomass Production by L. edodes Strain L200 on Acacia Sawdust Medium at 26 ± 1°C ...... . .. ... ..... . ..... ..... ........ ..... ... ........... 133
5.7 Eruit Body Production by 1. edodes Strain L200 on C-serum Supplemented Rubber Sawdust Medium at Fluctuating Temperature of 25°C (16h / day) / 15°C (8h / night) ............ ....... 136
5.8 Fruit Bodies Production by L. edodes Strain L200 on C-serum Supplemented Acacia Sawdust Medium at Fluctuating Temperature of 25°C (16h / day) / 15°C (8h / night) ................... 138
5.9 Metal Elements Content (in ppm) in the Rubber (Hevea) and Acacia Sawdust .... ............ ......... ....... ... ....... . ................. ...... ...... ..... 140
6.1 Duration (days) Needed for Strains L38, L272 and L2161 to Fully Colonize the Substrate .... ...................................................... 157
6.2 Time ( Days) Required by 1. edodes for First Primordium Emergence on Production Bags after Transferring to Fruiting Room . . ......... . . . .. . . .......... . . ...... . . . . .. . . . ........................................ ... . . . . . 162
6.3 The Development Rate of Primordia to Fruit Bodies of L. edodes in Two Types of Substrates ........... .................... ...... .......... ............ 164
6.4 Yield from First Flus� Second Flush, Total Yield and Biological Efficiency (B.B.) of 1. edodes Strains L38 and L2161 on Rubber Sawdust and Grass Type C Substrate ................ ............................ 166
XIX
Page
Hl Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain L 14 at Three Incubation Temperatures (Text�Table 3.1) '" 209
H 2 Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain L 22 at Three Incubation Temperatures (Text-Table 3.1) ... 209
H 3 Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain L 34 at Three Incubation Temperatures (Text-Table 3.1) . .. 209
HA Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain L 38 at Three Incubation Temperatures (Text-Table 3.1) '" 209
H 5 Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain L 200 at Three Incubation Temperatures (Text� Table 3.1) . 210
H6 Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain LINA at Three Incubation Temperatures (Text-Table 3.1) . 21 0
H7 Days Needed to Reach the Edge of Petri Dishes by L. edodes Strain M 3 at Three Incubation Temperatures (Text-Table 3.1) .. , 210
H.8 Visual Scoring of Mycelial Density of Full Grown L. edodes Strain L 14 on PDA at Three Temperature Sets (Text-Table 3.2) . 210
H.9 Visual Scoring of Mycelial Density of Full Grown L. edodes Strain L 22 on PDA at Three Temperature Sets (Text-Table 3.2) . 211
H.lO Visual Scoring of Mycelial Density of Full Grown L. edodes StrainL 34 on PDA at Three Temperature Sets (Text-Table 3.2) . 211
R.ll Visual Scoring of Mycelial Density of Full Grown L. edodes Strain L 38 on PDA at Three Temperature Sets (Text-Table 3.2) . 211
R.12 Visual Scoring of Mycelial Density of Full Grown L. edodes StrainL200 on PDA at Three Temperature Sets (Text-Table 3.2) . 211
H.B Visual Scoring of Mycelial Density of Full Grown L. edodes Strain LINA on PDA at Three Temperature Sets (Text-Table 3.2). 212
H.14 Visual Scoring of Mycelial Density of Full Grown L. edodes Strain M 3 on PDA at Three Temperature Sets (Text-Table 3.2) . 212
H.1 5 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 2 (3 Months) Grown on 200g of Sawdust and Grains Media at 2 5 ± 1°C (Text-Table 3.3) . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . ... .... 212
xx
H.16 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 12 (3 Months) Grown on 200g of Sawdust and Grains
Page
Media at 25 ± IOC (Text-Table 3.3) ............ .................................. 212
H.17 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 13 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± IOC (Text-Table 3.3) .............................................. 213
H.1S Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 14 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± 1°C (Text-Table 3.3) .............................................. 213
H.19 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 17 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± 1°C (Text-Table 3.3) .............................................. 213
H.20 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 22 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± IOC (Text-Table 3.3) .............................................. 213
H.21 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 31 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± 1°C (Text-Table 3.3) .............................................. 214
H.22 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 38 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± 1°C (Text-Table 3.3) .............................................. 214
H.23 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 200 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± IOC (Text-Table 3.3) .............................................. 214
H24 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 272 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± IOC (Text-Table 3.3) .............................................. 214
H.25 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain L 300 (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± IOC (Text-Table 3.3) .............................................. 215
H.26 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain UNA (3 Months) Grown on 200g of Sawdust and Grains Media at 25 ± 1°C (Text-Table 3.3) .............................................. 215
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H.27 Glucosamine (Mycelial Biomass) Content (in mg) of L. edodes Strain M3 (3 Months) Grown on 200g of Sawdust and Gra ins
Page
Media at25±1 °C(Te xt-Table 3.3) . . .. . . .. .. .. . ... ....... ...... . ... .. . ... .. .. . .. 215
H.28 Visual Scoring of Mycelial Density of L. edodes Strain L 2 on Sawdust an d Gra ins Me dia After Three Months Incubat ion at 25 ±l °C (Te xt-Table 3.4 ) .. .. ... . . .. . ... ... ...... . .. .... .. .... .. ....... ..... .... ...... . 215
H.29 Visual Scoring of Mycel ial De ns ity of L. edodes St ra in L 12 on Sawdust and Grains Media After Three Months Incubation at 25 fP C (Te xt-Table 3.4 ) .. .. .... ..... . .. . . . .. . .. .. .. .. . .... .. . . . . .. . . . . . . .. .. .. . . . . .. . 216
H.30 V isual Scor ing of Mycel ial De ns ity of L. edodes Stra in L 13 on Sawdust and Grains Me dia After Three Months Incubation at 25 ±l °C (Te xt-Table 3 .4) ............... ................. ........ ............ . . . ........ 216
H.3 l Visual Scoring of Mycel ial Dens ity of L. edodes Strain L 14 on Sawdust an d Gra ins Me dia After Three Mon ths Incubat ion at
25 ±l °C (Te xt-Table 3.4 ) . .. ... ...... . ... .. . . ....... ....... ................. .... .. . . '" 216
H.32 Visual Scoring of Mycel ial Dens ity of L. edodes Strain L 17 on Sawdust and Gra ins Media After Th ree Months Incubation at 25 fl OC (Te xt-Table 3.4 ) . .. . . .... .......... . ...... .... ..... . . .. .. .... .......... .... . . . 216
H.33 Visual Sco ring of Mycel ial Dens ity of L. edodes Stra in L 22 on Sawdust and Grains Me dia After T hree Months Incubation at 25 ±l OC (Te xt-Table 3.4 ) .... .. ..... .. .. . .. ...... ... ... .. . .. .. ..... ........ ....... ... .. 217
H.34 Visual Scoring of Mycel ial Dens ity of L. edodes St rain L 31 on Sawdust an d Grains Me dia After Three Mon ths Incubation at 25 fl oC (Te xt-Table 3 .4 ) . . .. . .. .. .. . ... . . . . . . . . ... . . ... .. . ... ... . . . .. .... ..... . . . ... .. 217
H.35 Vis ual Sco ring of Mycelial Density of L. edodes Strain L 38 on Sawdust and Grains Media After Three Months Incuba tion at 25 fl oC (Text -Table 3.4 ) .. . .. .. .. . ............ ...... ................... .. .. . .. ........ 217
H.36 Visual Scoring of Mycelial Dens ity of L. edodes Strain L 200 on Sawdu st and Grains Media After Three Months Incubation at 25 floC (Te xt-Table 3 .4 ) . .. . . .. . . . . . . .. .. . .. .. .... . . . . .. ... .. . . . . . . .. .. . . . . . . . . . . . ... . 217
H.37 Visual Sco ring of Mycel ial Dens ity of L. edodes Stra in L 272 on Sawdust and Grains Me dia After Three Mon ths Incubation at 25 floC (Text-Table 3.4 ) . . .. .. .. .. . . .. . . . ... . .. .. .. . .. .. .. ... ...... .... . . . .. . ... ...... 218
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Page
H.38 Visual Scoring of Myce1ial Density of L. edodes Strain L 300 on Sawdust and Grains Media After Three Months Incubation at 25 ± l °C (Text-Table 3.4) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 18
H.39 Visual Scoring of Mycelial Density of L. edodes Strain LINA on Sawdust and Grains Media After Three Months Incubation at 25 ± l °C (Text-Table 3.4) .............................................................. 2 1 8
H.40 Visual Scoring of Mycelial Density of L. edodes Strain M 3 on Sawdust and Grains Media After Three Months Incubation at 25 ±l°C (Text-Table 3.4) . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 18
H.4 l Biological Efficiency of L. edodes Strain L 2 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) ..... 219
H.42 Biological Efficiency of L. edodes Strain L 1 2 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) . . . . . 2 19
H.43 Biological Efficiency of L. edodes Strain L 13 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) . . . . . 2 1 9
H.44 Biological Efficiency of L. edodes Strain L 14 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) ..... 2 1 9
H.45 Biological Efficiency of L. edodes Strain L 17 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) ..... 220
H.46 Biological Efficiency of L. edodes Strain L 22 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) . . . . . 220
H.47 Biological Efficiency of L. edodes Strain L 3 1 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3 .5) . . . . . 220
H.48 Biological Efficiency of L. edodes Strain L 38 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) ..... 220
H.49 Biological Efficiency of L. edodes Strain L 200 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) . . . . . 221
H.50 Biological Efficiency of L. edodes Strain L 272 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3 .5) . . .. . 221
H.5 1 Biological Efficiency of L. edodes Strain L 300 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) ..... 22 1
H.52 Biological Efficiency of L. edodes Strain UNA Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) ..... 221
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Page
H.53 Biological Efficiency of L. edodes Strain M 3 Fruited at Three Different Sets of Fructification Temperature (Text-Table 3.5) . . . . . 222
H.54 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 2 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
H.55 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 12 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . . . . .. ... . . . . .. ... . . . . . . .. . . . . .. . . . . . . . . . . 222
H.56 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 13 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222
H.57 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 22 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . .. .... . . .. .. . .. ... . . . . . . . . . . . . . .... . . . . . . 223
H.58 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 38 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223
H.59 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 200 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3 .9) . . . . . . . . . . . . . . .. . . . . . . .. . . . . . .. . . . . . ... . . . . 223
H.60 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 272 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . . . . . ... . . . . . . . . . . . . . . .. . . . . . .... . . .. . . . . . 223
H.61 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain L 300 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) . . . . . . . .. . . . . . .. . . . . . . . . . . ... . . . . . . . . . . . . . . 224
H.62 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain UNA After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) ..... . . ...... .. ... . . . . . .. . . . .. . . . . . . . . . . . . . 224
H.63 Time (Days) Needed for the First Primordium to Appear for L. edodes Strain M 3 After Transfer to Fruiting Room at Three Sets of Temperature (Text-Table 3.9) .. . . . . . . . . . . . . . . .. . . .... . . . . .... . . .. . . . . . . . 224
H.64 Percentage of Primordia That Developed into Fruit Bodies in L. edodes Strain L 2 at Three Sets of Fruiting Temperature (Text-Table 3.10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 224
XXIV