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

ii

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.

v

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

XlI

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

X11l

Page

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

XlV

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

XVlll

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

XX1

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

xxii

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

XX11l

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