001437

CENTRAIJ Lll〕RARY、MA11lDOL UNIVERSITY

BASELINE FOR THE CULTIVAtt10N OF NATIVE OIL RICH

(BOTRYOCOCCUS BRAUNII), AS THE POTENTIAL SOURCE

THE BIOCRUDE OIL PRODUC丁 10NS IN THAILAND

,WADEE VONGPRASE肝 〔Bosc.田dow)

A THESIS SuBMIttTED IN PARTIAL FULFILLMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

MASttER OF SCIENCE

(TECHNOLOGY OF ENVIRONMENTAL MANAGEMENT)

IN ttHE

FACULTY OF GRADUATE STUDIES

OF

MAHIDOL UNIVERSITY

1986

COPYRIGHtt BY FACULTY OF GRADUATE STUDIES

MAHIDOL UNIVERSIttY

ALGA

OF

BY

The Thesis Supervisory Cormitee for

RAYWADEE VONGPRASERT

unanlmously approve the thesis entitled

BASELINE FOR THE CULTIVAT101 oF ‖ATIVE 01L RICH ALCA

(30TRYOCOCCuS wONII), AS THE POTE‖ TIAL SOURCE OF

THE BIOCRUDE 01L PRODuCT10‖ s I‖ THAILAND

and recommend the candidate tO submit the thesis to

the Faculty Of Graduate studies, Mahidol university

for the ‖ASTER OF SCIENCE degree on August 217, 1986.

縮 鰍fSomsak

MajorSansukh,

Advi sor

.?*...Y,.?a*.*!*,*Prima Yongmanitchai, lrl.Sc.

Co-Advi sor

… 〃 r

Monthree

Dean

Faculty l

Mahi dol

fl. F-+-.1^^ .........i

RungJarat Hutacharoen, M.Sc.

Co-Advi sor

of Graduate Studi es

Uni versi ty

Chul asamqya, M.D., ph.D.

The Thesis Defense Gormitee for

RAY‖ADEE YONCPRASERT

unanimously approve the thesis entitled

BASELINE FOR THE CULTIVAT101 0F NATIVE 01L RICH

(30TRYOCOCCuS BRAUN11), AS THE POTE‖ TIAL SOURCE

THE BIOCRUDE 01L PRODuCT10NS I‖ THAILAND

and agree that she has satisfactorlly defended her thesisand be awarded the degree of Master of Sclence (Technology of

Envi ronmental llanagement) frorn lrlahidol Unl versi ty on

nugust i2i, 1s86.

Ａ．ＡＬＧ

鰤

Somsak Sansukh,

l,lember

“

nthree chulasanaya,M.D。 ,PhoD。

Dean

Faculty Of Graduate studies

Mahidol university

t l.|q^-.-r.--Thanakorn Uan-0n, ph.D.

Dean

Facul ty of Envi rorunent

and Resource Studlesilahidol Uni versi ty

QにしThanakorn Uan-on, ph.D.

Chai man

X. p,,<l,)-Rungii arat Hutacharoen, il.Sc.lilenber

.ft**...Y.try.*.r$*;Prlma Yongmanitchai, l,l.Sc.Me+ber

/

B10GRAPHY

Name: RAYWADEE VONGPRASERT

Date of Birth: November 26, 1958

Place of Birth: Bangkok, Thail and

Education lnstitutions Attended:

1966 ‐ 1975: Padungdarunee sch001, Bangkok

Certificate of Mathayomsuksa III (March, 1975)

1975 ‐ 1977: Triam udom suksa sch001, BangkOk

Certificate of Mathayonsuksa v (March, 1977)

1977 - 1980: Facu]ty Of science,

Chulalongkorn universi ty, Bangkok

Bachelor of science (Biology) (March, 1980)

ACKNOl.lLEDGEMENTS

First of all, I would rike to express qy gratefulness to, the ratedean of Facul ty of Environment and Resource studies, AssociatedProfessor Dr. Nart runtawiroon for bringing my attention and hisguidance to the study.

I am most sincerely grateful to Associated professor Somsak Sansukh,Ms. Prima yongmaritchai and Assistdnt professor RungJaratHutacharoen, ny thesis advisor and co-advisors for their kindlycontributions to encouragements, useful criticisms, editing themanuscripts and many valuable suggestion throughout the study.

Grateful appreciation is extended to Dr. Thanakorn uan_0n, thepresent dean, for his valuable suggestion, encouragement and helpfulcri tici sms.

Most sincerely, I wish to express my thankfurnessNitayangkura and Dr. pongtep Antarikanonda for theirrecormendations for laboratory facil ities.

to Dr. Siripornsuggestions and

My special gratefulness isde Chimie Bioorganique etkindness in analysis thenatural collection) and many

I wish to thank Institute of Food(IFRPD), Kasetsart university and

Department of Agricu'l ture forfaci I i ties.

extended to Dr. C. Largeau, Laboratoire0rganique physique, France, for his

sample of Botryococcus braunii (frominformations and suggestions.

Research and product Devel opment

Agricul ture Toxicol oqy Division,the supporting in laboratory

I am also indebted to every persons in Department of Biology,Facul ty of science, prince of Songkhla university and Mrs. phayaoIntasuwan, the lecturer of Faculty of Science, srinakarinwirojUniversity (at songkhla) for their kindness and facil ities duringthe collections of B. braunii there.

My thank is also to Mr. Sittipong Nutsatit for skilled wordprocessor in preparation the manuscripts.

Sincere appreciation is expressed to my friends and especially thesepeople who provided encouragements and facilities.

Ms. Muntana Kumlaykasingka

Mr. Kridsanaruch TheerarajMs. Jintana Santaveesook

Mr. Suphaphon Tepcharaum

Mr. Somboon Phoopat

I'ls. Varsi tee Nana

Most of all, I would like to express my utmost gratitute to my

parents for their care, encouragement and assistance throughout theentire period of study.

itefotnur0rui

do{niar:fnrn

Qo?a

aocotrtdddiain

f,nrrilnrn

uuzlr.rf,u5rulirfu Rr: rfi r c t #grrarniratiit uCl! riu

lruffuq'ottulrf, ( Eolrysgs uraunii ) rColf,

rrJgunri,tniatiiuirornds0fiialurh : rnrrhu

ur'rdte r:zi artirJr: rOig

lnsrirrniuurfiarfir ( rnaluladnrtuinrtduuaadau)

:os;rdn:rer:d arifaf uruqt

ursl :ur s\rur0riu

{ire;rrrrrerrti isc{a rpr rciql

2529

rnfiaria

frzog'rsrar Botrlzococqrs braunii crndrsri'r,as,.rrlnsrdudutalraiun:'qn

tituntrt ra:r:firdiuro biocrucre oil msusn #alfti unialgal culture drcsuitjliluartfnt lazru rfu}rlilunr: rirc r{usfuaarilura;sonfo\rrJfifiinr: tJiulo biocnrde

otr dlirrnl'zorjrsirnjrefirJ:;u1o 2dllrsu{rrrnurisasi:J:cnoudeifiqf,o bot:.rococcene

uns ieobotryococcene lunrrlonlngliS micropipette washLng technique dsli1n:

arnrrdurnirrils a riaarslirarrortuirf,un:carrdaeu4ldnullfrfia zoospore dorirur

islu rfu large g?een ce].I c?rr{rra1t rir= riiJsargluriosrJffiar:luearldaeuquruzh

large green ce1l {'urr:a rclq rf,uhliuluorirsilrlerornr: rriuudsenirufiu 4 r0r liodnrr tc,!qrf,ghdria1tfl mean bidrass doublLng tine 0.6s{uuecdrbiocruale oilIt\tttt 11.oaX lauuSnf,nurirtluiu \rtJarutas:=u: declining relative grorrth dslf,rear

tjttulo r7 fglsnrr rtrr= r{s.rii rdosurcrnnr: rlr; rduu1utrrrD1r12 Kratz anar tllrers

qrrornr:qr riutfiudgnliluar: rirc rduruonfosrJfirlhnrtlu modifiear rocking tray dr

{ltio,rr..l? Kratz and uyers duarniLilruire ro?q rf,u1"uidqrlauf mean biqnass

doubling time 1.18 lu uecf biocrutte oil {\rqi6\r rs.as,g zauufifaa#slrrsnr: riu.,

r0urzar s fu uilurrsnrsnurirunrr ."r: rdrsuoafosrfirnarrlas padclle lvheer basr.n

doli rf,rstrr'rornr: riurf,o irro.'r.'- reatz and nyers iui..tnli rciq rf,urefr0 mean

bionass doubling time a.? {u uesf,dr biocrude oil rfus o.s7 dsilqllunrtfnyrueclunrdd"li rarge grreen cerl rranr:uunlidnrrt rn:r=rirjltlro cruae protein ;?c

isnu'ir ir crude protein trs1ef,o ss.sz7 lasritrrflau;.rliornnr: rrr= rdusraa paddre

wheel basin dslirzar s {slunrr rir= rfurluge:ornr: Kratz and llyers luanrrdnr:

rnr: r{gsnrgluiosrJfirllnr:uozuanrios fif,lnr:Its tnodified rockLng tray fir crude

plotein itrnirf,a ae.szx lauritufnufslunrr r{srr a lu ua; eo.asx laarirrnrrnurirrlu

nrrriss a lulage'1rtl1u{r:olr1r tts g fisrfue= rfuir targe $.een cel1 lruriolfleriedtr'rtra#s a afle dsiuo{fiuiiarosqa:arrr: jzs ranrnr: riu rdurua= ralori#litgnr:ur: r{us

丁hesis title Baseline for the cultivationof nati ve oi I rich al ga

( Botryococcus br:auni i ), as

the potential source of thebiocrude oil productions inThai I and

Raywadee Vongprasert

Master of Science(Technol ogy of Envi ronmental

Management)

Assoc. Prof. Sornsak Sansook

Ms. Prima Yongmanitchai

Assis. Prof. Rungjarat Hutacharoen

August 22, 1986

Name

Degree

Thesis Supervi sory Cormitee

Date of Awarding the Degree

Abstract

Thg samples of Botryococcus braunii from prince of SongkhlaUniversity reservoir were used in determining biocrude oiI contentand isolation to make unialgal culture for study the possibility incultivation under indoor and outdoor conditions. Biocrude oiIcontent was determined as 20% of dry weight; botryococcocene andisobotryoccoene were identified as the major conponents. The threedifferent medium under natural and control conditions inducedzoospore formation whlch developed to the large green cell inisolation by micropipette washing technique. The cell grewsuccessfully under indoor and outdoor conditions. The fourdifferent media were used under the I aboratory control condition andKratz and Myers media showed the fastest growth with mean biomassdoubl ing time of 0.65 day and maximum biocrude oil content of 11.04%of dry weight on the late of phase of declining relative growth

which took about 17 days for cultivation. The same series of mediaas in indoor cultivation were used under outdoor cultivation bymodified rocking tray. Kratz and Myers media al so exhibited thelowest mean biomass doubling time of 1.1g days and the highestbiocrude oil content of 13.g5% of dry weight within 9 days ofcultivation. In contrast, the small amount of biocrude oil contentof 0.5% was obtained fron the cultivation in paddle wheel basin,using only Kratz and Myers media, under outdoor condition with thelowest mean biomass doubling time of 3.7 days. crude proteincontent was also determined in case of obtaining the large greencel I and the highest crude protein content of 5s.57% of dry weightwas obtained from the cultivation by paddle wheel basin at the fifthday of cultivation where as the indoor cultivation and outdoorcultivation by modified rocking tray in NS III media showed theI ower contents of 49.67% of dry weight at the forth day ofcultivation and 40.23% of dry weight at the second day ofcultivation respectively. Therefore, the large green cell exhibitedboth of the maximum productions which depended on media, cultivationperiod and culturing unit.

f.

TABLE OF CONTENTS

丁IttLE

ACKNOWLEDGEMENT

ABSTRACT

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURES

CHAPTER l: INTRODuCT10N

CHAPttER 2: LIttERATURE REVIEW

。l Background Of BOtryococcus braunl]

2。 2 Physiolo91Cal States of BOtryococcus braunii

2。 3 Scope and objectives of the Research

CHAPTER 3: EXPERIMENTAL PROCEDURE

3.l Algal C01lection

3。 2 1sol ation

3。 2。 l Micropipette washing technlque

3。 2.2 Media

3◆ 2。 3 1ncubation cOnditions

3.3 1ndoor cul tivation

3。 3。 l Cul tivation equipment

3。 3。 2 Media

3。 3。 3 Cul ture conditiOn

PAGE

１

　

　

　

２

　

　

　

２

　

７

　

８

・Ｏ

ｎ

　

ｕ

ｎ

・２

　

‐３

　

‐３

‐３

・５

3。 4 0utdoor cul tivation

3.4。 l Modified rocking tray

3。 4.2 Paddle wheel basin

3.5 Determlnation of Botryococcus braunll

Growth

3.6 Harvesting

3.7 Determination of Biocrude 0il3.8 Determination of Crude protein

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Algal Collection

4.1.1 The occurances of Botryococcus brauniiin Prince of Songkhla University,sreservoi r

4.1.2 Morphology of Botryococcus braunii4.1.3 Biocrude oil and crude protein

contents of Botryococcus braunii

TITLE PAGE

15

15

16

18

19

19

19

22

24

０

　

　

　

　

０

２

　

　

　

　

２

4。 2

4.3

Isolation and IsolatesGrorcth and Productions of

26

BotryococcuS 31

31

36

42

44

braunii During Indoor and Outdoor Cultivations

4.3.1 Indoor cultivation4.3.2 Outdoor cul tivation

CONCLUSION AND RECOMI"IENDATIONS

5.1 Concl usion

5.2 Recormendations for Further Studies

CHAPTER 5:

TIttLE

APPENDIX l

APPENDIX 2

APPENDIX 3

APPENDIX 4

REFERENCES

PAGE

46

47

51

52

54

LIST OF ttABLES

TABLEPAGE

I Distribution of living B. braunii in various continents 42 Distribution of B. braunii from fossil records 63 Physical conditio*lnll-..urances of B. braunii in zr

Prince of Songkhla University,s rese"rlf"-4 Biocrude oil and crude protein contents of B. brauni i 26

collected from prince of Songkhra universitvt6*i"5 State of B. braunii fron isolation in different media zg

and conditions6 Biocrude oil and crude protein contents of B.

four different media under indoor condition7 Biocrude oil and crude protein contents of B. braunii in 3g

four different media under outdoor condittol-oGE u.,to 0ctober 1985 by modified rocking trays

8 Biocrude oil and crude protein contents of B. braunii in 41Kratz and tlyers media under outdoor conditi;;uringDecember 1985 to January 19g6 by paddle wheel basins

braunii in 35

LIST OF FIGURES

FIGURE PAGE

I Indoor cultivation unit 142 Modified rocking tray n3 Paddle wheel basin fi4 Green colonies of B. braunii collected from prince of 23

Songkhl a Uni versity's reservoir5 Oil droplets of B. braunii, the local strain 23

6 Brown colonies of B. braunii collected frorn prince of 25

Songkhl a Uni versi ty's reservoir7 Large green cells obtained from the isolation 27

8 Diagrarmatic surmery of asexual reproduction of 30B. braunii from the study

9 Growth curves, biocrude oil contents and crude protein 32

contents of B. braunii in four different media under indoorcondi tion

10 Difference of cell colors during the cultivation period 34

under indoor conditionll Growth curves, biocrude oil contents and crude protein 37

contents of B. braunii in four different media underoutdoor condition by modified rocking trays

12 Growth curyes and variation in pH, biocrude oil content 40

and crude protein contents of B. braunii in Kratz and

l'lyers media under outdoor condition by paddle wheel basins(from December 1985 to .January 19g6)

CHAPTER 1

INTRODUCTION

l'lithin the limits of present technoloqy, liquid hydrocarbons haveestabl ished themsel ves as the preferred fuel for many purposes,especial ly al I transportations. l{hi I e many al ternati ves such asalcohol, hydrogen, LpG, solar cel l, etc. have been suggested, butal I have sone I imitation in tenns of energy density, safety, or easeof handling that make them less attractive than the hydrocarbonfuels. l,loreover, the 0pEC price rises of 1973-1974 have emphasizedthat the liquid fuel can no longerbe treated as an inexhaustibleenergy resource available in expandable supply (l{ake and Hil len1980). so, it is necessary to reexamine other sources of rawhydrocarbon material s which can serve as the al ternati ve energysources.

Practlcal ly almost. al I of the energy in present util ization isfossilized photosynthetic energy (Calvin l97g). Therefore thesuggestions of cultivation of energy plants which naturally convertsol ar energy to be such of hydrocarbon components, are made by manyworkers (cal vin 1979, campbel I 1983, Ber r l9g4). The most efficientoil plant is Euphobia lathyris, a shrub, which contains 5% or so ofoil extracted by hexane and g5% is typical ly composed of terpenoidswith c3g structures (carnpbel I 1983). It is not only macroscopicland-plants which possess the ability to photosynthetic lrydrocarbonchains. 0ther groups of plants, especially algae, can producehydrocarbons. Shifrin and Chrisholm (19g0) suggest that I ipids(including lrydrocarbon) from phytoplankton are suitable for refininglnto conventional I lpld fuer s. For the outstanding al ga,Botryococcus braunii which the remains of its colonies are sourcesof trydrocarbons ln many geological deposits (AaronSon et al. 19g3),contains about 83ff and 76% of dry welght, respectively, of lipid andhydrocarbon (Maxwell et al. 1969). It is obviously seen thathydrocarbon contents of this specles are nuch higher than those ofland plant. This alga is also suggested by Basedow in 1925, Bowley

in 1944 and Hilren in 1976 to be grown as a source of hydrocarbons(Hil len 1976). The extracted oil from B. braunii is cracked andhydrogenated, it yierds a drstirlate .oniirting of 611. petrorfraction, 15% aviation turbine fueI, 15% diesel fraction, plus 3%reSidua1 011 〔wake and Hillen 1980).

In Thailand, B. braunii is discovered in both nature and man_madewater bodies. Tansakul and Sunbhanich (1gg1) reported that thisspecies appeared abundantly ln prince of Songkhla university,sreservoir.in March and september, 1gg0. AccordinglJ, Intasuwan(1982) found the mentioned arga during.tury to 0ctober, 19g2 inThale Noi (portion of the Songkhla lagoon, phatthalung province).

B. braunii would be one of the valuable genetic resources ofThailand in the future. But no other informations have yet beenstudied, this study will, therefore, take the efforts in carry outthe sounding ddta about this Iocal strain.

CHAPTER 2

LITERAttURE REVIEW

2.L Background of Botryococcus braunii

BotryococcyS praunii, an unusual]y high hydrocarbon_producing al ga

(Maxwell et al. 1968, Brown and Knights 1969), is a colonial member

of the ch10rophyta and in the fol10wipg place Of taxonomic losition

(Belcher and Fogg 1955, Prescott 1973):

Division: ChlorophytaCIass: Chlorophyceae

. 0rder: Chlorococcales

FamiIy : Dictryosphaeriaceae

Genus: Botryococcus

Existing of native form, is usually green, brown or orange colonies,containing ellipsoid cells, radiately arranged at the periphery ofirregularly shaped, usually dark-colored masses of mucilage, freefl oating, col onial muci I age much fol ded and extended into tough,foany strands, often forming.colonial complexes by interconnectingstrands of nucilage, chloroplast single, cup-shaped or discoid,containing a naked pyrenoid like body and in young active coloniesproduce large nunbers of evenly spaced, disk-shaped starch grains,large quantities of oil often present (Fritsch lg2l, Blackburn 1936,Prescott 1962).

Mul tipl ication of cel I s by I ongitudinal di vision only, wherebylncrease of cell-aggregates is brought about, these later becomingseparated by the development of elongated processes of the envelope;vegetable propagation by fragmentation of the older colonies(Fritsch L9271.

Thi s al ga aprears to be wi del y di stri butedecosystem as ol igotrophic and eutrophic fresh

in such of aquaticwaters, brackish and

saline ponds, Iakes, or reservoirs on all continents (Table 1). Itis extremely abundant under various conditions. Though the specificconditions of this alga are so varied that nothing can be concludedof its ecological determination (t{ake and Hil len 19g0).

B. braunii was primarily investigated more than hundred years agoowing to its implicated involvement in the fonnation of a variety ofhydrocarbon-rich sediments such as boghead coal, lignite, torbanite,coorongite etc. ranging in geological time from the precarbrian and

Cambrian to the present and recent past (Tabl e Zl. Bertrand andRenault believed that the "yel1ow bodies" of oil-producing mineralsof boghead coals called Pila were of alga origin but cannot provetheir theory (Blackburn 1936). In 1930, Bertrand only suggestedthat Pila was formed from Botryococcus (Blackburn 1936). Finally,Temperley (1936), from a detailed microscopic study of "living" B.

braunii and the boghead coal, concluded their results accordingly toBertrand's suggestion. Traverse had surveyed the occurences ofBotryococcus in lignites and Tertiary sediments. He hold the viewthat certain Paleozoic oil-bearing rocks derived their oil contentfrom this alga and pointed out that the same genus produced saltysediments in modern brackish and fresh water lakes and very likelywas important in the Cenozoic also (Maxwell et al. 1968). Moldowan

and Seifert (1980) reported the discovery of botryococcane inenormous abundance of 1.4 and 0.9% from Duri and Minas crude oiIs,respectively, in Sumatra.

Maxwell et al. (1968) demonstrated that the oily substanceimpregnating the brown colonies was composed of unsaturatedhydrocarbons constituted 76% of dry weight. Therefore in thefollowing years papers appeared focusing on llquld conpositions and

some physiological. characteristics. A few years after the oilcrisis, Hillen (1976) and l{ake (1976) proposed this alga to be

'lenergy crop" for liquid fuel, numerous studies had been widely done

on biosynthesis of hydrocarbons, sites of accumulation ofItydrocarbons, ul trastructural informations, and observations on itsnatural habitats.

Table I

Distribution of living B. braunii in various continents

Conti nent Si te Type of tlater

Europe lakes, ponds, water fresh watercontainers such as

butts, tanks

Africa lakes fresh waterAsia lakes fresh water, saline waterAustralia lakes, reservoirs fresh waterNorth America lakes, ponds, reservoirs fresh water, saline waterSouth America lakes fresh water, brackish

water

Source: Blackburn (1936), Aaronson et al. (1983)

6

Table 2

Distribution Of Bo braunii from fossil records

Conti nent Type of Fossil I'laterial

Europe Lignite (Denmark)

Boghead coal (France, USSR)

Torbanite (Scotland)Africa N' Hangellite (East Africa, l4ozambique)Asia Lignite ( India)

Boghead coal (pakistan)Coorongite (Siberia)petroleum (Indonesia)

Australia Coorongite, torbaniteNorth America Boghead coal (Alaska)

Lignite (0regon, Texas, Vermont)South America Marahunite (Brazil)

Source: Wake and Hillen (1990), Aaronson et al. (19g3)

2。 2 Physio]ogical States of Botry9cocFり S braunii

Brown and Knights (1969) concl udedphysiol ogical states of B. brauni i,

that there were threecharacterized by their

hydrocarbon contents, as the follorings:

(1) Green active state colonies were both found in nature andculture collections. For the culture collection strains (e.g.

Cambridge, England; Gottingen, German and Austin, U.S.A.)seemed to have originate frorn the same isolate by Droop in 1950(Berkaloff et al. 1984). The characteristics of these strainsusually appeared in small green colonies consisting of fer,rerthan 15 cells and in sorrc cases of only two or three (Berkaloffet al. 1984). These colonies contained about ll-44% of dryweight of hydrocarbons (Brown and Knights 1969, t{ake Lgl6,Casadeval I et al. 1985). These hydrocarbon contents were a

mixture of olefins, mainly dienes and trienes ranging from c17-C33, and the major component being the CZg, C31, and CZIdiolefins (Gelpi and Oro' 196g). The bulk of g. brauniihydrocarbons was synthesized and stored in the successive outerwalls (about 95% hydrocarbon content) surrounding the cells andbuilding up the matrix of the colonies (Largeau et aI. 19g0a,Largeau et aI. 1980b).

l2l Brown or orange resting state colonies, only in naturalcondition, contained the unusual isomeric triterpenoidsbotryococcene and isobotryococcene of fonnula c34Hg (Maxnellet al. 1969) which represented 3t_16% of the dry weight(Maxr+ell et al. 1968, Broun and Knights 1969, t{ake and Hil len1980, rake ind Hillen 198r) and was 90% botryococcene. Thestructural formul a of botryococcene was determined.by cox etal. (1973).

This state had not yet been obtained in laboratory cultures; infact, old cultures of culture collection strains generallyaccumulated carotenoids and turned red, similar to the resting

8

state. but they stil I exhibited about the same hydrocarbonpattern as the green colonies (Largeau et al. 1980a, Aaronsonet al. 1983).

(3) Large green cells, losing the characteristic "murberry" habitof Botryococcus colony, contained very small amounts ofhydrocarbons with retention times similar to c27 and c29 (t.lake

1976)- This. state, firstly reported by Brown and Knights(1969), was induced in the laboratory by inoculating brownresting state colonies from Loch Lomond (Stirl ingshire) and0akmere (cheshire), Engl and into fresh medium under,constantconditions of temperature, day length and light intensity. And

also Hake (1976) worked with the naturar samples of B. brauniifrom Victory reservoirs, and got I arge green cel I s (t.lake percomm.) from isolation with greatly decreased concentration ofhydrocarbons.

Although B. braunii could show a high efficiency in biomassproduction such as its bloom in many lakes and reservoirs(l'laxwe'l I et al. 1968, l,lake and Hi I I en 1980, I,rake and Hi I I en

1981, Aaronson et al. 1983) but in laboratory-grown cultures,it exhibited a slow growth (population doubling time of about a

week) (casadeval I et al. 1985). A I arge improvement was

carried out when Casadeval I et al. (1995) increased B. brauniigrowth, with a mean biomass doubling time of 2.3 days, bycul turing under "air-l ift" conditions. In this present time,investigations on growth and hydrocarbon production of thisalga have still been undertaking by French workers.

2.3 Scope and Objectives of the Research

The present work was intended to investigate the basis basel ineabout the potential in hydrocarbon productions of B. braunii, thelocal strain of Thailand, both in nature and under laboratoryconditions. Samples of B. braunii were col lected from such of

9

reservoir in the southern as Prince of Songkhla University'sreservoir where this alga appeared dominantly. In isolation of thisIocal strain to make unialga culture for laboratory cultivation,various procedures were used to maintain the colonial state. But incase of obtaining large green cell as previous works (Brown and

Knights 1969, I{ake 1976), crude protein content was al so determinedin order to observing its usable for food or feedstock. Forlaboratory cultivations was carried out by using the alga culturetechniques prepared by Institute of Food Research and productDevelopment (IFRPD) Kasetsart University at Bangkok where numerous

works about algae cultivations were quite successfully.

According to aforementioned, the objectives of the research are as

the fol lowings:

(1) To isolate and make unialgal culture of this alga forcultivation and stock culture for further study

(21 To study the possibility in culturing the alga both in indoorand outdoor conditions

(3) To study the biocrude oil and crude protein contents of thespecies obtained from nature and laboratory culture.

10

The study were designed

CHAPTER 3

EXPERIMENTAL PROCEDURE

in step by step as the following diagran:

(Li ving Sampl e)

+Isolation

$

）ＯｆＩ⊥▽

Ｏｆ

3.1 Algal Collection

Desirabl e al gal col onies were col I ected by pl ankton net meshing

100 um. The net was dragged at the spillway of the Prince of

Songkhl a Uni versity's reservoir, Songkhl a province. The fresh

sample was kept in the 1-litre bottles at low temperature about 4oC.

The dried sample was made by air dry and folloned by heat at 40oC in

the oven and cool up at room temperature in desiccator.

J Algal Collection(Dried Sampl e)

Cul tivati on

Determ i nati on

Determi nati on

Biocrude 0il

Crude Protein

( Uni al ga Cul ture)

0utdoor Cul ti vation

１■

3。 2 1solation

3.2.1 (James 1978)

This technique was purposed to separate and transfer the desirablecolonies by sterile pasteur pipette through a series of sterilefluid to tubes of sterile liquid media or sterile plate of agarmedia.

3.2.2 Media

Three selected media, used in this experinent, were as the following:

(l) Modified chu 13 double-strength medium was a defined mediumwhich was studied by chu as the most suitable medium for B.braunii (chu L94zl. This medium was widery used by ma;;workers (Brown and Knights 1969, Hake 1976, Aaronson et al.1983, Casadeval I et aI. 1995)

(21 Bristol's solution medium with soil extract was used as themedium for preservation B. brqunii in the curture coilection ofUniversity of Texas tUTEXXper comm.). This medium was amixture of a defined mediun (Bristol,s solution medium) (Starr1978) and an undefined medium (soil-water medium) (Starr 197g)

(3) Soil-water medium was an

to use for maintaining(James 1978, Starr 1978).

the Prince of Songkhlapreparation this medium.

undefined medium .which was suggestednormal morphology of desired algae

The soil collected from the edge ofUniversity's reservoir was used in

Pl us 1 and 2 ml of Hutner,s A_5 microel ement sol ution (Gel pi et al.1970) for media I and 2 respectively, adjust the pH with 50% NaOH to6.7 and sterilize these medium by steaming at loooc without pressureforlh(PayertgTt).

t2

Except medium 3 which was usual'ly used as liquid medium, media I and

2 were used both in liquid media and plate of agar media.

The agar plate was prepared by mixing 1.5% (w/v) Difco Bacto agar

into the medium, autoclaving at 121oC for 15 min, then pouring the

fluid about 25-30 ml into Petri dishes. For tubes of liquid media

was prepared by pipetting about 5 ml of sterile media to the tube

covered wi th cotton Pl ug.

3.2.3 Incubation conditions

The two different conditions for isolation vrere as the following:

(1) Natural condition:

The experiment was carried out under room temperature and

natural light at Ecological Resources Laboratory, Faculty of

Environment and Resource studies, Mahidol University

(21 Control condi ti on:

The specimen was al lorued to grow in incubator under temperature

at 3OoC, light intensity about 2, 3, 4,5 klux and a light-dark-cycle by t6 h light and 8 h dark.

The fol 1 owing experiments of cul ti vation were carried out atInstitute of Food Research and Product Development (IFRPD),

Kasetsart UniverSity. Three kinds of cul ture units were used forthe cultivation of the cultures under control led laboratorycondition and outdoor condition.

13

3.3 Indoor Cul tivation

The first and smal lest culture unit under control Iaboratorycondition which the principle of operating system was close to "airI ift" batch cul ture unit of Casadeval I et at. (1985). The I atterresulted large improvements in total growth and hydrocarbonproduction of B. braunii. This culture unit of IFRPD was used forpreliminary investigations of algal cultures and preculturing theinocul um for outdoor cul tivations.

3.3.1 Cul tivation equipment

the equipment consisted of rectangular polyacrylate containers withcul ti vation vessel s i I I uminated from one si de, and wi th a gas

distribution system on top. The cultures were grown in cylindricalglass tubes with conical bottoms. The tubes, holding 200 ml medium

each, were 340 mm long and 31 mm inner width. They were closed bydouble bored silicon-rubber stoppers, for supplying and releasinggas. Both the gas inlet and outlet beared a small spherical glass-wool filler which prevented of bacterial or other infections (Fig.1) (Payer 1971, Bhumiratana and payer 1973).

3.3。 2 Media

Except soil-water medium which suited for isolation when normalmorphology was required or long tenn preservation of stock culture(James 1978), the other two media from 3.2.2 incl uding with anothertwo media were used in this experiment. Therefore the four mediawere as the fol lowing:

(1) Modified Chu 13 double-strength medium(21 Bristol 's sol ution medium

14

Ｄ

Ｄ

　

Ｄ

　

Ｄ

　

レ

Figure 1 Indoor cultivation unit

(3)

(4)

Kratz and I'lyers media used by Gal pi et albraunii (Gelpi et al. 1970) contained theof the elements of this investigationsNS III medium was used for cul turing many

(Payer 1971).

15

. in their work on B.

highest concentration

green a'lgae at IFRpD

Plus 1 ml of Hutner's A-5 microelement solutions (Gelpi et aI.1970)formedium 3. The pH of all media was adjusted to 6.7 either with50% NaOH or 2N HCl. And fresh preparation media were used in thisexperiment.

3。 3.3 Culture conditiOn

The culture vessels were thennostatedly cultivated at 30+loc in a

rectangular basin; illurninated by 5-40 tJ fluorescent tubes (lightintensity about 10+0.2 t<lux); suppl ied of air enriched with L-?%c02. The light and dark cycle was 16 and g h respectively (payer197L, Bhumiratana and payer 1973). L glt of fresh al ga was used asi nocul um.

3。 4 0utdoor cultivation

Two kinds of culture units, modified rocking tray and paddle wheelbasin, h,ere used under natural condition. The former had run forinvestigating growth and production of biocrude oil of B. braunii inthe same series of culture media as used in laboratory condition,and only one medium was used for cultivation in the latter which wa.s

a preliminary mass production unit.

3.4.f Modified rocking tray

Thi s cul ti vation uni t, consi sti ng of 6 open shal I ow rectangul arbasins of 0.206 ,2 il luminated surface and each basins contained

16

usual ly 5 I of medium of about 2.5 cm depth (side wal I of basin was

about 10 cm height). This tray performed up and down movement with

a 3-second rocking period in order to make turbul ence in the

cultures (Fig. 2). For the outdoor cultures pure C02 were supplied

through porous gas di stri buti on tubes resul ti ng i n C02

concentrations which saturated during photosynthesis (Payer 1971,

Bhuniratana and Payer 1973).

The four different media, used in this experinent, were:

(1) Modified Chu 13 double-strength medium

l2l Bri stol 's sol ution medi urn

(3) Kratz and Myers media

(4) NS III mdium.

Fresh medium preparation and tap water were used and the pH was

adjusted to the range of 6.7-7.0 with ei ther 50% NaoH or 2N HCl.

During cultivation period after taking sanpl e about 20 ml fordetermination of dry weight from the basin and measuring the

rernaining volumn of water, then rrater was added up to the initialvolumn of 5 l. The inoculum was about 2 g/1 of fresh alga.

3.4.2 Paddle wheel basi n

A prel iminary mass production l{as simulated in the basin,constructed from PVC, of t.2 n2 surface area where of contained tOO

I of the medium (Fig. 3). The turbulence was provided by a rotatingpaddle wheel circulated the medi um continuously along an oval ditchrith paddle speed of 35 rpm. The culture was supplied with pure C02

through porous gas distribution tubes (Payer 1971, Bhumiratana and

Payer 1973). only Kratz and I'lyers media was sel ected in the

experiment and the pH was adiusted to about 6.7 wi th 2N Hcl . Fresh

medium preparation and tap yrater were used in this experiment.

Depth of cul ture suspension was maintained constantly (approximately

8 on) during the cultivation period. The inoculum was prepared from

I aboratory tube unit and outdoor modified rocking tray.

，′

１■

Figure 2 Modified rocking tray

Figure 3 Paddle wheel basin

18

3.5 Detennination of Botryococcus braunii Groxth

In a groring culture, dry weight and pack volume of cells per volume

of cel1 suspension (of medium) measured with time, and thisincrease, termed growth (Stein 1973).

Practically growth was measured as an increase in cel 1 dry weight(mg/l ) in indoor cul tivation and determined by centrifigationmethod. Duplicate 10 ml sample of cel I suspension rras centrifugedat 10,000 rpm for 10 min, dried at I05oC for 2 h, and weighed aftercool ing up to room temperature in desiccator (AoAC 1975).

Considering about the effect of evaporation under outdoor condition,so it was essential to measure the volumn of the suspension and

determination of growth as an increase in g/nz which was cal culatedas the fol 1 owi ng:

surface area (m2)

During the exponential phase, the grorrth rate accel eratedcontinuously and can be represented by the growth equation accordingto Kratz and li,lyers ( Antari kanonda 1980) :

l og1gltl1/Ng = kt

where k

NO, Nt

Specific growth rate constant, expressed in log1Ounits of increase per d;The numerical val ues for dry weight measured duringexponential phase at time 0 and time t respectively;t is the time interval between time 0 and time t

From this, generation (doubling) time was computed from the specificgrowth constant:

Generation Time, Td = 0.301/k day per division

19

3.6 Harvesting

An adequate cell suspension for determinations of biocrude oil and

crude protein was harvested by centrifugation (about 10,0m Fpo, 10

min), washed twice with water. Then the cell was dried at 4OoC inthe oven for about 5 days and cooled up to room temperature indesi ccator.

3.7 Determination of Biocrude 0il

The 1 g dried alga was placed in a solvent washed extraction thimblein the Soxhlet apparatus which was allowed to reflux until the freshextract sol ution of hexane (Largeau et al. 1980a, Campbel I 1983) was

colorless (l'laxwell et al. 1968). After removal of the solvent by

rotary evaporator, the residue (biocrude oil) was weighed.

3.8 Determination of Crude Protein

Micro ttel dahl method {Bail ey

the percentage of crude proteln

1967) was sel ected for the study and

was then cal cul ated as fol I ows:

% Crude Protein =

Normality of H xmlofH used x MI,l nitrogen x 6.25 x 100ght samp

20

CHAPTER 4

RESULTS AND DISCuSsloN

Based on the mentioned methodol ogy, the evident details can be

concl uded as the followings:

4.1 AIgal Col I ecti on

4.1.1 The occurances of Botryococcus braunii in Prince ofUniversity's reservoir (man-made waterbody)

The appearances of Botryococcus braunii in this place were similarto previous reports by Palmer (1980), Wake and Hillen (1980), t{ake

and Hillen (1981), and Aaronson et al . (1983) in that this alga was

generally found in lakes and reservoirs. The blooms of B. brauniioccurred in many countries ( t'lake and Hillen 1980, I,take and Hillen1981, Aaronson et al . 1983) and caused considerable problerns in thewater supply system of Darwin River Reservoir in Australia (ilake and

Hillen 1980) in contrast this alga in prince of Songkhla

University's reservoir was only the dominant species, floating inthe surface waters as small specks, and no problems were noted to be

in this reservoi r.

Whereas in this study the alga was found dominantly in April, June(wet season) 1984, and January (dry season) 1985 (Table 3), Tansakuland Sunbhanich (1981) reported that it appeared abundantly in l4arch

and September. These different times of appearances of this a'lga

might depended on some environmental factors, supporting growuh ofthe alga, which varied inconstantly in a year. But during the firstcollection, the. shorcr occurred almost everyday, the very heavyrains also appeared shortly before the second collection and it was

only rainy season in ThaIe Noi which this species was found( Intasuwan 1982).

うこ

Table 3

Physical conditions and occurances ofSongkhla University's

B. braunii in Prince ofreservo]r

April 1984

( observati on)

l,let Season

(June 1984,

lst collection)

Dry Season*

(January 1985,

2nd collection)

pH

Temperature

of water (oC)

Presence ofB. braunii

6.5-730-31

dominant species(Zooplanktons were

al so found on thelate of collection)

both green and brown

6.7 - 7

27-3L

dominant species(Microcystis sp.

al so appeared on

the late ofcol I ecti on )

brown with smallernumber of green

dominant

spec i es

Colony types green

The existing condition of the water of this reseryoir conductedas presentation in Appendix 3.

22

These evidents might create some conditions suited for this algalgrowth as suggestion of tlake and HiIIen (1980). In addition, thesouthern part of Thailand had the highest average rainfall of about2,400 rm and highest rainy day of about 240 days/year (Buri Lgl7l.Therefore, the effects of rain on growth of this alga was suggestedto be the interesting subject for further study.

I'lhen consi deri ng about the pH val ue of thi s water body, i t was

slightly acidic to neutral which was not suitable for another algalgrowth (Wake and Hillen 1981) but this alga could grow successful]yi n natural waters from pH 4.5 to pH 8.2 ( Bel cher 1969 , I,lake and

Hillen 1981). The temperatures of water during the collections(Table 3) and in Thale Noi (30.5oC) (Intasuwan 1982) nere in thesame range and closed to the temperature in the Darwin RiverReservoir (32oc) and Menton (26-310c), both in Australia (lilake and

Hillen 1980).

The other organism which appeared after being a dominant species ofB. braunii in this place, fn the first collection was zooplanktonand in the second one was a bl ue green al ga, Microcysti s sp.Aaronson et al. (1983) mentioned that peridinium sp. depletednitrogen content in Lake Kinnerat, Isarel, for its bloom before B.

braunii bloom in late September. A similar evident about these twoalgae in Prince of Songkhla University's reservoir was reported byTansakul and Sunbhanich (1981). But during this study did not findPeridinium sp..

It was noted that during the second collection (dry season), thisalga mainly brown although lesser number of green colonies, could be

found in the smal I reservoir of Srinakarinwiroj university atSongkhl a .

4.1.2 Morphology of Botryococcus braunii

This strain from Prince of Songkhla University's reservoir exhibited

23

Figure 4 Green colonies of B. braunii collected from

Prince of Songkhla University's reservoirMagnification x 10.

Figure 5 0i1 droplets ofMagni fication x

B. brauni i , the I ocal strai n

40.

E

●

―

24

characteri stic more c1 osely rel ated to Bl ackburn' s and Fri tsch' s

descri pti ons ( Fri tsch Lg27 , Bl ackburn 1936 , Fri tsch 1965 ) ' The

green colonies consisted of a number of pear shaped green cells

densely embedded within a transparent mucous envelopes which were

connected by interconnecting strands of mucilage (Fig. 4). ltlany oil

droplets were squeezed out by the pressure of cover glass under

microscope ( Fig. 5) . The presence of short arched pseudocil ia

arising in a tuft from the outer and of the cel'l were also observed

(Fig. 5) as Carlson's record (Fritsch L927, Fritsch 1965).

For the brown colonies, irregular lobe like colonies rvith tough

(orange colored) mucous envelope were absented of interconnecting

Strands of mucilage, So their Sizes were smaller than the green

co]onies (Fig. 6).

The cell s appeared to multiply by longitudinal division, whereby

increase of cell-aggregates was brought about, these latter becoming

separated by development of elongated precesses of the envelope;

vegetative propagation by fragmentation from the older colonies

(autospore) (Fig. a).

4.1.3 Biocrude oil and crude otein contents of Botryococcus braunii

Biocrude oil and crude protein contents of the second collection(Table 4) which appeared more brobrn colonies were slightly higher

than the first one. For the sample of the second collection v,as

also kindly analyzed by Dr. C. Largeau who found that the crude

hexane extract (which contained external hydrocarbons, i 'e' more

than 90% of total ones) accounted for 24% of dry weight'

Botryococcene and. isobotryococcene were the main product of this

Species ( per corm. ) . The val ues of cnude protei n content were

considered verY low.

Figure 6 Brown colonies of B. braunii collected fromPrince of Songkhla University,s reservoirMagnification x 40.

25

26

Biocrude oil and crude

. from Prince of

Table 4

protein contents of B. braunii collectedSongkhl a University's reservoi r

Wet Season

(June 1984,

lst Collection)

Dry Season

(January 1985,

2nd Col l ecti on )

% Blocrude oil

% Crude proteln

content

content

19。 10

10.85

21.64

11.64

4.2 Isol ation and Isolates

Owing to its rather big size (about o.5-l mm)of colonies, the

chosen method fOr this study was micropipette washing technique

(James 1978).

After washed colonies were inoculated into such of different media

under natural condition (Table 5) about a h,eek later in agar plate,the large green cel I (Fig. 7) was observed under microscope. Andthe same state of B. braunii, being composed of green mats at thebottoms of the tubes (in liquid), was found after 2 weeks in alImedi a .

The pH of the two defined media (modified Chu 13 doub l e-strengthmedium and Bristol's solution medi um) were adjusted to 6.7 which was

the detected pH of prince of Songkhla University,s reservoir, whilethis value of soi l -rrater medium was in the range of 6.6 - 6.9. Thetemperature of 33-36oC and light intensity of about 1500-2000 luxvere detected during the experiment under natural condition. Thetemperature was higher than that in prince of Songkhla University'sreservoi r.

Figure 7 Large green cells obtainedMagnification x 40.

from the isolation

27

メニ:ト

.3

「● F督

●

●

カ

:T

1季・に′

=●

28

Table 5

state of B. braunii

isolation in different media and conditions

Soll―‖ater Bristol:s Solution Medium Modified Chu 13 Double―

Media Medlum with Soll Extract Strength Medium

Liquid Li qui d

Condi ti on

Agar Plate

(soll extract

agar)

Agar

Plate

Natura l I arge

green

celis

I arge

green

celIs

'I arge

green

cel Is

I arge

9reencelIs

l arge

9reencelIs

Control 1 arge

green

cells

I arge

green

cells

l arge

green

cells

I arge

green

cells

I arge

green

cel Is

29

However, the temperature of 30oC (the detected temperature from thereservoir) was al so used in incubator under different I ightintensities (2, 3, 4, and the maximum light intensity of incubatorof 5 Klux) of contror condition and the large green cerl was alsoobserved at the same period of natural condition.

The results of these experiments suggested that these various mediaand conditions courd not induce this arga to form the autospore asin the nature. In addition, zoospore, frageilate agent of asexuarreproduction (Fig. 8) which always appeared when the surroundingcondition changes (Smith 1951, pringsheim lg67), was also observedafter a day of incubation. I|ost of algae, the agent turned to thenonnal form of the parent cell but in this experiment zoosporeornitted if motile phase and formed the large geeen cell. Therefore,this alga not only was a poljrmorhpic species, but also exhibited thedifferent patterns of reproduction owing to environmentar factors.

For the cells obtained from these experiments were simirar to thedescriptions of Brown and Knights (1969) and t{ake ( Lg76, per conm.).

30

′ ´

AutosPorr ll n'nature I

Colonl!l lorm

:lll

Zooapora

laborrlory condillon I

Ls.go groor call

夕⇒→⑤③

Flgure 8 Dlagramnatic surmery of asexual reproducti on

B. braunii from the study

Of

く惨

１■

つ０

4。 3 Growth and Productivities Of Botryococcus braunii During Indoorand Outdoor Cul tivations

The investigations on culturing of large green cel.l, the isolate,Irere carried out as followings:

4.3.1 Indoor cul tivation

The growth curves of Botryococcus braunii (fig. g) in parallelcultures under control condition occured irmediately withoutdistinct lag phase. The different states of B. braunii cultured bycasadevall et ar. (1995), inocuration or.rponlntffi[-rowing ceilsof green active state and fairly high initial concentration, andBelcher (1968), inocuration of a suspension of ord red colonies,shew the same resurts as in this experiment. Antarikanonda (19g0)mentioned two reasons for exp]aining this phenomenon which were:

(1) The growth characteristics of the organism itself, and(2) The culturing method, for exampre-the physiorogical conditionsof inocurum used, the size of inocurum etc. and the ratterreason agreed with Fogg,s suggestion (1975).

The fastest growth of B. braunii was supported by Kratz and l,ryersmedia, containing the r,GF.oncentrations of mineral nutrients,with mean biomass doubl ing time of 0.65 day and duration ofexponentiar phase about 2 days. For this phase in the other medialasted about 4 days with mean biomass doubling times (days) of 1.42,1.65 and 1.75 for lrls III medium, Bristol ,s solution .medium andmodified chu 13 doubre-strength medium respectivery. r{ake (1976)reported that the doubring time of the rarge green cerl in modifiedChu 13 double strength was only 30 h (1.25 days) and growth rate wasexponential below the point of the dry weight of 100 ng/1. But inthis study, the exponentiar phase appeared untir the dry rveightabout 1000 ng/r . The di fference of the resur ts depend on theculturing methods. Decline of growth after the dry weight of

32

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口■●Ｊｏ“。

●●ら贅０

′

0｀ 1● ■2 14 10 20 22 24

Dロッ5 0r Ctliture

F19ure 9 Growth curves, b10crude oll contents and crude prOtein

contents of Bo braunil ln four dlfferent medla under

indoor cOndit10n ′

(― )dry We19ht, (o引0)b10crude oll content and (0卜・・● )crudeprotein content of Mbdified Chu 13 double_strength medium

(D_a) dry weight, (ロー_4コ )biocrude oll cOntent and (■ 卜・・H日)crudeprotein content of Bristol=s solution medium

(― )dry Weight, (△「

―・ Δ)b10crude oll cOntent and

“

L‐・・¬▲)crudeprotein content of NS III medium

(― )dry Weight, (、「

―」▼)blocrude oll cOntent and (▼ に 」ア)crudeprotein content of Kratz and hlyers media

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一CENTRrヽ 1.LIBRARY

001437

IIAIIID0L ir-N I V ERSITY1000 mg/l might be due to decreasing concentration of nutrients and

mutual shading at increasing population densities (Antarikanonda

1980). For the latter evidence influenced strongly on the shortestduration of exponential phase of Kratz and I'tyers media.

The cell pigmentation varied obviously during the cultivation period(Fig- 9). The cell appeared light green at the beginning, the colorgrew darker and the cell enlarged during decline of growth and

finally it turned orange.

samples 1-4 (Table 6) correspondingly to physiological states were

subjected to determine biocrude oil and crude protein contents.Biocrude oil contents at sample 3 (about the end of phase ofdeclining growth rate) in all media were the highest and ranged fromLL.u% of dry weight in Kratz and r'lyers media to 9.67% of dry weightin NS III medium. In contrast, crude protein content at samp'le 1

(about the end of exponential phase) shorved the maximum especiallyin Ns III medium of 49.67% of dry weight and decreased markedlyuntil sample 4 (during the stationary phase) dropped to the minimum.

The preferential protein synthesis during the first phase (sample 1)

of the cul tures resu'l ted from acti ve cel 'l s i n photosynthesi s and

media containing high concentrations of nitrogen (Round 1973,. Fogg

1975, Trainor 1978). After the decreasing of nutrients, the growthdeclined (sample 2l and under nutrient stress condition of thisexperiment, the cell, being inactive in photosynthesis and low innitrogen content, enlarged and changed its color to orange (by

accumulation of carotenoid). As had been suggested, these cultureswere under nitrogen limitation (Stewart L974, Fogg 1975, Trainor1978). And during the late of decline of growth (sampre 3), themaximum biocrude oil contents obtained. The timing of such a

process was rpre close to the large accumulation of lipids conmonlyobserved in previous studies (Lewin 1962, Belcher 196g, Fogg 1975,Materassi et al. 1980, Shifrin and Chrisholm lggl).

Regarding the maximum biocrude oil content of the large green cell,

llo・ O tt a′ ぐ5

33

チ

Figure 10 Difference of cell colorsunder indoor condition

during the cultivati

34

ln period

―「―

』Ｈ

Ｈ

□rrt

35

Table 6

31ocrude oll and crude protein cOntents

different media under ind00r

of B. braunii in fourcondi ti on

Media Modified Chu 13

Doubl e―Strength

Bri stol ' s

Sol uti on

NS III Kratz and i,lyers

′ Blocrude Crude

Sampl es■ oll Protein

(%) (%)

Blocrude Crude

Oil Protein

(%) (%)

Blocrude Crude

01l Protein

(%) (%)

Blocrude Crude

01l Protein

(%) (%)

1

2

3

4

6。 13

8。 71

10.57

7.67

26.33

24.88

20.51

14.68

4.53

7.48

10.02

5。 83

28.10

26.54

23.07

16.73

5。 44

6.32

9.67

4.00

49.67

42.86

39。 67

13.23

2.88 33.78

8.65 26。 32

11.04 20.74

4.80 10.25

See Figure 8 forexponential phase;

sampl e 3, late ofphase.

the location of sampl es

sample 2, middle of phase

phase of decl ining relative

1-4。 Sampl e l, late of

of declining relative growth;

grOwth; samplo 4, stationary

36

this value has more than twice as compared with the content inEuphObia l athyris, the most efficient oil plant (Campbell f9g3).However, the biocrude oir contents of this investigation courd notcompare with the results of Brown and Knights (1969) and Uake(1976), finding very ritile hydrocarbon contents, because of thedifference of the quantities of products from anarysis.

4。 3.2 0utdoor cul tivation

After a precultivation under laboratory conditions the large greencells were investigated under natural climatic influences.

The cells arso grew successfuily in four different media underoutdoor condition by modified rocking trays but took the shorterperiods of cultivations (16 days) than under indoor condition. Thefastest growth (ftg. l0) appeared in Kratz and tlyers media with meanbiomass doubling time of r.rg days, higher than indoor condition.AccordinglJ, the mean biomass doubling times (days) of NS III mediumand Bristol's sorution medium were about 1.9g and 2.11 respectivery,but in modified chu 13 doubre-strength medium, the rower varue of1.52 days was obtained. The exponential phases last about 2 daysand no lag phases were detected in all media.

Four samples (Table 7) were also harvested accordingly to theprevious investigation on indoor cultivation and the similar resultswere obtained. The maximum biocrude oil contents, a little higher ascompared with these values under indoor condition, were at sample 3of Kratz and l,lyers media (f3.95%) and other nredia except modifiedchu 13 double-strength medium where sample 2 showed the maximumvalue. However, these values occured under nutrient stress (duringlate of phase of declining relative growth or early of stationaryphase) in all media. In regard to crude protein contents, thehighest values were at sample 1 in all media, especially in NS IIImedium 140.23%1, and reached the minimum in the stationary phase ofsample 4.

37

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ム、、、、、、、、、、こ`｀

1ヽ、、

▼ ― ...__._ .. 7

ヽ、

o I 2 ' 1 5 5 7 3 9 to t't

"0.;"r:1r.1t"Figure 11 Growth curves, biocrude oil contents and crude protein

contents of B. braunii in four different media underoutdoor condition by modified rocking trays

(G{) dry weight, (G-O) biocrude oil content and (G-{) crudeproteln content of Modified Chu 13 double-strength medium

(r.{) dry weight, (G--o) biocrude oiI content and (3,.{)'crudeprotein content of Brlsto'l ,s solufion medlum

(H) dry weight, (&-d) biocrude oil content and (&--l) crudeprotein content of NS III medium

fV-+) dry weight, (\F--V) biocrude oil content and (V-..{) crudeproteln content of Kratz and i,tyers media

＾´】

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38

Biocrude oi l and

outdoor condi ti on

Table 7

crude prOteln contents of

from September to octOber

B. braunll ln different media under

1985 by modified rocking trays

Media Modified Chu 13

0oubl e―Strength

Bri stol ' s

So'l utionNS III Kratz and Wers

81ocrude Crude

Sampl es' oll Protein

(%) (%)

31ocrude crude

01l Protei n

`Z〕

`%)

Blocrude crude

Oil Protein

(%) (%)

3iocrude Crude

01l Protein

(■ ) (%)

1

2

3

4

6.07

9.18

7.43

2.36

29.67

23.31

20。 84

16.62

4.97

6.39

7.55

5。 80

26.60

25。 12

24.99

18.88

3.88

5.32

11.07

7.56

40。 23

36.75

31.95

24.89

7.58 31.35

9。 66 30.46

13.85 29.89

10.65 24。 28

See Figure 10 for the location of sarnpl es 1-4. Sample l, late ofexponential phase; sampl e 2, middle of phase of declining relative growth;sampl e 3, late of phase of declining rel ati ve. growth ; sampl e 4, stationaryphase. For sampl es 2-4 of modified chu 13 doubl e-strength medi um were lnearly, middle and late of stationary phase.

39

During the cultivation period (from September to 0ctober 1985), the

cel I s were heal thy and no other organi sm except bacteri a

contaminated the cultures. The high temperatures of suspensions in

these shallow basins yrere about 31 to 4OoC.

For the cultivation of the large green cells in paddle wheel basin,

a prel iminary mass production uni t, Kratz and l'lyers media,

supporting the best growth and maximum of biocrude oil production in

this investigation, were selected for the experiment. The

exponential phase occured inmediately without distinct lag phase

(fig. 11). The former phase 'last only 2 days with mean biomass

doubl i ng ti me of 3 .7 days , the sl owest val ue i n thi s study.

Biocrude oil content decreased dramatically at the beginning of the

culture and reached the lowest value of 0.50% in the end of

cultivation period. In contrast, crude protein content did not

decrease according to growth curve and gradually rose from 35.39% to

55.57% of dry weight at the fifth day of cultivation (Table 8). Itwas markedly noted that the large green cells were still small and

did not enlarge during the cultivation period in cold season from

December 1985 to January 1986. However, near the late of cultivationperiod, these small cells aggregated to form a pellet the same as

the two previous experiment. The suspension temperatures of 25-33oC

r',ere detected. The culture was not contaminatedly by any other

organi sms such as other al gae, zoopl ankton and fungi ) except

bacteria but cause no problems during the cultavation period.

The high turbulance of 35 rpm was about 2 times as that of modified

rocking tray (3-second rocking period or 20 rpm). Richrnond et al.(1980) proposed that the higher rate of stirring decreased the

I ight/dark cycle and so the growth rate increased. In the

experiment even though the growth curve reached the stationary phase

but it could divide itself, the very small cell appeared, therefore,the very low biocrude oil content was detected.

A regu'lar increase of pH value was observed during exponential phase

owing to the complex effects of nitrate assimilation (Trelease and

40

目■●２●

ｏ４り

『０

一■

　

Ю

（″）　●“ｏ●口ｏｕ　一”Ｏ　ｏＯ口“●●““

（絆〕

●口ｏ口●●ｏ

●一０●０“ヽ　●●●“Ｕ　　６。

一ヽ

――

―

―

―

‐

‐

‐

（Ｎ日ヽ”〕　“〓“Ｈｏ〓　ヽい０

アロ‐―

‐．‐‐．‐―．‐‐‐‐．‐‐

●―

―

．‐■¨‐ｉ‐

‐

．‐

‐

7v---------v 7.n

--lv ?.5

Daf! of sul turc

Flgure 12 Growth curve ('9---g ) and varlation in pH (F.-._v ),blocrude oll content (\F___-V ) and crude proteincontent ( F._-{ ) of B. braunl i in Kratz and l,{ye rsmedia under outdoor cord-t tiii-ilpaaat

" wheel basins(from December 1985 to January 1986)

gl2l

41

Trelease 1935' Lewin 1962, venkataraman 1969) and c02 consumption in

lrmediately risen pH value resulted from out of C02 supply.

Table 8

dark reaction and the sl ight decrease untilperiod was the effect of dissolved C02. But

Blocrude oll and crude protein

Kratz and Myers media under

December 1985 tO January 1986

the end of cul tivationat the seventh day, the

contents of B. braunii inoutdoor condi tion fromby paddle wheel basins

Days of cultivatlon % Bう ocrude oll

(by dry weight)

% Crude Protei n

(by dry weight)

1

2

3

5

7

9

11

12

3.07

1.63

1.58

0。 72

0。 87

0.58

0.60

0.50

35。 39

43.54

53.09

55.57

46.62

49.64

52.70

53.27

42

CHAPttER 5

CONCLUS10N AND RECOMMENDATIONS

5.1 Concl usion

In the first study about Botryococcus braunii in Thailand, only thealgae in Prince of Songkhla University's reservoir was undertaken.Although the two collections of algal samples, dominanilyappearances, were carried out in different seasons but the sameranges of pH of 6.5-7 and temperature of 27-3|oc were detected andthese conditions were crose rerated to the previous reports (Bercher1968 Wake and Hillen 1980, l{ake and Hillen 1981 and Intasuwan l9g2).Biocrude oil contents of the local strain were only about ?0% of dryweight and this value was the highest of this stu_dy. In addition,the 247 (dry weight) of this content, which main products werebotryococcene and isobotryoccene, was kindly analyzed by Dr. c.Largeau.

The large green cell, was obtained from the isolation bymicropipette washing technique. The util ization of the threedifferent media (soil-water medium, Bristor,s sorution medium andmodified Chu 13 double-strength medium) under natural (33_360C and1'500-2'000 lux) and contror (gooc and 2, 3,4, and 5 rux and lightand dark cycle of 16:g) conditions in indoor experiment could notinduce the zoospore to change to the natural form of the parentcell. ['loreover the experiment in outdoor cultivation which thelarge green cell was in full sunlight of continuous variation undernatural conditions had no effect to the changable form of the ceil.

The experiments in curtivation of B. braunii showed that the algacould grow successfully under control laboratory condition of 30tloc, light intensity of 10t0.2 kIux, air enriched wi th t-z% c02 andlight and dark cycle of 16 : g. The four different concentrationmedia (modified chu 13 double-strength medium, Bristol,s solutionmedium, Kratz and l,lyers media and NS III medium) with the a(iusted

43(.I.,)i]'II,\ I, LIRITA RY.',.AIilD01. Li\i

pH to 6.7 of the reservoir suppJ,)a?ttiH5crose varue of biocrude oircontent and it reached the maximum during the 'late of phase ofdeclining relative growth or the beginning of the stationary phase

which took about 17 days for cultivation and the maximum value was

11.04% of dry weight by culturing in Kratz and I'tyers media. The

highest crude protein content of 49.67% of dry weight occured duringthe exponential phase and it took about 2-4 days for cultivation inNs III medium. The value was lorrer than that of the high proteincontent algae of 50-65% (Sinchumpasak 1980).

Not only supporting the highest biocrude oil content, but Kratz and

I'tyers media also supported the fastest growth with mean biomassdoubli4g time of 0.65 day (15.6 hours). Therefore, it was said thatKratz and I'lyers media was the most suitable medium for cultivationthe large green cells under control laboratory condition of theexperiment.

For outdoor cultivation, the first-step experiment was carried outby modified rocking tray under natural condition during September toOctober 1985. The four different media as using in indoorcultivation, were also used with a 3.-second rocking period and pure

cOz supplement. The cell grew well especia'lly in Kratz and I'tyersmedia which supported the fastest mean biomass doubling time of 1.1gdays during the exponential phase and the maximum biocrude oilcontent of 13.85% of dry weight at the ninth day of cultivationperiod. The maximum crude protein content of 40 .23% of dry weightwas detected by cultivation in NS III medium at the second day ofcul tivation period.

The last experiment was carried out by paddle wheel basin undernatural condition during the cool weather from December 1995 toJanuary 1986. Kratz and l'{yers, the only one medium used in thisexperiment, exhibited the mean biomass doubling time of 3.7 days and

biocrude oil production of 0.5% of dry ryeight. In contrast, thehighest crude protein content of 55.75% of dry weight appeared atthe fifth day of during the cultivation period. The suspension

temperature of 25-33oC was

moving suspension with high

44

detected, pure C02 was supplied to thepaddle speed of 35 rpm.

The outdoor cultivation under the uncontrolled temperature and Iightintensity, growth and productions of the alga were not only limitedby media, but arso under the contror of the pattern of curtivationunit and the speed of turbulance. considering above biocrude oilcontent, the large green cell could grow under the small scaleculture unit of modified rocking tray where as the further stu_dyshould focus on the unsuccessful cultivation of paddle wheel basinand in contrast to crude protein content.

These experiments suggested that the rarge green ceil exhibited bothof the maximum productions of biocrude oil and crude proteindepending on the harvesting period. Therefore, the harvesting atthe ninth and fifth day of cultivation by modified rocking tray andpaddle wheel basin both in Kratz and l,lyers media showed the maximumbiocrude oil and crude protein contents of 13.g5% and ss.7s% of dryweight respectively.

5。 2 RecommendatiOns for Further studies

The unsuccessfu! experiment in isolation should be furtherstudied as the following:

１■

(a) The methodology in inducing g:under laboratory condition

braunii to form autospore

(b) The factors in inducing the large green cell to form thecolony which wiil be very interesting in curturing forhydrocarbon production because the large green ceil growfaster than the colonial form. The study of casadevall etal . ( 1985) suggested that the fastest mean biomassdoubling time of the latter was about 30 hours where asthe rate of the former was only 15.6 hours.

45

t2)

(3)

(4)

The information of the appearances of B. braunii and thecorrelative data about water qrutr:tilf-Iia otherenvi ronnental factors of prince of Songkhla University,sreservoir and other water bodies during the seasonalsuccession should be important data for the two options

The smal l cell containing low biocrude oil content bycultivation in paddre wheer basin under natural condition wasdue to unsuitable technique in cultivation. Therefore, thefurther study should focus on the variation of speed turbulance

The stUdy on the compositions of biocrude oil of B. braunii,the local strain, shoutd be carried out in order to utirizethis product appropiately in the future. ln addition, theuseful products such as carotenoids, steroids etc. are arsoundertaken

In regard to protein production, the large green cell grownunder outdoor condition in paddle wheel basin, contains arather high crude protein content in the range of 35_56% of dryweight. Thus, the analysis of nutritional val ue and theimprovenent on suitable mass production of the large green cellshoul d al so be-undertaken.

ψ～

46

APPENDIX 1: Micropipette Washing TechniQue (James 197g)

Either soft glass tubing (3 to 4 mm bore) or disposabre pasteurpipettes can be used to form a micropipette. lrlhile clasping the tipof a sterirized pipette with a pain of forceps, hord the grass in asmall bunsen burner flame until it begins to soften. In a motionthat coincides with the removal of the grass from the flame, themicropipette tip is stretched with smooth, rapid pul l. By varyingthe timing and strength of the pu1 l, the bore of micropipette can beadjusted to meet the size needed for any microorganism. Usingforceps, break the tip from the micropipette with a steady genile,outward pull in a straight line with the pipette to make a smootheven tip. Bending or crushing the tip produces a jagged edge whichis difficult to use. Attach a rubber bulb or piece of soft rubbertubing to the top of the pipette.

l'lhile looking through a dissecting microscope, place a small sampreof the collection containing the organisms in a depression of asterile spot plate, a depression sride, or a watch grass. At reast6 to LZ other depressions shourd be firred wfth sterire water ormedia. HhiIe watching through the dissecting microscope, more thetip of a micropipette over the cel l. careful Iy dip the tip into themedium, pick up the cell, and expel it into another depression ofsterile water or medium. At reast rz to 15 singre cerrs shourd beisolated in this way and carefurry washed 6 to 1,? times indepressions of sterire fruid. A new micropipette shourd be used foreach washing.

After sufficient washing, singleof eight tubes of sterile mediurn.

to stimulate algal growth.

cel ls should be expel led into eachThe tubes are placed under lights

47

APPENDIX 2: Cul ture ‖edia

(1) ModificatiOn chu 13 double_strength medium (Wake 1976):

KN03

K2HP04

MgS04・ 7H20

CaC1 2・ 6H20

Fe citrate

Citric acid

(2) Bri stol:s s。lution medium

Slx stock solutions, 400

solution contains One of

NaN03

CaC1 2

MgS04・ 7H20

K2HP04

KH2P04

NaCl

ml in v。 lume each,

the fol10wing:

10.0 9

1.Og

3.09

3.O g

7.O g

l.09

are empl oyed. Each

0.10 g/1

0.02 g/1

0.05 9/1

0。 04 9/1

0.01 9ノ 1

0.10 9/1

(Starr 1978):

(3)

10 ml of each stock solution is added t0 940 ml of distllled

watero TO this add a drop of l.o% FeC13 S° l ution。

Soll extract agar (starr 1978):

Bristolrs sol uti on

Soil extract(soiI-water supernatant)Agar

960 ml

40 ml

15 9

48

(4) Soi l-water medium (after pringsheirn 1946):

A suitable garden soil, which should not have too great humus

content and been recently fertil ized with commercia.lfertilizers, sultables to be of medium. The basic soil-watermedi um is made as follows:

(a) Place l′2 - 1′4 inch of garden soll in bottOm of test tube

(b) Add distllled water untll the tube is 3/4 full

(c) Cover with cOtton plugs, meta1 0r plastic caps, etc.

(d) Stream(not autocl ave)for l h on at least 2 consecutive

days.

(5) Kratz and MyerS nedia (celpi et al. 1970):

KN03

K2HP04

(6) Hutneris A-5 microelement (celpi et al. 1980):

Ｈ３８０ＭｎＣ‐枷¨＝。０３

‖gS04・ 7H20

Na cltrate

Ca(N03)2・ 4H20

Fe(S04)3・ 6H20

2・4H20

4・7H20

4・5H20

(85%)

4.O gノ 1

1・ O g/1

0。 25 9/1

0。 165 9/1

0.025 9ノ 1

0.004 g/1

2.86 9/]

1.81 9ノ 1

0。 222 g/1

0.079 9ノ 1

0.0177 9/1

49

(7) lis III medium (Payer 1971):

Stock solutions of the composition of NS III:

Chemical MW Amount to water M01arity m1/1o00 m1 0f medium

/

KN0

KH2P04

K2HP04・ 3H20

‖gS04・ 7H20

CaC1 2・ 2H20

NaCl

101.1

136.1

228.2

246

147

58.4

101.1 9 1000

1i::: :]「

000

62.0 9 1000

7.4 9 1000

116.8 9 1000

ml

ml

ml

ml

ml

1.0

1.5(P)

0.25

0.05

2.0

10

2

2

2

0。 1

Micro A

B

c

2

2

2

Preparation of Micro A, B, C see separate recipe as following

50

Stock Solutions of Microelements pAZ_6

Chemical; ‖w Dllut10n l DllutiOn 2 For Resul ti ng MolarNS III Element Conc.

Amount

(rg)

A。 1)KBr (119)・

K」 (166)で Li Cl (42.4)

H3803 (61.6)

A.2)Znso4・ 7H20

‖iS04・ 6H20

0° S04・ 7H20

CuS04・ 5H20

A12(S04)3・ 18H20

(NH4)6M07024・ 4H20

NH4V03 {117)

144

658

70

125

167

44

29

|::3111 1:f~::II卜 ;9:口 ml H20

Br (79.91 2.19-6J (126.9) 1.10-6Li (a.241 2.n'7B Ir0.8) s.10-7

zn (65.4) 2.ro-8Ni (s$.i) 1.1s-7Co (5$.9) 1.1g-8cu (63.5) 2.r0-8Ar (27.0; 2.1s-8t4o (96.0) 1.10-8v (s0.9) 1.10-8'

3) MnC1 2・ 4‖20 50-looo ml H20+3 ml Hc1 35%

Fe(N03)3・ 9H20

Titriplex III

(EDTA)

2 n\ /1 l.ln (54.9; 5.1g-7

C)

発コЮO mり は∝pれ dark卜 2耐 /1 Fe (55。 3)4。 10~5

51

APPENDIX 3

Determination of protein

(Micro Kjeldahl Method after Bailey, 1967 modified)

Reagents

(l) 0.02 NHzsO4 Titrisol or H2s04 for determination of nitrogen(21 4% Boric acid w/v(3) Tarchiro indicator(4) 50% NaOH w/v(5) 98% H2S04 for determination of nitrogen.

Procedure

(1) To 100 mI Kjeldahl flask add 0.6 g selerium mixture(2) Heigh sample 0.06 g (dependent on nitrogen content) in a

al uminium foi I dnd transfer into fl ask by using boi I eddistilled water

(3) Rinse 4 ml 98% H2SO4 slowly into the flask(4) Digest gently on the Ki.edahl digestion apparatus until the

solution is clear, cool to room temperature(5) Rinse, with 20 ml distill water and continue digestion for I h,

redigest with l-z ml conc. H2s04 and let it cool to roomtemperature

(6) Add approximately 30 ml distill water and 2-3 drops of Tarchiroi ndicator

l7l To 100 ml Erlenmeyer flask pipette l0 ml of 4% H3803 and 2-3drops of Tarchiro indicator into the boric solution

(8) Distill by adding 30 ml of 50% Na0H; the condensed nitrogen was

catched by 10 ml of 4% boric acid(9) Distil l for ?o min or ill l 40 ml condensate is obtained, then- rinse the tip of condenser with boiled distill water(10) Titrate NH+ in boric acid with 0.02 N H2so4 until end point.

Compare the color with blank.

52

APPENDIX 4

!g!q" qual ity of prince of tt[Uni versity's reservoir atJanuary 20, 1985

Prince of songkhla university's reservoir, a rock and earthfilr dam,was constructed in 1g6g and improved for extent vol umn capacity to438,995 m3. The catchment area was about 2.05 km2 from rrrao rnoHong. It has an average depth of 5 m and a maximum depth of 11 m.The purpose was only for domestic water supply.

The analysis of 4 water samples about 1

of this reservoir was carried out as the

m from and around the edge

fol I owi ngs:

Sampl e

Determination

N-Nitrate (ppm)

N-Nitrite (ppm)

N-Armonia (ugll )

Ortho-phosphate (ppm)

Alkalinity (rg/t CaC03)

Acidity (ng/l CaC03)

Chloride (ppm)

Sulfate (ppn)

Total suspended solidpH

Temperature

0.349

0。 030

0。 442

0.015

60

12

166

4

2

6。8

27

2。 230

0。 012

0.411

0。 005

40

8

166

3。 333

2

7

27.5

0.232

0。 020

0。 411

0。 020

40

16

111

2。 667

2

6.8

27

0。 430

0。 006

0.379

0.000

40

16

222

3。 333

4

6。 7

27

53

Analytical procedures

N-Nitrate by cororimetric with surphaniramide and N-(1-naphthyl ) -ethyl ene-di ami ne

N-Nitrite by Cadmium reduction methodN-Annonia by Colorimetric as indo_phenol blue0rtho-phosphate by Ascorbic method

Alkalinity by Titration method indicator end pointAcidity by Titration method indicator end pointChloride by yolumetric with Ag*, indicator end pointSulfate by Turbidity method

Suspended solid by Filtration method

From (l) Golterman, H.L., crymo, R.s., and 0hnstad, M.A.M. 197g

2nd ed. Blackwell Scientific publications, Oxford, London.

Ql Greenberg, A.E., Conners, J.J., and Jenkins, D. 19g0Standard methOds for the exam]nation of water and waste

Water 15th edo American Pub]ic Health AssociatiOn.

And the other reservoir which had B. braunii in this study,Srinakarinwiroj (at Songkhla) Universitv t iloir was constructedin 1970 with only a volumn capacity of about 40,000 ,3. Thecatchment area uras a small hill nearby.

54

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