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-4,
ETHANOLIC FERMENTATION OF D-XYLOSE
AND PINE TIOOD HYDROLYZATE BY
THE YEAST
PachYsolen tannoohllus
A thesls presented l-n pa:rtlaL fulftlnent of the :requi:rements
for the degree of Doctor of PhLlosophy ln Blotechnology at
I{as6ey Unlvenslty
TZE SEN I|ONG
t 984
lB.slnrcT
This thesis reports a study of the ethanolic fermentation of
D-xylose and wood hydrolyzate bo ethanol by the yeast pachysolen
tannophilus with a view to developlng an effective ug,e of renewable
hemi-cellurose hydrolysis products from Nry Zealand forest blomass
residues. 
.
Iniflal work briefly addressed the problsn of findlng a suitable
yeast frcm natural habitats suitable for the fermentation. Soon after
that work commenced literature reports suggested that prelimlnary
conversl0n of pentoses by enzymatic means was a possibility.
consequently, this aspect of converslon was considered and rejected.
one reason for thls was that literature waa drawlng attention to the
pentose f ermenting characteristics of paehysoren tannophilus.
Laboratory scale studies demonstrated the yeast pachysolen
fannophilus to be capable of ferrnenting the hexose and pentose sugars
present in the hydrolyzate. The yeastrs specific growbh rate in the
hydrolyzate could be lmprovect by neutrallzlng the lnhibibory substances
with 2 8/I of anhydrous sodiun sulphite. Ethanol has an inhibitory
effect on growth but can also be readily assimtlatecl by the yeast.
Fermentation studles with gyration speeds of 50, 1OO and 2OO
r.p.m. showed that oxygen was a crltical paramefer affectlng growth
and elhanol produetion. Bateh fermentat,ion expertments were pursued to
examine this oxygen phenonenon more closery. cell growth, substrate
upfake rate and cultwe pH responded strongly to the suppry of oxygen.
However, productlon of ethanol acecrnpanled cell growth only in Late
rr exponentl alrr phase.
Fermentatlon characterlstlcs were established under contlnuous
culture at an aeratlon rate of 0.37 L/I.min and values obtalned were a.S
follows; maxlmun specific growth rate, 0.046 h-1; blcrna.ss yierd, o.04
e/ei ethanol yleld, o.1T g/gi Ks varue, 13 e/L and Ki values, 0.5
e/L.
A redox pobentlal controlled chemostat study revealed that
steady-state culture poised at -50 mV exhlbited a 55fi lncreased ethanol
concentration and Bf decreased xylitol concentraflon over the val.ue
observed without redox conbrol.
With a knowledge of D-xylase fermentatlon as established in these
batch and chemostat experiments, it hraE possible to consider more
detailed aspects of the fermentation which would be applicable to
process develognent. Questions addressed lncluded which strain of
Pachysolen'tannophilus should be used, whab quantlty of inoculum was
necessary, what lnteractions existed between f ermentation variables.
Statistically designed experiments were enployed to answer these
questions. Empirical models so developed revealed that ethanoL yield
has a tinear relationship with initial substrate concentration. These
models have given some lnsight into how environmental factors affect
the ethanolic ferrnentation by this yeast and have also indicated the
optimal conditions required for an effective fermentabion of wood
pent os es .
These important fermentation process variables were established
and are expected to be useful ln moving the process from laboratory
scale as carried out here to a pilot plant scale of operations. The
values established were temperature, 28o or loler; initlal mediun ptt
for ethanol productlon, 5.6 to 5.8; substrate concentration used can
be up to 80 g/l of pentoses; minimun inoculum densiLy, 5.5 g/I ey
welght cells and NRRL Y-2461 was reccmmended as the best strain to
achieve the fermentation. The pre-treabment of fhe prehydrolyzate by 2
g/L of anhydrous sodiun sulphite was hlghly desirable in order to
enhance growth and fermentation rates.
The research has shown thab Pachysolen tannophilus ls capable of
fermenting pentose fractlon of wood hydrolyzate and that the optimal
condltions for this fennenbatlon w111 lead to signiflcant utillzation
of wood sugar. However, ln the completely mixed reactor systans used
in these experiments, the ethanol yielcts obtalned were not as
attractlve as those observed for hexose fenmentations under similar
conditions. Thls, it ls felt, points to the greater difficulty the
yeast experiences ln fenmentlng pentoses and lt also suggests the need
to lnvestigate the value of other reactor formats at some future date.
l- l-
17 December, 1984.
This is to certify that the work on which
the thesis, ETrIAnoLrc FERnENrNrroN aF tyxyhmg ,,;'tD prNE nlooD
nnROLYZAfE Et frIE YEASE Pachgsolen tanaopbiJus, is based
has not been accepted In whoLe or in part for any other
degree or diploma and all the help and assistance received
in the research on which the thesis is based, are fully
acknowledged.
Tze Wong.
Candidate.Ph. D.
ACrOr.mGEEttS
I wish to acknowledge the following :
my supervisors Dr. G.J. Manderson, Dp. V.F. Larsen and
Dr. R.H. Archer for thelr kind supervislon and guidance.
Dr.. J. Butcher, DF. K. Mackie and Dr. T.A. Clark, Forest
Research Insti.tute for their advice and provlsion of the
redox potential electrodes, the supply of acid hydrolyzate
and prehydrolyzate and other materials and for the analyses
of xylitol ln culture supernatants.
Dr. I.F. Boag for advice on the statistical experimental
desl gns .
Mrs. D. Steward for analyzing the sugar, xyIitol.
Mrs. K. Deverell for kind advice and asslstance during
my visit to the Forest Research Institute, Rotorua.
Mr. J.T. Alger, Mr, P. Shaw, Mr. D.W. Couling and
Mr. M.Stevens for their assistanee in building and
maintaining equignent and technlcal advice.
Miss Fiona Choy for typing this fhesis.
My wife, Kln , for her kind and moral support throughout
entire coLlrse of study.
- finally, to my family and many other people who
the preparation of this thesls.
bhe
helped durlng
This research was supported by a grant from the Nen Zealand Foresf
Research Instltute, Rotorua, New Zealand as Contract Nunber 149 between
the New Zealand Forest Service and Massey Unlverslty. This support is
sincerel y aeknowledged.
l-rl_
TABLE OF CONTENTS
page
ABSTRACT .... O 1
ACKI0WLEDGE}Tn{TS ....... 111
TABLE OF CONTENTS ..... . lv
LISf OF FIGURES Xi
LIST 0F TABLES ..... .......... . ......... ...... xvi
ABBREVIATIONS AND SYMB0LS ..... ..... o.. xxi
CHAPTER ONE : INTRODUCTION
CHAPTER TWO :
2.'I INTRODUCTION
REUIEII 0F LITERATURE
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5
5
52.2 MICROBTAL PENTOSE CoNVERSTONS . .. .. .. ... .... .. o.. . o.. o.. . . . .
2.2.1 Bacterla .....
2.2.2 MyCella1 Fungl .........o....r......o......r.....oo...
2.2.3 Yeasts a a a a r a. a a a aa a a a a a o o a aa
2.3 INDIRECT ALDOPENTOSE FERI"IH{TATION : ENZIME LINKED .... .. ....
PENTUTOSE FERMENTATION
2.3.1 Conversion of D-xylose to D-xy1ulose ... o o.. o.. o..... . 1 4
2.3.2 D-Xylulose Fermentatlon o o. . o o. ... .. .. .... o.. .. .. .. . .. 1 6
2.3.3 Isomerlzatlon Llnked Fernentatlon ............o.o 17
2.4 DIRrcT ALDOPENTOSE FER'IENTATION ...................o.ooo.... 1-g
2.U.1 Uslng Naturally Occurrlng leasts ....oo.o............. 19
2.U.2 Uslng a Yeast Mutant o..o...... .o.....o.........o 20
7
I
10
14
l-v
Page
202.\.3 Using Yeast Stralns Irnproved by
Recomblnant DNA Methodology
2.5 BIOLOGY OF Pachysolen tannophilus
2.5.1 Morphological Descrtptlon of Pachysolen tannophllug ... 21
2.5.2 Reproductlon of Pachysolen tannophilus ..... ... .. 21
2.5.2.1 Asexual (Vegetablve) Reproducblon 21
2.5.2.2 Sexual Reproductlon (Ascus Formatlon) ... o.... 21
2.5.3 Pnylogenetic Relatlonshlp of Pachysolen tannophllus ... 22
with other Yeast Genera
2.6 GROWI}I AND FERMB'ITATION CHARACTERISTICS OF
Pachysolen tannophi lus
2.6.1 Effects of pH on Growth and Ferurentatlon . ............ 23
2.6.2 Temperature Effects .. ..... ..... ...o... 23
2.6.3 Fermentatlon Substrates ... o,.....,........ o... o. o. r o. 24
2.6.U Aeratlon Effects .......... ..... 24
2.6.5 Inoculum Concentratlon . o ... ... ... ...... o.. ..... . 26
2.6.6 Effect of Inhlbltrs on Growth and Fermentatlon ..... . 26
2.6.6.1 Inhlbltory Substances ln Hydrolyzates .... o... 26
2.6.6.2 Ethanol Inhibltlon 27
CHAPTER THREE : MATERIALS AND METHODS
3 . 1 MATE RI ALS . . . . . . . . . . . . . . . . . . . . o . . . . . . . . . o . . . . . . o o . . . . . . . . . o .
3.1.1 Mlcroblologlcal Medla ............o......r....... 32
3.1 .2 Chromatographlc Materlals .. o. o....................... 32
3.1.3 GaSes ............... .......o....'............. 32
3.1.4 Chemlcals ............. o........... o. o................ 32
3.1 .ll .1 Sugars ................... o................... 32
3.1.4.2 Enzymes .. .... . ....... o. o... .... o. . ...... .. . .. 33
3.1.4.3 Other Chemlcals ....o..................o...... 33
3.1 .5 SOlvgnt . o. o............... r......................... . 33
3.1.6 Wood Hydrolyzate ...........o.............o........... 33
21
22
32
3.1 .7 Mlcro-organlsms
3. 1 .7.1 Bacterla
3.1 .7 .2 Yeasts
a a a a a a a o a o a a o a a a a a r aa aa a a a aa a a a o a a a a a
aa.aa aaaaa aaaa. aaaaaaaaa
Used
Page
36
36
36
3T
38
38
38
40
46
46
46
\T
47
49
49
49
49
50
CLEANING OF GLASSWARE 3T3.3
3.4 ANALYTICAL MET}IODS
3.5 ENZYME ASSAY o..o. ............................o........
3.5.1 Preparatlon of Cell-Free Extract .o..............o....
3.5.2 Enzyme ASSay . . o.. .. .. . . o...... ......... .. .. .. o ..... ..
3.5.3 Cystelne-Carbazole Test .o...o..............o...o.oo..
3.5.4 Enzyme Unit .. o... o......... o.o...o............. o... o.
3.6 CULTURE GRoI{TH CONDTTToNS
3.6.1 Shakg - Flask Culture ..o.....o.......o....'.....r....
3.6.2 Fermenter Culture .....
3.6.2.1 Equlpurenb - Fernenters and Blectrodes Used . . .
3.tI.3 Analysis of Sugars
3.q.q Column Chromatography .....r......o.................o. 43
3,4.4.1 Ion Exchanger Chromatography .. r........... .. o 43
3.4.4.2 Gel Flltratlon ..................o....o.....'. ll3
3.ll .5 Gas Liquicl Chromatography ..... .......... o..,.... .... . 44
3.4.6 High Performance Ltquld Chromatography
3.U.7 Polyacrylamide Dlsc Gel Electrophoresis ......... q4
3.6.2.2 pH Measurement
3.6.2.3 Dissolved 0xygen Tenslon (DOT) Measurement ... 50
3,6.2.4 Redox Potentlal Measurement .................. 54
3.7 DISCUSSION 0F METHODS .o.......... o.....'............... r. o. 54
vi
4.1
Page
CHAPTER FOUR : PRELIMINARY STUDIES 0F SCREENING FOn PE{TOSE
FERMN'ITING ORGAI.II9IS AND ENZYME LINKED
P Bi 11' IOSE FERI.IENTAT ION
Part I : SCf,EEIIIF Fm XTIIISE FHIE|IIIG mGrf,MS .. .
BT |EIIIE TRICNflT CT'LIUNE TEilIq'BS
4.1 .1 TNTRoDUCTToN .,. .. ..... . .. ......
4.1.2 EXPERTMH{TAL METHoDS ...o...,..,..............
4.1 .3 RESJLTS .....
It.1.3.1 Enrichment Cultures from Anaerobic Digestion ......
U.1 .3.2 Enrichment Cultures fron Decaytng Wood .....,.
4.1.3.3 Enrichnent Cultures from Garden Compost plus Sotl
4.1.4 DTSCUSSTON ..... .....
tl 
.1 .5 CONCLUSIONS . . o . .. . .. . . .. ... ...... o o o.. ... .. .. o.. o . o o.. .
u.2 Par*, II : EtiZ[G-YElSf Sf,StElSi SIIIDIES
4.2.1 TNTRODUCTToN ..... 62
\.2.2 EXPERTMENTAL METIIODS .......,.. o... ....,.... . o.. . o...... . 62
U.2.2.1 Culture and Extractlon of D-xylose Isomerase . . 62
4.2.2.2 Enzyme Puriflcatlon o.... ....o...... 62
4.2.2.3 Enzyne Sbablllty ............o.. 64
U.2.3 RESULTS .. .... r..... o................. o. o. o. t o.. o... t..
U.2.3.1 Screenlng of D-xylose Isomerase Produclrt8 Organlsms.
U.2.3.2 Extractlon of D-xylose Isomerase from . o....
Baclllus eoagulans NCIB 8870
U.2.3.3 Partlat Purlflcatlon of D-xylose Isonerase of .....
LactobacllIus fermenfum ATCC 9338
56
56
57
57
57
59
60
60
62
6q
6q
65
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Page
\.2.3.4 Study of D-xylose Isomerase Actlvlty ln Hydrolyzate. 80
4.2.11 DTSCUSSTON
U.2.5 CONCLUSIONS
5.ll DrscussroN
5.5 CONCLUSIONS
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82
.HAPTER FrvE ' :::;::il"::il'ff:-:"T::;;:":'
Synthetlc Medium and Wood Hydrolyzate
5.1 INTRODUCTION . o... . o o. o................ o.... o...... o..
5.2 EXPERIMENTAL MEf,HODS ............ o....... o..... o..
5.2.1 Shake Flask Experimgnts ...........ooo................
5.2.2 Ygast Cultures ...o.o,.o....o. ..... ........
5.3 RESULTS .o......o..................... ........o......
5.3.1 Screenlng of Yeasts for D-Xyloge Fermentabion. . . .. . . .
5.3.2 Effect of AgiLation Rates on CeIl Growth and Ethanol ..
Production
5.3.3 Study of the Fermentatlon of Wood Sugars to Ethanol
by Pachysolen tannoPhllus
5.3.4 Effect of Inoculum Slzes on Cell Growth, Fernentatlon .
Tlme, Ethanol Productlon and Speciflc Growth Rate
5.3.5 Effect of Oxygen Supply ln Shaken Flasks Culture on . ..
Cell Growth and Ethanol Productlon
5.3.6 Asslmllatlon of Ethanol by Pachysolen tannophllus 97
5.3.7 Effect of Anhydrous Sodlum sulphlte I Na2SO3 J ........
Treatment of PrehydrolYzate
83
83
83
84
Itl
84
84
86
88
91
95
a a o a o a a a a a a a a a a a aa o a a a a a a a a a o a o a a a a a a a a a a a a o a a a
a aaa aaaa aaaa oaaaaa a a aaa aoaaa aaa aa a aaaaaaa
101
10ll
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Page
CHAPTER SIX : EXPERIMENT 1 INTERACTIVE EFFrcTS OF SUBSTRATE 106
CONCENTRATION AND INOCULUM LEVEL
ON PE,ITOSE FERMENTATION IN TI{E
SYNTHETIC MEDIUM
6.1 INTRODUCTION ,.......... o 106
6.2 EXPERIilENTAL DESIGN
6.3 FERMB,ITATION CONDITIONS 109
6.4 RES.'LTS
6.5 DTSCUSSToN
6.6 CONCLUSIONS
7 .4 RESULTS
7.5 DTSCUSSToN
7.6 CONCLUSIONS
a.a r. a a aa aa aa aaa a.a a ao.a.a a a a a a aaa a aa a. aa a a a 118
't20
CHAPTER SEVEN : EXPERIMB,IT 2 - EFFrcTS 0F pH, TEMPERATURE AND ...
'l
NITROGEN SUPPLEMENTATION ON
SOFTWOOD PREI{TDROLY ZATE
FERMENTATION
T .1 INTRODTrcTION
7 .2 EXPERIMENTAL DESIGN .......... .. o... ............ o........ ..
7.3 FER!"IENTATION CONDTTTONS
12
121
12',1
125
. . . . . . . . . . o . . . o . . . . . . . . . . . . . . . . . . . . . o . . . o . . . . r . a 1 36
............... o. o.............. o...... a.. a o a a. 138
t-x
Page
CHAPTER EIGHT : FERMENTATION STUDIES 0F D-XIbSE USING ........ 139
Pachy solen tannophl lus
8.1 INTRODUCTION o..................................r...... 139
8.2 FER{H'ITATION CONDITIONS ........ o.. o........... o............ 1 39
8.3 RESULTS . o......... ...... o o....... o.. .......... o............ 1 40
8.3.1 Batch Growth and Fermentatlon Studles .o.........o.... 140
8.3.2 Batch Fermentatlon wlth nPulserr Aeration . .. o.... o.... 1 41
8.3.3 Contlnuq.rs Culture Studles .... .............. r. . . . . . .. 14?
8.3. q The Reclox Controlled Chernostat . ........... ......... .. 160
8.4 DISCUSSION o.... ..... .................o...... 166
8.5 CONCLUSIONS CONCERNINC D-XYLOSE FERI'IENTATION BI . ..... o. ... o 172
Pachysolen tannophi lus
CHAPTER NINE : GEIERAL CoNCIITSIONS .o........o............o... 17ll
x
TJST (F ETGUNESi
page
2.1 The Converslon of Pentoses to End-products 6
2.2 Pathways Used by Bacterla for the Formatlon 9
of End-products (A) the mixed aclds and
2,3-butanedlol (B) aeetone, butanol, butyrlc aclcl
and isopropanoL.
2.3 Pathways of Pentose Converslon to 1 1
D-xylulose-5-phosphate uslng Isomerizatlon or
0xidation-Reductlon SteP
2.\ Wood Sugars Metabollsm by Yeasts 1 3
3.1 A Calibratlon Curve of CeIl Count (X) versus 39
Dry Weight (W) for Pachysolen tannophllus IFO 1007
3.2 Standant Curve for Total Reduclng Sugar 4?
(O-Xylose) by Uslng Modlfled Somogyl and
Nelson Methocl.
3.3 Sbandard Curve for Ethanol Analysts 45
3.4 Cystelne Carbazole Method for Assay of D-Xylose 48
and D-XyluIose
3.5 The Assembled Fermenter ln Contlnuous Aerated 51
Cherpstat
3.6 The Assenbled Fetmenter ln Contlnuous Flor Systen 52
wlth Redox Controlled
3.7 A Schematlc Dlagraur of the Fermenter ancl lts 53
Anclllary Equlpnent for Contlnuotts Culture Studies
xl-
Flgure TlLle Page
4.1 Effect of Culture Age on D-xylose Isonerase and 66
Activity ln CelI Free Extracts of Baelllus
coagulans NCIB 8870 Harvested Throughout Growth Cycle
U.2 Time Course Study of D-xylose Isonerase Reaetlon 67
Equlllbrlum ln CeIl Free Extraets of Bacillus
coagulans NCIB 8870 tlarvested between 1 6 and
24 hours of Growth
4.3 Time Course Study of the Release of D-xylose Isomerase 68
from 20 hours Cells of Baclllus coagulans NCIB 8870
Using (A) 1 e/L of Cattonle Detergent and (B) Braun
Homogen iz er
q.q Chromatograph of the Lactobaclllus fermentum ATCC 9338 72
D-xylose Isomerase from the TO-n% Ammonium Sulphate
Fractlons on DEAE-Sephadex A50 Showing t}te Enzyme
Peak and Protein Peak(s)
4.5 Gel Filtratlon of the Laetobaclllus fermentum ATCC 9338 73
D-xylose Isomerase from the A50 Fractions 45-50 on
Sephadex G-200 Showirg the Slngle Enzyme Peak and
a Proteln Peak
ll.6 Absorptlon Spectra of Varlous Purlflcatlon 7 U
Fractlons from Lactobaclllus fermentum ATCC 9338 75
U.T Polyacrylamide Gel Electrophoresls of D-xylose ?8
Isomerase of Various Purlflcatlon Fractlons
ll.8 Lineweaver-Burk Plot of Data Collected from 79
D-xylose Igomerase 1n Partlally purlfied Extract of
Lactobaelllus fernentum ATCC 9338
5.1 Effect of Agitatlon Speecls on cell Growth and Ethanol 87
Produetlon ln Shaken Flask Cultures of
Pachysolen tannophllus IFO 1007
x l_l_
Flgure Tltle Page
5.2 Fermentatlon of Wood Sugars to Ethanol by 90
Pachysolen tannophllus wlth the Inttlal Sugar
Concentratlon of 20 e/I
5.3 Effect of Inoculum Slze on (A) Ethanol Productlon 93
and (B) Population Growth of Paehysolen tannophllus
rFo't007
5.q The Effect of Inoeulun Concentratlon 94
oi Speclfic Growth Rabe and Fermentation Time
to Two-thirds Maxlmum Ethanol Productlon
5.5 Effect of Otygen Supply on CelI Growth and Ethanol 96
Productlon in Shaken Flask Culture of
Pachysolen tannophllus IF0 1007
5.6 Assimllation of Ethanol by Pachysolen tannophllus 98
I Fo 100? ln (A ) \ g/L and (B) 16 g/L of
Ethanol wlth 6.7 g/L of Yeast Nitrogen Base at 30o
5.7 Effect of Sodlum Sulphlte Concentratlon on the 100
Speciflc Growth Rates of Three Different Stralns of
Pachysolen tannophilusln the Soft l{ood
Prehydrolyzate
6.1 Two-factor Central Composlte Rotatable Deslgn with 107
axlal 'starr polnts at coded dlstanee c = 1.414 from
the 0rlg tn
S and I respented the two lndependent varlables,
substrate and lnoeulum coneentratlons respectively
6.2 Plot of CeIl Growth and Ethanol Production against 110
Fernentatlon Tlme frcrn One Flask at the Centre Point
(0,0,0) of the ExPerlmental Deslgn 1
6.3 Residual Plots (A) Standardlzed Resl<luals versus 113
Predlcted Y (B) Standardlzed Reslcluals versus
Nornal Random Numbers for Parslmonous Equatlon for
Speclflc Ethanol Productlon Rate of Experlment 1
x rll-
Flgure Tttle Page
6.ll Contour Plots of Fermentatlon Tlme Observed ln 115
Shaken Flask Studies of Lachysolen tannophllus as
a Function of Substrate Concentratlon and Inoculum
Level ln Synthettc Medium
6.5 Contour Plots of Maximum Instantenous Ethanol 116
Productlon Rate ln a Shaken Flask Stuclles of
Pachysolen tannophilus as a Function of Substrafe
Concentration and Inoculum Level ln Synthettc
Mbd ium
6.6 Contour Plots of Final Cell Yle1d in Shake Flask 117
Studies of Pachysolen tannophllus as a
Functlon of Substrate Concentration and Inoculum
LeveI in Synthetlc Medlum
7.1 A Three-faelor Central Composite Design 124
H, T and N represent the three lndependent varlables
pH, Temperature and Nltrogenous Supplementatton
respeebively
The numbers are coded level of the treatment
comblnations relative to bhe centre polnt
7.2 Plot of Cell Growth and Ethanol Productlon against 127
Fermentablon Tlme frcm One Flask at the Centre Polnt
(0,0,0) of Pachysolen tannophllus NRRL Y-21160 of
the Experlmental Deslgn 2
7.3 A Conparison of Contour Plots of Maxinun Ebhanol 133
Corpentratlon as a Functlon of pH and Temperature
of Three Stralns of Pachysolen tannophllus ln Plne-
wood Prehydrolyzate
?.ll Comparison of Contour Plots of Speclflc Growth Rate 134
as a Functlon of pH and Temperature of Three Stralns
of Pachysolen tannophilus in Plne-wood Prehydrolyzate
xav
Flgure Tl tle Page
'1.5 A Comparlson of Contour Plots of Ylelds of Cell of 135
Three Strains of Pachysolen bannophilus as a
Functlon of pH and Temperature Growlng in Plne-wood
PrehydrolyzaLe
8.1 Time Course of Growth, Ethanol Production and pH 1U2
Change for the Batch Culture of Pachysolen
tannophllus at 28o and pH 5.1
8.2 Batch Fermentation of D-xylose to Ethanol by lhe 143
Yeast Pachysolen tannophilus at 28o and pH 5.1
under Varying Aeration Condltions
8.3 Ethanol Concentratlon as a Functlon of the 1 48
Substrate Consumed by Pachysolen tannophilus NRRL Y-2461
Grown ln a Chemostat
8.q Blomass as a Functlon of the Substrate Consumed 150
by Pachysolen tannophilus NRRL Y-21161 Grown in a Chenpstat
8.5 By-product (xylltol) as a Functlon of the Substrate 151
Consumed by Pachysolen tannophllus NRRL Y-2461 Grown
in a Chemostat
8.6 Ethanol and Xylltol Produced as a Functlon of 152
Blomass Growth for Pachysolen tannophllus NRRL Y-2461
8.7 Ethanol Produced as a Functlon of By-Product 153
(Xylttof) Produced by Pachysolen tannophllus NRRL Y-2116'l
8.8 Lineweaver PIot of 1/D versus 1/Sx for a Chemostat 154
Cul.ture of Pachysolen tannophtlus NRRL Y-2461
8.9 Blomass Yleld and Ethanol Corpentratlon as a 156
Funetions of D1lutlon Rate for a Chemostat Culture
of Pachysolen tannophilus NRRL Y-2461
8.1 0 Effect of Ethanol Production on Cell Growth 157
of Pachysolen tannophllus NRRL Y-2461
xv
Flgure Tltle Page
8.11 (A) The Speclf lc Rate of D-xylose Consurptlon vers,rs 159
Dllutlon Rate
(B) Reclprocal Plot of the Observed Cell Yleld 159
versus Dllutlon Rate
8.12 Plots of Ethanol againsb Cell or Sugar and Cetl 165
versus Sugar ln a Contlnuqrs Flow System with Serles
of Redox Controlled and Non-controlled
8.13 S0ggestect Fate of D-xylose durlng the Growth of 167
Pachysoten tannophllus under nPulsetr Aerated
Conditlons of the Culture
8.1t1 Proposed D-xylose Metabollc Scherne of Paehysolen
tannophilus under Oxygen-llmited Condltions
171
xvl-
page
ust (r llExsi
1.1 Carbohydrate Composttlon of llardwoods and 3
Softwoods
1.2 Sugar Composltions of Softwood (Pinus
radlaba) Acld Hydrolyzate Llquors
2.1 Ends Products Formed Durlng the Conversion of 8
Glucose and/or Xylose by Selected Bacteria
2.2 General Comparsion of Pentose ( D-Xylose ) 12
Metabollsm of Bacterla, Yeasts and Mycella1 Fungi
2.3 Ethanol Production from Xylulose by Yeasts 18
Grown tn Batch Culture
2.\ Comparison of Klnetlc Constants of Various Ethanol 29
Irrhibitlon Studies of Saccharomyces Stralns
3.1 Composition of t{ood Hydrolyzate Fraetlons 3tt
3.2 Cornpositlon of D1sblllatlon Resldues Following 35
the Ethanolle Fernentatlon by Saceharouryces
cerevislae of Llme Neutrallzed Wood
Hydrolyzate
ll.1 Fernentatlon Enct-Products ln Supernatants from 58
Enrichement Cultures Developed frqt Anaeroblc
Digester Sludge
\.2 Fermentatlon End-Products ln Supernatants from 58
Enrlchement Cultures Developed frst an Inoculun
of Rotted tlood Fragments
4.3 Fermentatlon End-Products ln Supernatants fron 59
Enrlchement Cultures Developed from an Inoculum
of Garden ConPost PIus Sotl
xvLa
Table TitIe Page
4.4 A Comparlson of the Enzyme Actlvtty Released into 69
Supernatanbs from Baclllus coagulans by Differenb
CeIl DlsruPtlon Mebhods
4.5 D-xylose Isomerase Actlviby and Proteln content 70
of Varlous Fractlons from Amslonlum Sulphate
Fractlonatlon
4.6 Puriflcatlon of D-xylose Igomerase of Lactobaclllus 76
f€rmentum ATCC 9338
tl.T A Comparatlve Sbudy of Km Values of D-Xylose 77
Isonerase from Lactobaclllus species
4.8 A Study of Partially Purified Lactobacillus 80
fermentum D-xylose Isomerase and 'sweetzymer
Activitles ln Plne Wood Acld Hydrolyzate
5.1 A Screenlng of Yeasts for D-Xylose Fermentation 85
Capabll lt les
5.2 tilood Sugars as Substrates for Growth and 89
Fermentatlon uslng f3gly,soIen tannophllus ln Shake
Flasks (50 r.p.m. ) at 30o C for 7 days
6.1 A Central Conposlte Rotatable Statisbically 108
Desgn Experlment to Study the Interactlons of
Substrate Corrcentratlon and Inoculum Concentratlon
on Ethanol Productlon ancl CeIl Ylelds
6.2 Full Regresslon Models for Experlment 1 112
7.1 A three-factor central courposlte statlstlcal 122
Deslgn for ExPerlment 2
7.2 Level of Independent varlables ln the cosrposlte 123
Deslgn Experlment 2
xviii
Table Title Page
7.3 Derlved Regresslon Models for the Straln IFO 1007 14
7.4 Derived Regresslon Models for the Straln 130
NRRL Y-2460
7.5 Derived Regression Mode1s for the Strain 131
NRRL Y-2461
7 .6 A Courparlson of the Tests of lack of Fit wlth 132
ahd without the Square-root Transfornatlon of
the Response Varlables of Pachysolen tannophilus
NRRL Y-21t61 Growlng on Prehydrolyzat'e
7.7 Reeommended Strain Used and Condltions for the 138
Ethanollc Fermentation of Prehydrolyzate Sugars
by Pachysolen tannophllus
8.1 Ethanol Fermentation by the Yeast Paehysolen 145
tannophilus Growing on D-Xylose at 28o and pH 5.1
under Varylng Aeratlon Conditlons.
8.2 Ethanol Productlon by Pachysolen tannophllus ln 146
Batch Culture wlth Anaerobic and Aeroblc Sparglng
Reglmes
8.3 Maintenance Energy and Growth Yle1d Coeffleents 158
Calculated from Flgures 8.11 (A) and (B)
8.4 Ethanollc Fermerntatlon of D-XyIose by Pachysolen 161
tannophllus NRRL 2461 under Contlnuq.rs Flow and
Redox Controlled Condltlon
8.5 Derlved Fermentatlon Characterlstlcs for 162
Pachysolen tannophllus NRRL 2461 ln Contlnuous
Culture wlth Controlled Redox Potentlal
x1x
TabIe TltIe Page
8.6 Volunetrlc Ethanol and Xylttol Productlvltles 163
and Speclflc Growth Bates of Pachysolen tannophllus
ilRnL 21161 ln Contlnuq,rs Culture wlth Controlled
Redox Potentlal
8.7 Comparlson of Yteld Ratlos of Pachysolen
tannophllus ln Contlnuo.rs Culture wlth and
nlthout Controlled Recbx Potentlal
r64
xx
rnBnrlrrTr(Is IID sDf,ll.s
g grarn or graviLatlonal force under centrlfugatlon
h hour
kg kllogran
I litre
M molar or mole
mg mllli gram
min mlnute
ml mllli litre
rM milli mole
mV mllli volb
ppb part per biltlon (1.e. ugll)
s second
r.p.m. revolution per mlnute
vvrn gas volune/medltn volume/nLn (L/L.mln)
v/v volune/volune (nll1)
w/ v welght/volrme G/L)
A.R. analytlcal reagent
ATP adenosine-5'-trlPhosPhate
AI{-DMCS acid washed - dimethyldicilorosllane
BOD blochenical oxygen dsnand (gL)
D dllution rate tn-l )
DEAE- dlethylamtnoethyl-
DF degrees of freedom
DOT dlssolved oxygen tenslon
dPldt rate of productlon forrnatlon (g/I.h)
ds/dt rate of substrate consmptlen (g/l.h)
E ethanol concentratlon (g/I)
F F-ratlo of mean stms of squareg
FFAP free fatty aclcl Phase
Ftof F-ratlo of MSLF to MSPE
Kl ethanol lnhlbltlon constant for growth (g/f)
Km the Mlchaells constant
xxi
Ks saturatlon constanb
log logarithm to base 10
ln logarlthm to base e
m maintenanee energy eoefflclent (g substraEe/g clry celI.h)
MRS de Man, RoSosa' Sharpe agar or broth
MS mean sun of squares
MSLF mean sun of squares due to lack of fit
MSPE mean sun of squares due to pure error
IARC mean sul of squares due to regresslon
MSRS mcan sum of squares due to resldual
N nmber of replicates
NADP nlcobinamlde adenine dinucleotlde phosphabe
Nc nunber of centre polnts in experimental design
Nf number of factorial polnts ln experimental cleslgn
Ns number of'start polnts in experimental design
Qp speeific product formation rabe (e/g.h)
Qs speclflc substrate constmptlon rate (e/S.h)
So lnitial sugar (D-xylose) concenbration (g/1)
Sx steady state value of D-xylose eoncentratlon (8/I)
Sxo xylltol concentration G/I)
SS stm of square
STDEV standard devlation
TCA trlcarboxylic acid cYele
W clry welght of cells (g/L)
X blcrnass concenbratlon (g/I)
I response variable or
observed growth yleld (g cells/g substrate)
Yg true gpowbh yleld (g cells/g subsbrate)
Y predlcted value of response varlable being modelled
I p/s product yield
I g/x product yleld
I x/s bicmass yleld
I xols xylltol yleld
I xo/x xylltol yleld
xxaf
> ts Sreater than
< ls less than
c coded dlgtance from orlgln of ',starf on axlal polnt ln a
central conposlte or central cmposlte rotatable deslgn
g represents the coefflclent ln the experlnental deoign
6o ls the I-lntercept or constant tern
u spclflc growth rate (1/X, dX/ft) (fil)
lhax spclflc growth rate (1lX. dx/dtl tfil l
o represents degrees tenperatuure ereressed on Oelslus scale
oooOOooo
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