Inhibition of the lactose to ethanol fermentation of Kluyveromyces marxianus Y113 and attempts at its alleviation through media improvement : a thesis presented to Massey University in fulfillment of the thesis requirement for the degree of Master of Technology

Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author.

INHf BITION Ot' TtlE ^I-,ACTOSE TO ETIIANOIT IrERI'lFlNTAl'f ON ^O-F

KLUYYEROMYCES I'ARXIAiVUS Y1-13 AND ATTEMP'TS A'T TTS ALLEVIAT'ION

I'IIROUGH MEDIA IMPROVEI-IENT

ABSTP.ACT

Intr j.tri-ti91 rtf the lar:tose to etharrol ^f ermt:ntation ctf ^{A.mal^-rr anus}

\-11-3 iias illr.r:stigated. ^{The use} of initial factose concetll.l"aLions cf, 150 g/Iitre or greate.r resulted in lessi ^biotnass accumulat.ir--,11,

lower etharrol prc>rlu<:tir-it1- and incontplete substrat-e utilisation.

KeeSrirrg

rhe initial Lactose

concentrat-ion

at 100 g/litre

^but

increasing the

^medium

osmolality

b)- up

to

5 times

r-ia the addition

::,t

non-utilised salt or

^maltose

resultecl irr substantiall y

reduced Lriomass accumulation and

slightl!

lower ethanol

productivitl'.

This sugqested

that

trigh nedium

osmolality inhibits ttre I'east in a

^non-

specific

^way by increasing the energy required

for ceII

maintenance

at the

^expense

of

biomass producti"on.

Keeping the intial lactose c'Jucentratir.rn at LIJO g/ litre but adding

irgr co 5% (by weight) ^{et.LranoL reduced} the amount and rate of bi.ontass

accumulaLion and Ied to incomplete substrate utilisaLion, ^{als ue11} as dranratically Iowering che ^amount of ethanol produced bl- ^the 1'east itself . The detrimental ^{ef f} ects of added et-hanol ^became

=ignif icarnt only ^{when more} than 2 Lo 3ex (b)' w"tnttt., h.ls; added.

I

II I I It

aI I

{

I

I

^ma:timum

alcohol

concentratiou

of

4

to

5% (by weight

It

Ln

aI1 cases, irrespectiye of t[e

coucentration of

!

:

irritj-aI1y.

^T|ese

results

^suggested

t[at the

ethano]

errergy metabolism

of the cell in

^some

specific

^ua merely increase

ttre

requiretnent

for cell

^naintenanc

)

was observed

ethanol

^added

intribited

^the

y and did

not

e

energy.

In t-he copcerrtl'ations tried sulrplernerrtatiou ^c.tf tl-re medium uith yeaf;t estract , magnersium, caf cium arrd chi t in aLl tail ed to ^produce ali' chapge j1 the pel-f ormance of the f er:ntentatiott. Supplemet'rtation xitir sti1l bortoms was found to be quite stronglt- inhibitory to t.ire

iermetrtatlol'l ^.

Denriltorai is;rt-ion of tfre ^{wtrey- permeat.e medium} rt:duced that ^t.he pr:r1ormance of t-lre fermenLation ^compared to that carried out ^on stanclar-d whel' permeate medium. K.nal^Ilanus \'113 $as able to

f er-ment a rnediurn cif def ined composition but the biomass ^growth, eLtranol productir-ity and lactose utilisation fiere not as good ^as Lhose 3chieyed using complex rnedia such as whey or lactose broth.

j-ncreasing ttle concentration of rrutrients in ^{the def} ined medium was

cf small benefit but the performance Has still weII below that ^seen on complex media.

ACKNOWLEDGEMBNTS

-

_Sincere

qratitude is

espressed

to

Dr A

J

llawson

for his thoughtful

and

careful

^guidance and

supervisic;n

during the

course

of

these studies.

I'he-help aild encollragement of the staff and sturlent.s of t-he Biot-echnologl' Department, Ilassey University

is gratefulll' acknowledged.

The suppor:t- atrd ellcriuragelnent

ctf

f latmates ^atrd

f

riends of

^ther author durirrg

tlre

course

of

these ^st.udies

is

^great-).y

apl-(jciated. liithout

^them

the task

uould

have been substatrt.ially Inore difficult.

Firrally the

auth<--rr wishes

to

express

sincere gratitude to his typist,

^Jenny

Terry,

whose estreme

skill

and speed saved him from

several

nervous breakdorins.

CONTENTS:

Abstract

Acknowledgemen t s

Contents

List of

Figures

List of

T'ab1es

i.. Introduction

2.

Ethanol l'roduct.i on f rom [,rhe1'

- Literature

^Rev:,ew

2.L

General Aspects

of

Ethanol Productiorr

2

-1.1

Int-roduction 2.1

-Z

Biochemistry

2-'1,.3

Microbiolog]' and Sutrstrat-es

2.2

Hhey

3s a Potential

Substrate

for

Ethanol Production

2.2,1

Int roduction

2.2.2

^{T1'pes and Composi}

tions of

rihel'

2.2.3

lfhey and lihel'

Derivatirre

Prcrduction

2.2.4

^l.r'he1'

Utilisation

2.2.4

.1

Introductlorr

2.2.1.2 Protein

Recoverl' and Use

2.2.1-3

Lactose Recoverl' and Use 2 -2.

4.1

Fermentation Processes

2.2.5

Ethanol Production From lthel' 2. 2.5

.I }licrobiologt'

2.2.5.2

Biochemistry

2-2.5.3 Effect of

T'enperature

2.2.5.4

^Ef f

ect of

^pH

2-2-5-5 Effect of

^Et.hanol Concentrarion

2 .2 .5

.6

^{Ef f}

ect of

l.ledia T'oni-ci t1'

2.2.5.7

^{Ef f}

ects of other Inhibitors

and Promoters

2.2.5.8

Commercial Aspects

2.3

^Summary

3. Materials

and lleittrods

3.1

Intaterials

3.1.1.

Orqanisms

J'

I

I I I

I

i2

I

I

I l.t.z

^Media

I

! :.

^r

.9

^chemicars

f

i 3.2

^Methods

3.2.I Biological

^Methods

,

_3.2.1.1,

Culture

Maintenance

3 .2.

I.2

Propagation and

fnoculation

3.2.t.3 Incubation

and Sampling

of

Experiments

3.2.I .1 Sterilisation of lledia

^{and Equlpment}

3.2.2 Analytical

^Methods

3.2

-2.2 pll

Measurement

3.2.2.3

Biontass }leasurement

3.2.2.

4

Lactose }leasurement

3.2.2.5

Ethanol MeasuremenL

4.

The

Effects of Osmolalitl',

Lactose,

SaIt

and Ethanol Corrcentration on Ethanol Production fr:om Lactose

{.1

Int.roduction

1.2

Lact-ose Concentration

1.2.I Introduction

1.2.2 Results

and Discussion

1.3

Medium T'onicity

{.3. L Introduction

1.3.2

Results and Discussion

4.4

^Et.hano1 Concentration

4.4.1 Introduction

4.4.2

Results ^{and Discussion}

4.5

^Summary

5. Effects of

^Medium

Nutritional

St.atus

on

Ethanol Production from Lactose

by

K-marxianus Y1l-3.

5.

L Introduction

5.2 Effects of

Yeast

Extract

Concentration

5.2.1. Introduction

5-2-2

Results and Discussion

5.3

Comparison

of

^Whey Permeate and Demineralised ^Whey Permeate

for the

Production

of

Ethanol.

5.3.1 Introduction

5.3.2

Results and Discussion

3

5.4 Effects of

Supplementation

with StiIi

Bottoms

5 .4.

L Introduction

5.4.2

Results and Discussion

5.5 Effects of

Supplementation

with Chitin.

Calcium

or

Magnesium

5.5,-1 Introduction

5.5.2 Results

and Discussion

5.6

Attempted Development

of a

Defined Medium

for

use

with K.

marxianu,s Y113

5.6.

L Introduction

5.6.2 -

Results and Discussion

5.6.2.1,

Amino

Acid

Requirements

5.6.2.2

Vitamin Requirements

5.6.2.3 TriaI of

Defined Medium

for

Lactose Fermentation by

K.

rnarxianus Y113

5.6.2.4

Attempt.ed Improvement

of the

Def ined Medium f or Growth

of K.

marxianu^s Y113

5.7

Sunrrnary

of Nutritional

Studies

6.

Conclusions

Re ferences

Appendix

A. Calculation of

Fermentation Parameters

Appendix

B- Estimation of

Concentration and

Uncertainties

Appendix

c.

E:l::i;l:ll:""t osmorarity to sorute

2 2

LIST OF ^FIGUT{ES

2.I ^Chemical Derivatives frotn ^Ethanol

2.2 Ernbden-Ileyerhof-Parnas tllycolytic Pathway

2.3 ^New Zealarrd Annual ^Wtrey I'roduction; l-975 to 1989

2.1 l,letabolism of ^{IJactose Bef} ore Entry into the ^{E.M. P.}

Glycolytic

^PattrwaY

5 Structure of Lipid Bilal'er

^}lembrane concentratiotr

6

Comparison

of

Dj

stilLat-ion

Cost.s arrd Ethan<;I

(loncentrat 1ot1 .

4 .I IJiomass Concentration Vs ^I ime; Rltrt ¹ 4.2 i:t-hauol Conceutt:ation Vs Time; Run ¹ 1.3 Lactose Concentration \is f ime; ^{Run 1} 4.I Biornass C<>ttcentration Vs Time; Run ² 4.5 tjt-hano1 Concentration ^Vs 'I'ime; Run ²

-l . 6 I..rct-c.rse Coucetttratiotl ^Vs Tine; ^{Rut-t 2} 4.7 tiiomass ConcenLration \is I'ime; Run ³ 4.8 Ethanol Concentration ^Vs Time; Run ³ 4.9 Lact-c:se ConcertLration \is Time; Run ³ 4.10 Biomass Concentration ^Vs Time; Run ⁴ 4.11 Ethanol Concentratiorr ^Vs Time; Run ⁴ 4 -LZ tsiomass Corlcentratiou ^{Vs 'f} ime; Run ⁴ 4.13 Ethanol Concentratiou ^Vs I'ime; P.un ¹

4.14 Lactose Concentration ^Ys fime; Run ⁴ 4.15 Biomass Concentration ^Vs Time; Run ⁵ 4.16 Ethanol Concentr:aliou Ys Time; Run ⁵ {.17 Lactose Concentratjon ^{Vs T'ime; Run 5}

{.18 cornparison of Ethauol Productiotl in ^{Loo g/} litre Lactose Broths

4.19

Comparison

of

Lactose Consuption Broths

lt.20

Comparison

of

Ethanol Production Broths

4.2I

Comparison ^<>f lractose Consumption Brot-hs

in

¹⁰⁰

g/1itre

l,actose

in

1.50

g/Iitre

Lactose

in

150

9/litre

^Lactose