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 Tech

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

I I I I I I t a I I { I

I

ma:timum

alcohol

concentratiou

of

4

to

5% (by weight

I

t

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

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

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

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

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.

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