Final Major Project Report 2011

Retardation of Yeast Autolysis 1
University of Technology, Jamaica
School of Engineering
The retardation of yeast autolysis in the fermentation of beer
in partial fulfillment of the requirement for the
Bachelor of Engineering Degree
Kesha K. Hemmings
Lori-Ann S. Vincent

University of Technology, Jamaica
School of Engineering
The retardation of yeast autolysis in the fermentation of beer
in partial fulfillment of the requirement for the
Bachelor of Engineering Degree
July 16, 2011
Kesha K. Hemmings
Lori-Ann S. Vincent
Project Supervisor
Faculty Projects Coordinator
Date

Acknowledgment
First and foremost we want to take this opportunity to express our most sincere gratitude to all
individuals who provided assistance in the completion of this research.
During this project we have received invaluable assistance from most of our colleagues
for whom we have great regard, and we wish to extend our warmest thanks to Mr. McAnuff and
Mr. Baker of the Chemical Engineering Department for their help in the acquisition of the
necessary literature and layout of results which formed the base of this study.
The researchers extend appreciation to Ms. Scarlett and Ms. Muir from the Health and
Applied Science Department for conducting the cell viability analysis, which was a crucial part
of the project.
Gratitude must also be extended on the Ministry of Energy and Mining, Mines and
Geology Division for accurate analysis to determine the initial concentration of Zn2+
and Cr3+
present in the wort sample in order to conduct our experiment.
Special thanks to Mr. Dawkins of the Molecular Biology Department at the University of
the West Indies, Mona Campus for providing the necessary guidance to better understand certain
aspects of the project.
Last but not least, Mr. Stewart, the Fermentation Operator from Red Stripe who provided
us with starting materials (wort and yeast) for our experiment and additional data that played a
vital role in this project.

Table of Contents
List of Tables ................................................................................................................................................6
List of Figures...............................................................................................................................................7
Abstract.........................................................................................................................................................9
Executive Summary....................................................................................................................................10
El Resumen Ejecutivo.................................................................................................................................12
Chapter One ................................................................................................................................................14
Introduction.............................................................................................................................................14
Background.............................................................................................................................................14
Problem Statement..................................................................................................................................16
Purpose of the Study ...............................................................................................................................16
Objectives................................................................................................................................................16
Research Questions.................................................................................................................................17
Significance of the Study.........................................................................................................................17
A Feasibility Study on the Retardation of Yeast Autolysis during the fermentation of beer......................19
Chapter Two................................................................................................................................................21
Review of literature.................................................................................................................................21
Introduction.............................................................................................................................................21
Types of Yeast .........................................................................................................................................22
Fermentation Phases ..............................................................................................................................22
Factors Affecting the Rate of Fermentation............................................................................................25
Yeast Metabolism....................................................................................................................................33
Basic Nutritional Requirements for Yeast...............................................................................................35
Various Minerals in Grains ....................................................................................................................45
Presence of Anti-nutrients.......................................................................................................................46
Formation of By-Products ......................................................................................................................47
Yeast Management..................................................................................................................................47
Assessment of Fermentation Parameters................................................................................................48
Summary of literature review..................................................................................................................53
Chapter Three..............................................................................................................................................54
Methodology ...........................................................................................................................................54

Introduction.............................................................................................................................................54
Specific Procedures ................................................................................................................................55
Preliminary Wort Analysis......................................................................................................................55
Preparation of Nutrients.........................................................................................................................55
Chromium Chloride Selection.................................................................................................................56
Nutrition for Maximum Cell Viability.....................................................................................................57
Specific Gravity Analysis ........................................................................................................................59
Cell Viability Analysis.............................................................................................................................59
Chapter 4.....................................................................................................................................................61
Results and Data Presentations..............................................................................................................61
Results.....................................................................................................................................................61
Chapter Five................................................................................................................................................98
Discussion of Results ..............................................................................................................................98
Introduction.............................................................................................................................................98
Discussion...............................................................................................................................................98
Summary and Conclusion .....................................................................................................................104
Limitations................................................................................................................................................106
Recommendations.....................................................................................................................................106
References.................................................................................................................................................107
Appendix ..................................................................................................................................................116

List of Tables
Table 2.1 Different phases of Beer Fermentation........................................................................................................24
Table 2.2 List of vitamins, their importance and optimal concentrations ...................................................................37
Table 2.3 Carbon and nitrogen sources for growth and metabolism of yeast..............................................................44
Table 2.4 Cmparative nutritive value of cereal grains ................................................................................................46
Table 3.1: Different concentration levels of chromium chloride.................................................................................56
Table 3.2 Random selection of concentration levels for each variable in each flask...................................................58
Table 4.1: Preliminary Results ....................................................................................................................................61
Table 4.2: Effects of Cr3+
concentrations on yeast viability, percentage alcohol content and attenuation ..................62
Table 4.3: Effects on % viability and alcohol produced with constant Zn2+
and biotin.............................................65
Table 4.4: Effects on % viability and alcohol with constant Zn2+
without biotin......................................................68
Table 4.5: Effects on % viability and alcohol produced with constant concentrations of Cr3+
and biotin....................71
Table 4.6: Effects on % viability and alcohol produced with constant Cr3+
without biotin.........................................75
Table 4.7: Effects on the duration of fermentation and % viability with constant Zn2+
and biotin.............................79
Table 4.8: Effects on the duration of fermentation and % viability with constant Zn2+
without biotin. ....................83
Table 4.9: Effects of the % viability and duration of fermentation with constant Cr3+
and biotin...............................87
Table 4.10: Effects of the % viability and duration of fermentation with constant Cr3+
without biotin. ....................91
Table 4.11: Effects of specific gravity on the duration of fermentation. .....................................................................95

List of Figures
Figure 2.1: Different phases of microbial growth kinetics ..........................................................................................24
Figure 2.2 Effects of ethanol on brewer‟s yeast viability ............................................................................................27
Figure 2.3 Metabolism of glucose in yeast under aerobic and anaerobic ....................................................................33
Figure 2.4 Yeast metabolism of glucose......................................................................................................................35
Figure 2.5 Structure of biotin.......................................................................................................................................38
Figure 2.6 Effects of chromium on ethanol and biomass concentration......................................................................43
Figure 2.7 Effects of chromium on ethanol and biomass concentration......................................................................44
Figure 2.8 Hydrometer with measuring cylinder use to measure the specific gravity.................................................49
Figure 2.9 Refractometer use to measure specific gravity...........................................................................................50
Figure 2.10: Staining of yeast cells using Methylene Blue..........................................................................................51
Figure 4.1: Effects of Cr3+
ions concentrations on yeast viability. ..............................................................................63
concentration on the percentage alcohol content and attenuation. ....................................64
on yeast viability at constant Zn2+
concentrations and biotin............................................66
on percentage alcohol content at constant Zn2+
concentrations and biotin.......................67
concentrations without biotin. ....................................69
on percentage alcohol content at constant Zn2+
concentrations without biotin.................70
Figure 4.7: Effects of Zn2+
on yeast viability and alcohol content when Cr3+
( 100 mg/L) with biotin. ......................72
( 150 mg/L) with biotin. ....................73
( 300 mg/L) with biotin. .....................74
(100 mg/L) without biotin. ...............76
(150 mg/L) without biotin. ...............77
(300 mg/L) without biotin................78
on the duration of fermentation when Zn2+
(2 mg/L) with biotin. ..................................80
( 3 mg/L ) with biotin. .................................81
Figure 4.15 Effects of Cr3+
(4 mg/L ) with biotin...................................82
(2 mg/L) without biotin. ..............................84

Figure 4.17 Effects of Cr3+
(3 mg/L) without biotin................................85
(4 mg/L ) without biotin. ............................86
Figure 4.19: Effects of Zn2+ on the duration of fermentation when Cr3+ (100 mg/L) with biotin.............................88
on the duration of fermentation when Cr3+
(150 mg/L ) with biotin..............................89
( 300 mg/L) with biotin..............................90
(100 mg/L) without biotin. ..........................92
(150 mg/L) without biotin...........................93
(300 mg/L) without biotin. .........................94
Figure 4.25 Specific Gravity trends for each percentge attenuation............................................................................96
Figure 4.26 Overall representation of the percentage viability in each flask...............................................................97

Abstract
Autolysis of yeasts is of great concern in many fermentation processes, as it not only
decreases the percentage viability, but also reduces the quantity of alcohol and the quality of beer
produced. Thus, this study seeks to retard the self digestion of cells that occurs during the
process.
The effects of varying concentrations levels of chromium chloride (CrCl3) and zinc
sulphate (ZnSO4) in the presence and absence of biotin on yeast viability were assessed. This
assessment was conducted by determining the viability of the organism at the end of the
fermentation process.
Concentrations of chromium chloride ranging from 100 to 300 mg/L (at increments of 50
mg/L) with constant concentration of Zn2+
(4 mg/L) and biotin (0.6 mg/L) were carried out in
different flasks (containing wort), to determine the optimum concentrations of CrCl3. The
optimum concentrations were then varied with Zn2+
ranging from 2 to 4 mg/L (at an increment of
1 mg/L) in the presence and absence of biotin. Lager yeast with an initial viability of 92% was
used and the process was carried out under anaerobic conditions at a constant temperature of
20 ºC.
It was evident from the obtained results that Cr3+
concentration at 300 mg/L and Zn2+
concentration at 3mg/L resulted in maximum improvement of the fermentation process,
production of biomass (89.7%) and alcohol content (5.7%).

Executive Summary
Saccharomyces cerevisae is a molecular genetic organism which is categorized as a
species of budding yeast and is commonly used in the fermentation of beer. During beer making,
the microorganism facilitates the conversion of carbohydrates (sugars) into alcohol. However,
yeast is sensitive to physical and chemical stresses and its degree of sensitivity influences
autolysis, which is the destruction of cells by its own enzymes. The essence of this project is to
retard the occurrence of autolysis during the beer making process by supplying the yeast with
additional nutrients to improve yeast performance and its longevity.
This assessment was conducted by determining the viability of the organism at the end of
fermentation. As a result, three main objectives were undertaken: to quantify the viable yeast
cells by determining the optimum concentration of chromium chloride in the presence of zinc
sulphate and biotin at their optimum concentration, to achieve the highest viability by varying
concentrations of zinc sulphate and chromium chloride in the presence and absence of biotin and
finally to examine the effects of these concentrations on the duration of fermentation, apparent
attenuation and alcohol content.
Initially, a pre-analysis on the wort (unfermented beer) was conducted before the
investigation could be carried out. The aim was to determine the initial concentration of Zn2+
and
Cr3+
(which was < 0.1 mg/L). The importance of this analysis was to estimate the quantity of
each nutrient that must be added to achieve the experimental concentrations.
Concentrations of chromium chloride ranging from 100 to 300 mg/L (at increments of 50
mg/L) were carried out in different flasks (containing wort) with optimum concentration of Zn2+
(4 mg/L) and biotin (0.6 mg/L) to determine the optima concentrations of CrCl3. After

fermentation, cell viability analysis was carried out to obtain the three optimum percentage
viabilities that should be used for further investigation.
The optimum concentrations of chromium chloride (77.8,78 and 82.9%) were then varied
with Zn2+
ranging from 2 to 4 mg/L (at an increment of 1 mg/L) in the presence and absence of
biotin. This was carried out in eighteen flasks (containing wort) with a standard (no additional
nutrients). Prior to fermentation, the specific gravity of the wort was recorded with the use of a
Brix refractometer. This analysis was done once a day (24 hour time interval) until the end of
fermentation (this is where constant specific gravity readings were observed). Other parameters,
such as the alcohol content and percentage attenuation were determined.
These results depicted which flask (containing the specified nutrient concentrations) as
shown in Appendix D, is capable of producing the highest (%) alcohol content and attenuation.
Additionally, a cell viability analysis was carried out, to further conclude which flask contained
the optimum concentrations that improved yeast performance.
Optimum concentrations were achieved when Zn2+
was at 3 mg/L, Cr3+
at 300 mg/L in
the presence of biotin at 0.6 mg/L. This was a clear indication that at these concentrations,
brewers will be able to maximize the quantity of alcohol produced with minimal input thus,
decreasing the overall cost of production.

El Resumen Ejecutivo
Saccharomyces cerevisiae es un organismo de genética molecular que se clasifica como
una especie de levadura en ciernes y se utiliza comúnmente en fermentación de cerveza. Durante
la fabricación de cerveza, el microorganismo facilita la conversión de los hidratos de carbono
(azúcares) en alcohol. Sin embargo, la levadura es sensible a las tensiones físicas y químicas y
su grado de sensibilidad, influencias autolisis, que es la destrucción de las células por sus propias
enzimas. La esencia de este proyecto es para retardar la ocurrencia de autolisis durante el proceso
de elaboración de la cerveza mediante el suministro de la levadura con nutrientes adicionales
para mejorar el rendimiento de la levadura y su longevidad.
Esta evaluación se llevó a cabo mediante la determinación de la viabilidad de organismo
en la final de la fermentación. Por lo tanto, tres objetivos principales se llevaron a cabo: para
cuantificar las células de levadura viable, al determinar la concentración optima de cloruro de
cromo, mientras que presencia de sulfato de zinc y biotina en su concentración optima, para
lograr la máxima rentabilidad mediante la variación de las concentraciones de sulfato de zinc u
cloruro de cromo en la presencia y la ausencia de biotina y para examinar los efectos de estas
concentraciones sobre la duración de fermentación, la atenuación aparente y contenido de
alcohol.
Inicialmente, un pre-análisis sobre la hierba (cerveza sin fermentar) se llevó a cabo antes
de la investigación podría llevarse a cabo. El objetivo fue determinar la concentración inicial de
Zn2+
y Cr3+
(que era < 0.1 mg/L). La importancia de este análisis fue estimar la cantidad de cada
nutriente que se debe agregar para alcanzar las concentraciones experimentales.

Las concentraciones de cloruro de cromo que van desde 100 a 300 mg/L (en incrementos
de 50 mg/L) se llevaron a cabo en diferentes recipientes (que contengan hierba) con una
constante de Zn2+
(4 mg/L) y biotina (0.6 mg/L) para determinar concentraciones optimas de
CrCl3. Después de la fermentación, el análisis de la viabilidad celular se llevó a cabo para
obtener los tres viabilidades optimo porcentaje (77.8, 78 y 82.9% respectivamente). Esto indica
que las concentraciones experimentales deben ser utiliza dos para análisis adicionales.
Las concentraciones óptimas de cloruro de cromo fueron variadas con Zn2+
de 2 a 4 mg/L
(con un incremento de 1 mg/L) en presencia y en ausencia de biotina. Esto se llevó en dieciocho
frascos (que contengan hierba) en la adición den una norma (sin nutrientes adicionales). Antes de
la fermentación, la gravedad específica del mosto se registró mediante un refractómetro Brix.
Este análisis se realizo una vez al día (24 horas de intervalo) hasta el final de la fermentación
(gravedad específica constante). Otros parámetros, como el contenido de alcohol y la atenuación
se determinan con estas gravedades específicas.
Estos resultados se muestra que frasco (que contiene las concentraciones especificadas en
nutrientes) como se muestra en el Apéndice D, es capaz de producir el mayor o el menor
porcentaje (%) de contenido de alcohol u la atención. Además un análisis de la viabilidad celular
se llevó a cabo, para concluir más que el frasco contenía concentraciones optimas para mejorar el
rendimiento de la levadura.
La concentración optima se logra cuando Zn2+
fue de 3 mg/L, Cr3+
a 300 mg/L y en la
presencia de biotina en 0.6 mg/L. Esta es una clara indicación en estas concentraciones, los
cerveceros podrán aprovechar al máximo la cantidad de alcohol que se produce con una mínima
aportación por lo tanto, disminuir el coste total de producción.

Chapter One
Introduction
Background
Over the years alcoholic industries have become of increasing importance to the world‟s
economy, due to annual rise in the consumption of beer. According to Heath (1995), beer is
considered to be the world's most widely consumed alcoholic beverage that undergoes three
main processes: malting, brewing and fermentation. It is principally made from four ingredients
each of which imparts its own flavour and characteristics to the finished product. These include a
starch source, yeast, hops and water. The most common source of carbohydrate used is malted
barley; this provides fermentable sugars that are utilised by the yeast via fermentation thus,
producing alcohol. Hops add to the aroma and flavour of the beer and water provides a medium
for the fermentation process. There are different types of beer that can be produced depending on
the starting ingredients and the duration of the fermentation process (maturation). However, it
can be categorized as either lager or ale beer since the main distinction is determined by the type
of yeast used.
Yeast is biologically classified as a fungus in which there are different strains for
different purposes. The first eukaryote whose genome was fully sequenced is Saccharomyces
Cerevisiae and is commonly used in the fermentation of beer (Goffeau et al., 1996).
Robert (2003) explains that there are two main classification of the Saccharomyces specie that
are exploited by brewers, those that are top fermenting yeast (ale yeast) and bottom fermenting
yeast (lager yeast). The difference in the strain of yeast used is what defines the unique beer
characteristics and is said to be a critical component to many brewers.

In addition to yeast contribution to the style of beer produced, it has been established that
it is the key factor in the fermentation process (Boulton & Quain, 2001). However, yeast is
sensitive to instantaneous changes in physical and chemical parameters, and its degree of
sensitivity influences autolysis. Autolysis is said to occur when endogenous enzymes which
consist of mainly proteases and ribonucleases digest the intracellular components in the yeast
cell. Thus, not only result in hydrolyzes of macromolecules, but also the degradation of light
weight molecules which mainly consist of nucleotides, amino acids and peptides. It is also
characterized by the loss of cell permeability, alteration of cell wall porosity and the subsequent
leakage of intracellular products into the environment (Fleet & Hernawan, 1995).
The rate of fermentation and the amount of alcohol produced from a precise quantity of
raw materials are of vast importance. As a result, emphasis on plant economics, unit operations
and quality control mechanisms has heightened to increase yield, efficiency and profitability.
Hence, methods to maintain healthy and viable yeast cells are of extreme importance in the
industry (Ingledew & Synder, 2009).

Problem Statement
Monitoring and controlling yeast viability is of utmost importance in order to achieve a
high-quality finished product at a low economic cost. However, industrial fermentations that
exploit yeast cells are confronted with a multitude of chemical, physical and biological stresses.
These may impair cell functions further leading to the occurrence of autolysis, thus hindering the
progression of fermentation. As a result, this project seeks to determine the optimum
concentration of nutrients necessary to prolong the life span of yeast.
Purpose of the Study
To optimise yeast performance during the fermentation of beer to retard the occurrence of
yeast autolysis.
Objectives
This project aims to:
1. Quantify the viable yeast cells to determine the optimum concentration levels of
chromium chloride (CrCl3), in the presence of biotin (vitamin B7) and zinc sulphate
(ZnSO4) at their optimum concentration.
2. Compute and evaluate the percentage cell viability, at varying concentrations of
chromium chloride (CrCl3) and zinc sulphate (ZnSO4), in the presence and absence of
biotin (vitamin B7).
3. Examine the effects of zinc sulphate (ZnSO4), chromium chloride (CrC3) and biotin on
the rate of fermentation, apparent attenuation and percentage alcohol produced.

Research Questions
1. What concentrations of chromium chloride (CrCl3) gives maximum percentage viability,
in the presence of biotin (vitamin B7) and zinc sulphate (ZnSO4) at their optimum
concentration?
2. How does the varying concentrations of zinc sulphate (ZnSO4), and chromium chloride
(CrCl3) in the presence and absence of biotin (vitamin B7) affect yeast viability and
percentage alcohol produced?
3. How is the rate of fermentation affected by the presence of these nutrients and the
percentage viability?
4. Which nutrient combinations and their respective concentration resulted in maximum
percentage viability overall?
Significance of the Study
The main objective of brewers is to maximise the quantity of alcohol produced with
minimal input. However, the amount of yeast required for pitching and its physiological state is
of great importance. Nevertheless, the addition of nutrients to the fermentation medium will not
only increase the tolerance of yeast at high alcohol concentration levels, but also improve yeast
viability, thus decreasing the amount of yeast require for inoculum development. Therefore,
succession of this project will not only increase alcohol production, but also reduce the amount
of yeast require for a brew. Hence, the overall cost of production would decrease with increasing
plant profitability. This would be beneficial to stakeholders, shareholders, employees and
consumers.

Key Definitions
Autolysis: The destruction of tissues or cells of an organism by the action of substances, such as
enzymes, that are produced within the organism.
Brewing: The production of beer through the steeping a starch source in water followed by the
addition of yeast to initiate the fermentation process.
Enzymes: Proteins that participate in cellular metabolic processes with the ability to enhance the
rate of reaction between biomolecules.
Fermentation: A biological process in which sugars are converted into cellular energy and
thereby produce alcohol and carbon dioxide as metabolic waste products in the presence of yeast.
Fermentable sugars: Carbohydrates that are easily utilised by yeast during fermentation.
Malting: A process which involves the germination of grains by soaking in water. The grains are
quickly halted from germinating by further drying with hot air.
Maturation: The holding stage (post fermentation) where the beer is conditioned (i.e. removal of
diacetyl) prior to filtration and packaging.
Metabolism: The chemical processes that occur within a living organism in order to sustain life.
Metabolize: To undergo the chemical changes of metabolism.
Saccharomyces cerevisiae: Molecular genetic organism that is categorized as a species of
budding yeast.
Wort: The sweet liquid that comes from the mashing of grains, it is referred to as unfermented
beer.
Yeast: Unicellular micro-organism classified in the fungi group which is used as an agent to
ferment sugars.

A FEASIBILITY STUDY ON THE RETARDATION OF YEAST AUTOLYSIS DURING THE
FERMENTATION OF BEER
Scientists disproved that living organisms were responsible for the fermentation process;
they claimed that it was merely a chemical and physical reaction. As a result, no connection was
drawn between the existence of yeast and the phenomenon of fermentation. Briggs et al. (2004)
states that three scientists Cagniard-Latour, Kützing, and Schwann discovered independently that
yeasts are living organisms and they play a major role in the fermentation process. The
investigation also shows the proliferation of the organism, thus refuting the theory of
“spontaneous generation of life” which was introduced by Anaximander and Anaximenes.
Lavoisier (1789) and Gay-Lussac (1815) established the chemical formula for the
conversion of glucose to produce carbon dioxide and ethanol. The formula led to a fairly
accurate description of the overall process (Williamson et al, 1994).
Glucose Ethanol + Carbon Dioxide
C6H12O6 (aq) 2 C2H5OH (aq) + 2 CO2 (g)
However, the importance of yeast was still left unknown. It was not until Pasteur in 1876
published his paper entitled E Â tudes sur la bieÁre which showed results of vigilant microscopic
examination of beer fermentation. He observed that the growth of the yeast cells were in fact
responsible for the fermentation process (Briggs et al., 2004). It was at this point the viability of
yeast was acknowledged, which led to further investigations on its significance during
fermentation.
Yeast
Yeast

A direct relationship was drawn between yeast viability and the rate of fermentation. As a
result, many studies seek to indentify conditions that are optimal for the yeast growth and
metabolism. However, there are several factors that affect the rate of fermentation namely:
 Temperature
 pH
 Amount of yeast that is pitched
 Concentration of by-product formed
 Nutrients used for medium formulation
 Initial oxygen requirements
 Substrate concentration
 Yeast viability and vitality
 Accumulation of toxic substances
 Stress factors such as alcohol level and osmotic pressure
Although yeast is capable to ferment naturally, the requirements for its optimal performance
must be met to give maximum product yield. Thus, it is important that these conditions along
with others are monitor and control. However, appropriate media formulation is usually
challenging for many brewers.

Chapter Two
Review of literature
Introduction
In this section of the research paper, a brief overview of the life cycle of yeast was
discussed. It focuses on four phases that the micro-organism undergoes when it is introduced into
the medium. However, the kinetics of microbial growth is affected by several factors which
should be considered to bring forth a successive process. With regards to the different
fermentation phases, yeast performance is significantly influence by wort composition, as each
nutrient plays a vital role in the maintenance and metabolism of the cell. As a result the
formulation of the media must be proportionally balanced in order for the yeast to function at its
optimum during the fermentation process. However, in some cases the presence of anti-nutrients
reduces the assimilation of these nutrients. Additionally the formation of by products in many
instances can also have a negative effect on yeast metabolic activities as well as the characteristic
taste and flavour of the beer. Nevertheless, refinement of the process can minimize the presence
of these inhibitors thus, increasing not only the rate of fermentation but also improving yeast
viability.

Types of Yeast
As previously mention the different types of beer can be categorized as either lager or ale.
Ale beers are produced from top fermenting yeast known as Saccharomyces cerevisiae, where as
lager beer are produced from bottom fermenting yeast Saccharomyces pastorianus (formerly
referred to as Saccharomyces carlsbergensis or Saccharomyces uvarum). However, there are
many different strains of brewer‟s yeasts from the two groups, each of which defines the specific
type of beer and its flavor that is produce (Palmer, 2001). From a brewers perspective there are
several phenotypic differences between lager and ale yeast. The main distinction between the
two species is their fermentation temperature. Lager yeasts can ferment at a temperature range of
7-34 ºC with an optimum growth temperature of below 30 ºC while ale yeasts ranges from 15ºC-
39.8 ºC with an optimum temperature of above 30 ºC (Boekhout & Robert, 2003). The
temperature that is utilized is dependent on the specific characteristic taste that the brewer desire.
Nevertheless, ale and lager yeasts are said to follow identical fermentation phases even though
they produced different styles of beer.
Fermentation Phases
Generally yeast growth follows four phases, the lag period, growth, fermentation, and
sedimentation phase when they are inoculated into a growth medium. The lag phase reflects
the time required for inoculated yeast cells to adapt to their physical and chemical growth
environment (pH, temperature and sugar content of the wort). The duration of this phase is
not only dependent on the growth conditions, but also the type of inoculum, and the
components used in media formulation (Walker, 1998). After the adaptation phase and
sufficient reserves are built up within the cells the growth phase or respiration phase begins.
In this phase, the yeast cells utilises the oxygen in the wort to oxidize a variety of acid

compounds, resulting in a significant drop in pH and sugar content (as much as 50% of the
initial) and an exponential rise in the microbial population This phase is also evident from the
covering of foam on the wort surface and air bubbles due to the liberation of carbon dioxide
gases. During this phase metabolism becomes anaerobic as the level oxygen decreases and the
production of carbon dioxide increase (Jacobson, 2006).
After the oxygen has been depleted the fermentation phase then follows. Fermentation
of beer is an anaerobic process as a result; any remaining oxygen in the wort is removed out
of solution via carbon dioxide bubbles produced by the yeast. In this phase the yeast is
actively consuming the sugar in the wort and is distinguished by reduction of wort specific
gravity and the rapid production of carbon dioxide, ethanol, beer flavours and aroma. Most
beer yeasts remain in suspension for 3 to 7 days, after which flocculation and sedimentation
will commence (Goldammer, 2008). The final phase of the population history is death phase,
this is period in which the microbial population begins to decrease significantly and autolysis
of the cells occur due to build up of toxic substances and depletion of nutrients (Jacobson,
2006; Stanbury et al., 2003). It is also important to note that the physiological condition of the
yeast cells is a contributing factor for the duration and outcome of the fermentation phases
(Walker 1998).

Figure 2.1 showing the different phases of microbial growth kinetics
Esser et al. (2002) mention that when describing the phases of fermentation, brewers
usually observe the surface of the fermentation broth. Table 2.1 below gives a detailed
description of each fermentation stage.
Table 2.1 showing the different phases of Beer Fermentation
Fermentation stage Surface look
Initial stage Beginning of covering with white foam
Low krausen Increase of fine bubble foam to a creamy cover
High krausen High foam with coarser bubbles, most
intensive phase
Collapsing krausen Foam looks browner and decreases
Collapsed foam Dirty brown layer collapsed foam
Source: ( Esser et al., 2002)

Factors Affecting the Rate of Fermentation
According to Waites et al. (2001), the main factors affecting the rate of fermentation are
the quantity of yeast pitched, yeast cell viability and yeast quality, the dissolved oxygen level in
wort prior to pitching, concentration of assimilable nitrogen, concentration of fermentable sugars
and the fermentation temperature. Additionally, Briggs et al. (1999) also stated that the choice of
yeast strain, the size and geometry of the fermentation vessel, pH and the tolerance of yeast cells
to stress factors such as osmotic pressure and ethanol concentration must be considered.
Moreover, based on research conducted by Stoicescu & Bonciu (2006) wort properties such as
the wort gravity, lipid content and concentration level of nutrients such as minerals and vitamins
are contributing factors to the fermentation process. Furthermore, sterilisation of apparatus,
medium and maintaining aseptic conditions during the process will influences not only the rate
of the fermentation but also the purity of the product produced (Stanbury et al., 2003).
Concentration of Fermentable Sugar, Osmotic Pressure and Wort Specific Gravity
Mac William (as cited in Hui et al., 2006) stated that carbohydrates make up 90-92% of
wort solids, in which the fermentable sugars is 70-80% of the total carbohydrate. The three major
fermentable sugars are glucose, α-glucosides maltose and maltotriose. It was declared that, the
rate of fermentation will increase proportionally as the concentration of these simple sugars
increases. On the other hand, as the yeast metabolises these sugars, the rate will decrease.
According to Guidici & Solieri (2009), if sugar concentration is above 50%, the osmotic pressure
becomes excessively high and inhibits yeast growth. The increase in osmotic pressure causes the
water inside the yeast cell to diffuse out in order to equalise the concentration. This will cause
the cell to become dehydrated thus resulting in yeast autolysis.

Lager and ale beers are mostly produced by high-gravity brewing because the
fermentation of high specific gravity leads to the relative overproduction of acetate esters which
is vital for the complex flavour of beer (Saerens et al., 2008). In addition, Lea & Piggott (2003)
mention that fermentation of low-density wort results not only in low alcohol content but also in
low flavour content. The density of the wort is proportional to the concentration of fermentable
sugars present in the wort. Therefore, the specific gravity declines over the course of
fermentation due to a decrease in fermentable carbohydrates and an increase in ethanol
concentration.
Ethanol Concentration
Ethanol is the major product resulting from the action of yeast on sugars present in the
wort during fermentation. Beaven et al. (as cited in D‟Amore, 1992) stated that ethanol
concentration can have separable effects on the specific rate of yeast growth, cell viability and
also the rate of fermentation. He investigated the effect of ethanol concentration on brewer‟s
yeast viability which is summarized in Fig. 2.2 shown below.

Figure 2.2 Effects of ethanol on brewer‟s yeast viability.
Note: Saccharomyces uvarum (carlsbergensis) lager strain 3021(O) and Saccharomyces cerevisiae strain 3001(●)were
preincubated with various concentrations of ethanol in PYNmedium for 20 minutes at 30 ºC.
In the diagram, it was observed that the lager strain was more sensitive to the ethanol
concentration after twenty minutes of incubation at temperature of 30°C than the ale strain. In
addition, it is concluded that the inhibition of cell growth and viability was observed to increase
with increasing ethanol concentrations. However, the ability of yeast cells to convert fermentable
sugars into carbon dioxide and alcohol is dependent on the enzymes present. Several enzymes
are involved during the conversion process where each is primarily responsible for a particular
step. Alcohol destroys enzymes and yeast cells if in high concentrations. This will occur at
different levels for different strains of yeast. According to Sachan (2008) brewer‟s yeast cannot
withstand beyond 5% to 6% alcohol by volume.

Amount of Yeast used to pitch the Fermentation
The inoculation rate or pitching rate is usually described as the amount of yeast per unit
volume. According to N‟Guesssan et al. (2008), at the beginning of fermentation, the sugar
consumption rate increased with inoculation rate. Monk et al. (as cited in Lea & Piggott, 2003)
expound that sufficient yeast culture is needed to complete the fermentation under adverse
conditions. However, too much yeast can result in the rapid fermentation and or yield of
alcoholic beverage with disproportionate aroma of fresh yeast. Additionally, if insufficient yeast
is used the fermentation rate will be slow and may result in “stuck fermentation”. In general, the
amount of yeast needed is highly dependent on the original gravity of the beer, the volume of
wort, the yeast viability and the fermentation temperature. Nachel (2009) mentions that high
gravity worts (gravity reading of 1.056 or higher) have a greater need for more yeast to be added.
The writer further explained that for every gravity increase of 0.008 above 1.048, the yeast
volume should double.
The Level of Oxygen Dissolved in Wort and Lipid Content
Oxygen is required for the synthesis of essential yeast plasma membrane lipids, these are
synthesized at the start of the fermentation process and their amount is usually determined by the
level of dissolved oxygen in the wort (Boekhout & Robert, 2003). Compounds such as sterols
and unsaturated acids play a key role in maintaining the structure of the cell membranes. Lipid
content and composition in the brewing process enables quality control of the final product.
According to Bravi et al. (2009), lipids have a beneficial effect on yeast growth during
fermentation as well as deleterious effects on end-product quality. Haukeli et al. (as cited in Hui
et al., 2006) explain that lipids in the form of unsaturated fatty acids and ergosterol tend to

increase in concentration as long as oxygen is present. However, not all worts contain sufficient
unsaturated fatty acids to support yeast growth therefore, adding lipids to wort might be an
alternative. Boekhout & Robert (2003) mentioned that the synthesized sterols will determine the
biomass yield and as a direct consequence, the rate of fermentation. They also mention that the
more initial dissolved oxygen is present in the wort, the better the growth of the yeast and faster
the fermentation rate. Waites et al. (2001) further elaborated that a minimum of 10 (ppm) is
usually aimed for in wort prior to fermentation and if the oxygen level is inadequate, yeast
growth and ethanol production are usually impaired.
Concentration of Assimilable Nitrogen
Nitrogen deficiency is the most prevalent cause of sluggish fermentations and can reduce
fermentation rates significantly. Lack of nitrogen diminishes yeast metabolic activity, as well as
the biomass yield (Varela et al., 2004). It is evident that nitrogen content plays a vital role in the
fermentation process. According to Fumi et al. (2009), nitrogen compounds in worts are
fundamental for brewing processes and beer quality. These compounds affect the rate of
fermentation and the formation of active flavour compounds. In wort, the main nitrogen sources
for yeast metabolism are individual amino acids, small peptides, and ammonium ions. The
nitrogen sources formed from the proteolysis of barley malt proteins during malting and mashing
are collectively known and measured as Free Amino Nitrogen (FAN). Ingledew and O‟Connor-
Cox (as cited in Lekkas et al., 2007) expound that adequate levels of FAN in wort ensure
efficient yeast cell growth thus increasing the rate of fermentation. Furthermore, Ingledew &
Synder (2009) mention that usable nitrogen available helps the yeast cell to maintain the integrity
of its genetic make-up produce structural proteins and produce functional enzymes to enable the

cell to undergo metabolism. This nitrogen is sourced from the medium either from the grain or
via external addition.
Yeast cell viability and yeast quality
Bouix & Leveau (2001) explain that yeast performance in alcoholic fermentation depends
directly on yeast activity which can be seen as function of cell viability and the physiological
state of viable cells. To optimize the quality of fermentation, it is essential to predict yeast
vitality rapidly so that corrective actions may be taken before the yeast is pitched in the brewery
or during fermentation. In general, temperature, alcohol concentration and other factors that were
discussed can affect cell viability thus resulting in autolysis of yeast cells. According to Sanchez
(2009), cell viability significantly decreases when exposed to high temperatures and the yeast
cell numbers decreased an average of 90% with each month of storage at 40°C. Furthermore, the
activity of the yeast should be under optimal conditions in order to achieved viable yeast cells.
Yeast Strain
Pratt et al. (2003) emphasized that the selection of a yeast strain with the required
fermentation and recycling characteristics is critical. They also explain that the yeast strain will
influence the rate and extent of fermentation, the flavour characteristics and the overall quality
and stability of the finished beer. It is imperative that the strain selected be suitable under certain
conditions during fermentation, this includes a capacity to withstand high osmotic pressures and
elevated ethanol levels. Evans & Hamet (2005) defines fermentability as the ability to turn sweet
wort into alcohol and therefore different yeast strains attenuate wort differently thus affecting the
outcome of the rate of fermentation.

Size and geometry of fermenting vessel
The geometry of the fermenting vessel must not only facilitate the operations that have to
be performed inside the equipment, but also be suitable for the fermentation characteristics of the
chosen yeast strain and appropriate for the quality of the beer that is to be produced.
Furthermore, it plays an important role in fermentation as it has been observed that as the height
to-diameter ratio of the vessel increases, the rate increases (Boulton & Quain, 2001).
Fermentation Temperature and pH
Temperature is a major factor that primarily affects yeast growth (viability) and the
metabolic rate causing an overall effect on the rate of fermentation (Boulton & Quain, 2001). If
the temperature at which the yeast is added to the wort is too low the initial fermentation rate will
be slow. On the other hand if the temperature is too high, the yeast will experience a heat shock
which can result in autolysis. Yeasts will grow over a temperature range of 10°C-37°C in a
neutral or slightly acidic environment and tend to grow best between pH 4 to pH 6. However,
different yeast strains have an optimal temperature range that aids in its growth (Featherstone &
Tucker, 2011). According to Boulton & Quain (2001), most brewing yeast strains have a
maximum growth temperature within the range of 30 to 35°C, suggesting that very rapid
fermentations could be achieved.

Sterilization of materials and apparatus
Almost all fermentation processes have a contaminant-free environment to obtain
maximum and high purity product. As a result, fermentation vessel, medium, air supply, all
materials and apparatus must be sterilized and an aseptic environment should be maintained
throughout the fermentation process.
Viable foreign organisms can be removed whether by using saturated steams (autoclave),
dry heating (using an oven), radiation, chemicals or by some physical procedure such as filtration
before the media is introduce to the fermenter (Stanbury et al., 2003). It is also essential to
sterilize all the equipments and materials that maybe used for storage and culture or medium
formulation (Beakers, Conical Flasks, Bottles, Petri Dish). These can be sterilized with sodium
or potassium metabisulphite solution and then rinse with boiled water to remove any residual
sulphite (Azam-Ali, 2008). Acid wash are also used this is where 10% of acidic solution (HCl or
H2SO4) is used to rinse apparatus and materials after they have been washed with warm soap
water.
Sterilization of the medium
A culture medium is one that is design to support the growth of microorganisms or cells.
Formulation of the medium involves the incorporation of various nutrients such as vitamins,
minerals (metallic ions). In other words, these media are complex to facilitate the growth and
reproduction of microbes to obtain maximum product yield of high purity. As a result it is
important that the sterilization process employed does not degrade its quality and quantity. On
the other hand, the technique should eliminate or reduce foreign microbes (contaminants) as
much as possible. The recommended holding temperature for batch sterilization of a
fermentation medium ranges from 101 ºC - 130 ºC for 20-30 minutes (Stanbury et al., 2003).

Additionally during the fermentation process it is important that aseptic conditions are
maintained and the entre of contaminant are prevented. Calcium hydroxide solution can be used
to prevent the entre of contaminant into the fermentation media thereby creating an anaerobic
sterile environment (Ebbing & Gammon, 2010).
Yeast Metabolism
Metabolism is the biochemical assimilation (also known as anabolic pathways) and
dissimilation (refer to as catabolic pathways) of nutrients by a cell. Anabolic pathways involves
reductive processes leading to the production of new cellular materials while catabolic pathways
are oxidative processes that remove electrons from substrates or intermediates that are used to
generate energy. The energy created supplies the cell with energy for transport, movement and
synthesis of reactions (Jacobson, 2006). The different types and pathways of yeast metabolism
that can occur are dependent on whether the process is aerobic or anaerobic and also the type of
substrate. Figure 2.3 highlights the processes involves in both conditions.
Figure 2.3 showing the metabolism of glucose in yeast under aerobic and anaerobic

According to Pommerville (2011), glycolysis involves the metabolic pathway that
converts an initial 6-carbon substrate (glucose) into two (3-carbon) molecules of pyruvate.
During conversion, there are eight intermediates formed, each of which are catalyzed by a
specific enzyme. In addition, glycolysis yields a net release of 2 molecules of ATP through
substrate-level phosphorylation and 2 molecules of NADH (Konhauser, 2007). Bhagavan & Ha
(2011) states that, the conversion of pyruvate to alcohol occurs in two steps. In the first step, the
pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase, which utilises thiamine
pyrophosphate (TPP) as a coenzyme. Solomon et.al (2004) expound that in the second step,
NADH produced during glycolysis transfers hydrogen atoms to acetaldehyde, reducing it to
alcohol by alcohol dehydrogenase. The figure below illustrates this complex process followed by
the reactions that take place during alcoholic fermentation.

Figure 2.4 showing the yeast metabolism of glucose (Jacobson, 2006)
Basic Nutritional Requirements for Yeast
The growth and proliferation of microorganisms such as the yeast Saccharomyces
cerevisiae are controlled in part by the availability of nutrients (Werner-Washburne et. al, 1993).
As result, the medium that is used for the fermentation process must contain the basic nutritional
requirements that will retard yeast autolysis and produce maximum yield of product. There are
two classifications of essential nutrients, micro-nutrients (vitamins and minerals) and macro-
nutrients for example carbon and nitrogen. All microbes require nutrients along with water and
energy sources (such as oxygen if needed) for growth and reproduction (Stanbury et al., 2003).
However, the most suitable concentration of each nutrient that is needed for yeast performance is
often difficult to achieve especially on a large scale basis.

One of the major concerns of yeast in the alcoholic industries is its tolerance to increasing
concentration of ethanol and by-products (built up of toxic substances) which is considered
detrimental to the yeast cell. However, with the addition of nutrients such as metallic ions and
vitamins, the life of the yeast can be extended thus, increasing the rate of fermentation and the
quality of beer produced. Despite the several factors that affect the fermentation process,
Ingledew & Snyder (2009) states that, the most important factor during fermentation is
maintaining adequate yeast viability by the implementation of proper nutritional requirements.
However, the form and concentration of nutrients require for maximum product yield, is highly
dependent on the type (Ale and Lager) and strain of yeast used to carry out the process (White,
2001).
Nutrients used in Fermentation Processes
According to Stanbury et al. (2003) the source of nutrients used to create the medium should
meet as many criteria as possible these include:
1. Maximum yield of product or biomass per gram of substrate used.
2. Maximum concentration of biomass or product.
3. Permit the maximum rate of product formation.
4. Minimum yield of undesired product.
5. Consistent in quality and be readily available throughout the year.
6. Is should cause minimal problems during media making and sterilisation.
7. Minimum problems in other aspects of the production process especially aeration and
agitation, extraction, purification and effluent treatment.

There are various types of nutrients that are used for different purposes dependent on the type of
products (metabolites) that is to be produced. Additionally the Mode of Operation (batch or
continuous) and the stage on the Microbial Growth Curve (exponential or stationary) that result
in the production of the desire by-product, is influence greatly by the nutrients that are used to
formulate the media.
Vitamins
A vitamin is an organic compound required as a nutrient in minute amounts by an
organism (Lieberman & Bruning, 1990). They are said to be essential nutrients as they facilitates
in the synthesis of adaptive enzymes that are necessary for fermentation of carbohydrates.
Ingledew & Snyder (2009) highlights few vitamins that are commonly used in the fermentation
process, their importance and optimal concentrations.
Table 2.2 showing the list of vitamins, their importance and optimal concentrations
Brewer‟s wort is usually rich in vitamins such as biotin, thiamine (B1), calcium
pantothenate, nicotinic acid, riboflavin, inositol and pyridoxine. However, the vitamins require
for yeast growth and metabolism varies widely with the type and strain of yeast used for the
fermentation process. Although brewer‟s wort is a rich source of most of these growth factors
(vitamins) and deficiencies are rare, there is an absolute requirement for the addition of biotin

(vitamin B7). Its absence in many cases result in stuck or incomplete fermentation as it facilitate
in the metabolic activities of the yeast (Taylor & Eaton, 2005; White, 2001). Additionally,
Kulkarni et al. (2011), biotin also plays an important role in the production of high alcohol
concentration.
Figure 2.5 showing the structure of biotin
Minerals
Yeast requires a number of metallic ions for maximum by-product formation, optimum
growth, and tolerance to environmental conditions. These can be enhanced once the appropriate
concentrations of ions are added to the medium prior to fermentation. According to Taylor &
Eaton (2005) ionic species:
1. Plays both an enzymatic and structural role these may include potassium, phosphorus,
magnesium, and calcium.
2. Function as the catalytic centre of an enzyme such as zinc, cobalt, manganese and
copper.
3. Act has an activator or stabiliser of enzymes for example magnesium and potassium.
4. Glucose Tolerance factor such as chromium.

This project will highlight the major anion (such as phosphorus) and cations (including
magnesium, calcium, zinc, copper and iron and chromium) that play a significant role in yeast
viability during the fermentation process.
Effects of Phosphorous
The concentration of phosphorus compounds, both inorganic and organic, appears to be
independent of the physiological state of aerobic cultures, and does not reflect changes in
metabolic activity such as decreased rate of growth and multiplication (Katchman & Fetty,
1954). However, Taylor & Eaton (2005) noted that, phosphorus is vital to yeast cells as it is
important in its structural formation such as:
 Phosphomannan and phospholipids
 Nucleic acids (DNA and RNA).
 Phosphorylated metabolites (for example, ATP and glusose-6-phosphate)
Effects of Magnesium Ions
Walker & Maynard (1996) dictates that, magnesium not only plays a major role in both
the growth and metabolism of the yeast cell, but also exerts a protective effect on yeast cultures
subjected environmental stresses during the fermentation process. Additionally, magnesium is
the most abundant intracellular divalent cation in yeast cells and acts primarily as an enzyme
cofactor.
Moreover, experimental results showed that ethanol production is directly dependent on the
availability of magnesium ions at various concentrations. Birch & Walker (2000) investigations

illustrates that, elevation of magnesium levels in the growth medium results in an increase in the
growth and metabolism of the yeast cells thus increase the concentration of alcohol produced.
Effects of Calcium Ions
Calcium ions is important for yeast flocculation, but is not thought to be required for
yeast growth and fermentation (White, 2001). It is involve in the membrane structure and
function but is usually in small concentration. The physiological and biotechnological
importance for the assimilation of calcium is due to its role as both a modulator of growth (cell
cycle) and metabolic response (Taylor & Eaton, 2005). On the other hand Nabais et al. (1988)
made a correlation with ethanol production and the concentration levels of calcium in a
fermentation medium using Saccharomyces cerevisiae. The investigation illustrates that, the
production of ethanol increases rapidly in optimal concentrations ranging from 0.75mM to 2.0
mM of calcium to a fermentation medium with a trace contaminating concentration of 0.025
mM. The experiment also highlighted that for lethal concentrations of ethanol, the specific death
rates were minimal for cells that were grown and incubated with ethanol in medium with an
optimal concentration of Ca2+
. It was maximal for cells grown and incubated with ethanol in
unsupplemented medium, and intermediate for cells grown in unsupplemented medium and
incubated with ethanol in calcium-supplemented medium. However, high concentration of
calcium may be inhibitory to yeast (*Walker, 1994). In addition, Chotineeranat et al., (2010)
states that the concentration ranging from 0.18% w/v Ca2+
to 0.72% w/v Ca2+
resulted in a
significant decrease in the rate of fermentation and ethanol yield.
Effects of Zinc Ions
Hornsey (1999) mentions that brewer‟s worts can vary in zinc content from 0.1-5 ppm
(mg/L) according to how they are prepared and usually the element is rapidly taken up by the

yeast. Deprivation of the mineral can prevent budding cells thus, trace levels are essential for
yeast growth and function of many enzymes activities. Zinc also contributes to various protective
functions such as proteinase attack, and promotes agglutination of yeast cells. Taylor & Eaton
(2005) affirmed that “zinc requirement for the growth of yeast cannot be met by other metal
ions.”
Vecseri-Hegyes et al., (2006) experiment noted that, addition of zinc increases the rate of
fermentation and that a concentration of 0.4 gave best results. Satyanarayana and Kuze (2009)
also investigated the influence of zinc on alcohol production during fermentation where
concentrations of 0.9, 1.5, 2.5, 14 and 26.5 ppm (mg/L) were tested. They observed that high
alcohol production was determined at 2.5 ppm (mg/L) of Zn2+
. Studies highlights that zinc not
only enhanced the ethanol tolerance of the yeast but also increases the ethanol production (Zhao
et al, 2009). Moreover, Vecseri-Hegyes et al. (2006) dictated that the presence of zinc ions
enhanced the uptake maltose and maltotriose while the sedimentation ability and heat sensitivity
of the yeast reduced, thus increasing production.
Zinc contributes to the structure of the cell, stabilization of enzymes, and stimulates
metabolic processes (Hegyes et al, 2006). Jacobsen et al. (as cited in Satyanarayana & Kunze,
2009) noted that when zinc levels are below 0.1 ppm it may result in slow and incomplete
fermentation which is termed “sluggish”. On the other hand, it cannot be in excess as this will
hinder the fermentation process. However, Magonet et al. (as cited in Satyanarayana & Kunze,
2009) stated that zinc is essential for alcohol dehydrogenase (ADH). It facilities the conversion
of acetaldehyde into alcohol at the end of fermentation which is important for maximum product
yield. Reilly (2004), also noted that zinc ions are usually located at the active centre of the
enzyme (ADH) and participates directly in the catalytic mechanism, by interacting with the

substrate molecule in a bond making or breaking step. Likewise Havkin-Frenkel & Belanger
(2008) dictates that high concentrations of acetaldehyde in wort usually depict a steady decline in
yeast viability. In the same light Jones (as cited in Boulton & Quain, 2001) explains that
acetaldehyde is highly toxic to yeast cells which can lead to the inactivation of enzymes.
Therefore, lack of zinc ions inhibits the function of the enzyme. Based on this observation,
Havkin -Frenkel & Belanger (2008) concluded that a shortage of zinc ions in the wort can lead to
excess acetaldehyde production resulting in stuck fermentation.
Effects of Copper and Iron Ions
Copper and Iron are essential nutrients for yeast growth they acts as cofactors of many
metabolic enzymes. However, the concentration of both minerals must be low as high levels can
be detrimental to the yeast viability due to their ability to transfer electrons (Freitas et al., 2003).
Iron and copper are particularly important because they participate in electron transfer reactions
which are critical for enzymatic activities.
Effects of Chromium Ions
Chromium can occur in various oxidation states from –2 to +6. However, the most
commonly found are 0, +3, and +6. Raspor et al. (2000) noted that “trivalent state of chromium
is thermodynamically the most stable and is commonly found in the living systems”. They also
highlight that (Mertz 1984; Ducros 1992; Hunt 1983) emphasized on +3 ions and their existence
in the biological system. As a result many investigations seek to examine the effect of Cr3+
specifically chromium chloride (CrCl3). Recent studies have concluded that Cr3+
may serves has
a glucose tolerance factor in yeast and that its biological role is primarily connected with
carbohydrate metabolisms (Vincent, 2007).

There are two types of brewer‟s yeast those that are used for the fermentation of beer and
those that are known as nutritional brewer yeast. However nutritional yeast was originally a by-
product of brewing beer which was term as just brewer‟s yeast, but has being modified for daily
intake of minerals and vitamin (Anderson, 2000). Hence, it is denoted as nutritional yeast. Yeasts
are often high in concentration of chromium chloride (glucose tolerance factor) which enhances
and control carbohydrate metabolism in mammals (Vinson & Bose, 1984; Vincent, 2007). Zetic
et al. (2001) investigated the effect of chromium using fresh baker‟s yeast (S. cerevisiae) with
30% of dry matter obtained under two conditions semi aerobic and static. The results obtained
revealed that the addition of optimal amounts of chromium chloride (CrCl3) into the basal
medium enhanced the kinetics of alcohol fermentations and stimulate yeast growth in all
experimental conditions. An illustration of their investigation is shown below.
Figure 2.6 showing the effects of chromium on ethanol and biomass concentration under semi
aerobic conditions.
Note: The dynamics of biomass and ethanol production in static conditions with 200 of CrCl3 and without the addition of
CrCl3 into the molasses medium (■, ▲) , biomass (medium without „■‟and with „▲‟ addition of CrCl3 ). (●, x). Ethanol (medium
without „●‟ and with „x‟ additional of CrCl3).

Figure 2.7 showing the effects of chromium on ethanol and biomass concentration under static
conditions.
Note: The dynamics of biomass and ethanol production in static conditions with 200 of CrCl3 and without the addition of CrCl3
into the molasses medium (■, ▲) , biomass (medium without „■‟and with „▲‟ addition of CrCl3 ). (●, x). Ethanol medium
without „●‟ and with „x‟ additional CrCl3.
In addition to Zetic et al. (2001) investigations on a similar experiment was recently done
by Abd-Elsalam (2011) and they also noted that additionally, chromium in the trivalent form is
an essential nutrient in carbohydrate, lipid and nucleic acid metabolism which was evident in
their investigation. As a result, it is evident from both investigations that the addition of CrCl3
enhanced the rate of CO2 production as well as the glucose utilization during alcoholic
fermentation. However, Zetic et al. (2001) states that the role of chromium in brewers is still not
fully understood.
Effects of Macro-nutrients
Carbon is the main energy source for microbial activities, it is renowned that most yeasts
employ sugars as their main carbon and hence energy source. There are many different carbon
sources that are in existence. However its utilisation is dependent on the product specification
and the type of yeast. With regard to nitrogen metabolism, most yeast is capable of assimilating

simple nitrogenous sources to biosynthesize amino acids and proteins. The table below shows
some commonly used carbon source via yeast (S. Cerevisiae).
Table 2.3 showing carbon and nitrogen sources for growth and metabolism of yeast (S.
cerevisiae)
Source: http://biochemie.web.med.uni-muenchen.de/Yeast_Biol/03%20Yeast%20Metabolism.pdf
Effects of Water
Water is a major component in any fermentation process, it not only contains minerals
but also facilitates in the biochemical assimilation and dissimilation of nutrients by a cell.
Minerals such as calcium carbonate, iron, sodium, magnesium, and copper along with others are
commonly found in water. Even though these minerals might be of significant, it is important
that careful analysis is preformed to ensure that they are within the specified range (Stanbury et
al., 2003).
Various Minerals in Grains
Grains that are used in the fermentation process are usually high in carbon and energy
source. However, vitamins and minerals are usually in trace amounts and as a result, addition of
external nutrients is often require for optimum yeast performance. Table 2.4 below shows grains
that are frequently used and the percentage nutrients in each.

Table 2.4 showing comparative nutritive value of cereal grains (Haard et al., 1999)
FACTOR Wheat Maize Brown
rice
Barley Sorghum Oat Pearl
millet
Rye
Available CHO (%) 69.7 63.6 64.3 55.8 62.9 62.9 63.4 71.8
Energy (kJ/100 g) 1570 1660 1610 1630 1610 1640 1650 1570
Digestible energy (%) 86.4 87.2 96.3 81.0 79.9 70.6 87.2 85.0
Thiamin 0.45 0.32 0.29 0.10 0.33 0.60 0.63 0.66
Riboflavin 0.10 0.10 0.04 0.04 0.13 0.14 0.33 0.25
Niacin 3.7 1.9 4.0 2.7 3.4 1.3 2.0 1.3
Lysine 2.3 2.5 3.8 3.2 2.7 4.0 2.7 3.7
Threonine 2.8 3.2 3.6 2.9 3.3 3.6 3.2 3.3
Met. & Cys. 3.6 3.9 3.9 3.9 2.8 4.8 3.6 3.7
Tryptophan 1.0 0.6 1.1 1.7 1.0 0.9 1.3 1.0
True digestibility 96.0 95.0 99.7 88.0 84.8 84.1 93.0 77.0
Biological value 55.0 61.0 74.0 70.0 59.2 70.4 60.0 77.7
Net protein utilisation 53.0 58.0 73.8 62.0 50.0 59.1 56.0 59.0
Utilization protein 5.6 5.7 5.4 6.8 4.2 5.5 6.4 5.1
Presence of Anti-nutrients
Grains are essential as they not only contain minerals and vitamins but also usable
nitrogen or Free Amino Nitrogen (usable FAN) that is important for the fermentation process.
However, they also contain anti-nutrients which reduce the absorption of valuable vitamins and
minerals. Phytates, tannins, saponins, and enzyme inhibitors are a few examples that result in the
inability of yeast cells to assimilate their required nutrients. Nevertheless, refining processes
(such as soaking of grains) can reduce the amount of anti nutrients that is present (Cabrera,
2002).

Formation of By-Products
By-products that are produced contribute to the taste, aroma, and other characteristics of
the beer which contributes to its uniqueness. On the other hand, these by-products can negatively
impinge on the flavour of the beer and reduces yeast metabolism if there is a lack of proper
quality control techniques (Goldammer, 2008). In many instances yeasts are thought of as a “bag
of enzymes” whereby each enzyme catalyzes different chemical reactions. In addition these
enzymes have specific mineral requirements to facilitate optimum performance. These chemical
reactions generate various compounds such as alcohol, flavour compounds, and energy for
growth and reproduction of cells (White, 2001). Additionally, the formation of by-products
during the fermentation process can also affect the yeast performance if they are not related to
the growth and synthesis of the microorganism (Stanbury et al., 2003).
Yeast Management
Eßlinger (2009) states that re-pitching the yeast instead of a single use, is a common
practice in the brewing industry. Yeast may be re-used 5-10 times, sometimes even more, before
fresh inocula are prepared (Waites et al., 2001). Therefore, one important aspect of yeast
management is to maintain the yeast quality from one fermentation process to the next. Before
the yeast is re-pitched, the process starts with harvesting the yeast that is obtained from the
previous fermentation process, following an optional acid-washing (to remove any bacterial
contaminants) or sieving and finally yeast storage and treatment. According to Smart (2003), the
storage of yeast is a critical step in yeast handling, as it should ensure that yeast cells are
maintained in a minimal metabolic state. Eßlinger (2009) further expound that to maintain the
nutrient reserves during storage it is necessary to reduce yeast metabolism by lowering the
temperature between 2 and 4°C. Waites et al. (2001) also noted that the viability of yeast used

for re-pitching should be at least 90-95%, otherwise subsequent fermentation rates are will be
sluggish.
Assessment of Fermentation Parameters
The fundamental understanding on yeast behaviour in a particular medium can be
assessed by different analytical methods depending on the parameter that is being investigated.
The results obtained should illustrate the effectiveness of the fermentation process in various
media. The progress of fermentation is usually monitored at 24 hr intervals and the cessation of
fermentation is affiliated by the specific gravity readings. The readings can also depict the
possible outcome of the amount of substrates consumed and the products being formed. The total
yeast cell counts and viable counts at the end of fermentation can be determined by the direct
microscopic methylene blue method.
Specific Gravity
Specific gravity is the measure of the density of a liquid relative to water (Williams,
2003). In general, the liquid that aids in the formation of beer is called wort. Its specific gravity
is always higher than that of water because it contains significant amount of dissolved sugars.
Furthermore, it can be observed that upon the completion of the fermentation process, the
specific gravity of beer is always less than when it started, due to the conversion of sugar into
alcohol. Boulton & Quain (2001) stated that the measurement of the reduction in wort specific
gravity is the most commonly used method of gauging fermentation progress. The specific
gravity measurements are used to determine the alcoholic strength and the amount of sugar
utilised by the yeast.

The determination of the alcohol level and the attenuation (which is the degree to which
sugar in wort has been fermented into alcohol) is dependent on the original gravity (OG) and the
final gravity (FG) reading. According to Holl & Schweber (2011), the original gravity and the
final gravity is the specific gravity reading of the wort and the finished beer respectively.
Usually, the alcohol level in beer is described as the alcohol-by-volume, ABV Miller
(1998) gives a simple formula to determine the alcohol content and the attenuation; the equations
are illustrated in the Appendix. The most popular methods used in the brewing industry to
determine the specific gravity of a liquid are the use of a hydrometer and refractometer.
However, the scaling on the refractometer is in Brix, which can easily be converted to specific
gravity; this equation is also illustrated in the Appendix section. Furthermore, the consistency of
specific gravity readings indicates that the fermentation process has ended therefore the overall
rate of fermentation can be established.
Figure 2.8 Hydrometer with measuring cylinder use to measure the specific gravity
Source: http://www.brewmorebeer.com/calculate-percent-alcohol-in-beer/

Figure 2.9. Refractometer use to measure specific gravity
Source: http://www.brewmorebeer.com/calculate-percent-alcohol-in-beer/
Cell Viability
Cell viability measures the fitness or health of the viable yeast cells that are pitched to
undertake effective fermentation. Yeast may exhibit good viability (in excess of 95%) but
promote poor fermentation because the cells are deficient in essential metabolites. The essential
metabolites for effective fermentation are associated with intra-cellular reserves, sterols and fatty
acids of membrane systems and yeast cell energy reserves in the form of ATP. The method used
to determine cell viability (and to a degree, the definition of viability) is often related to the
phenomenon studied. There are several techniques that can be used to test for yeast viability such
as, the use of vital dyes, biomass probe, slide culture, ATP (where the dead cells have no
energy) and plate culture (Smart; Priest and Campbell 2003). Cell viability may also be judged
by morphological changes or by changes in membrane permeability and or physiological state
inferred from the exclusion of certain dyes or the uptake and retention of others.

Assessment of cell viability
The reference analysis for viability is the plate count measurement method as it gives a
truer representative of the viable cells present. However, the primary drawback of this method is
that it is time intensive thus staining methods are commonly used to determine yeast viability.
The methylene blue test is universally use; with its application the viable cells remain colourless,
whereas dead cells are stained blue. The physiological basis of the test is that viable cells take up
the stain at a sufficiently slow rate for it to be oxidised to colourless. Conversely dead cells
cannot exclude the dye as a result they are stained blue. After which the viability can be
determined by preparing a suitable dilution of yeast or beer slurry and counting the total cells and
stained cells (dead cells) using a haemocytometer and a light microscope (Boulton & Quain,
2001).
Figure 2.10: showing staining of yeast cells using Methylene Blue (Boulton &Quain, 2001).

Once there is a distinction between live cells and dead cells the viability can be calculated
using the equation below:
However, this method has recently been questioned to give poor reproducibility and inaccuracy
in results with apparent viability below 90% (Lewis & Young, 2003). Additionally even though
it might not stain cells that are said to be viable it does not necessarily means that the cells is
healthy. As a result, it was investigated that this can be overcome by the addition of an alkaline
solution to the slurry (which would heighten the pH) for about 15 minutes at a temperature of
25°C (Sami et al., 1994). He noted that since entry of the dye is influenced by membrane
potential, the lowering of the external H +
concentration would favour the entry of the dye into
stressed cells, which normally give a false positive viability. Nevertheless, other dyes such as
methylene violet and trypan blue have recently been introduced as an improved staining
procedure that would provide more accurate results.

Summary of literature review
It is evident that the introduction of nutrients into the fermentation medium not only
increases the fermentation process but also improves yeasts metabolic activities there by yield
maximum alcohol production. It is especially seen that the importance of Zn2+
ions and biotin
further enhances the progression of such process.
Although many studies have investigated the effects of commonly listed nutrients that is
necessary for yeast viability. Its viability and mortality in the presence of chromium chloride is
still not fully understood. As a result this research seeks to investigate varying concentrations of
CrCl3 and evaluate its effects with different concentrations of zinc sulphate in the presence and
absence of biotin.

Chapter Three
Methodology
Introduction
In an effort to retard the level of yeast (lager) autolysis that occurs during the
fermentation process, additional nutrients was introduced into the medium (wort). The technique
selected was batch, and each vessel (flask) containing different concentration of nutrients were
monitored and controlled throughout the fermentation period. The process occurred under
anaerobic conditions and the temperature was maintained at 20ºC. During the fermentation
process, the specific gravity readings were recorded at set time intervals. This was to evaluate the
effectiveness of varying concentration levels of zinc sulphate (ZnSO4) and chromium chloride
(CrCl3), with and without the addition of biotin (vitamin B7). At the end of the process,
percentage cell viability, apparent attenuations and percentage alcohol produced were computed
to determine the optimum concentration of each nutrient.

Specific Procedures
Preliminary Wort Analysis
Prior to the addition of nutrients into the fermentation medium, preliminary investigations
were carried out on the wort to determine the initial concentration of Zn2+
and Cr3+
present. This
was important, as it gave an estimation of the quantity of each nutrient that must be added to
achieve the desire experimental concentrations. The analysis was carryout at Mines and Geology
Division using an Atomic Mass Spectroscopy (AAS) where a concentration of was
detected for each metallic ion. The specific gravity analysis was then performed and the pH was
noted. This was to ensure that the medium met the requirements for satisfactory yeast growth and
product formation.
Preparation of Nutrients
The concentration of nutrients that must be added to the wort was computed by
subtracting the amount that was detected via preliminary analysis from the total (experimental
concentration). Concentrations were in units of milligrams of sample per litre of water ( ). The
experimental masses for the investigation were 100mg, 150mg, 200mg, 250 mg, and 300mg of
chromium chloride while that of zinc sulphate includes 2mg, 3mg, 4mg and biotin (vitamin B7)
was 0.6mg. Masses were measured using an analytical balance each of which was then dissolved
in 1000mL of distilled water. The solutions not only allow for homogeneity (uniform
composition and properties) but also facilitate the ease of utilization of nutrients by the yeast.

Chromium Chloride Selection
A volume of 5mL of each nutrient solution was measured and poured into separate 125
mL conical flask. Five (5) different concentration of Chromium Chloride ,
and was added to five different flask with optimum concentration of biotin
( ) and zinc sulphate ( ).The volume was then made up to 125mL with wort. Table 1
below represents the above data.
Table 3.1: Different concentration levels of chromium chloride that was varied with optimal
concentration of Zinc ion and biotin (vitamin B7).
Flask # Cr3+
(mg/L) a
Zn2+
(mg/L) b
Biotin (mg/L)
1 100 4 0.6
2 150 4 0.6
3 200 4 0.6
4 250 4 0.6
5 300 4 0.6
a
Zn2+
and b
Biotin represents the optimum concentration in the wort (Synder et al. 2009).
During the fermentation process the specific gravity reading of each beer sample was
recorded every 24 hour. This was done not only to assess how the different concentration of each
nutrient in each flask affected the rate of fermentation, but it also provides an indication of the
end of the fermentation period.

At the end of the fermentation process a cell viability analysis was conducted and the percentage
viability for each flask was calculated. This was done to determine the three optimum
concentrations of chromium chloride that gave the highest viability percentage. These were then
used for further investigations.
Nutrition for Maximum Cell Viability
Once the three optimum concentrations of chromium chloride were determined, the
concentration levels of each variable (Zn2+
and Cr3+
) were randomly selected for each trial, some
were in the presence or absence of biotin. This was to evaluate which combination of nutrients
resulted in the highest viability count. Given the experimental variables and the concentration
levels, it was clear that a general factorial design is suitable for the arrangement of each
experimental run. The table below represents a full description of the experimental variables and
their levels.

Experimental design results
Table 3.2 Random selection of concentration levels for each variable in each flask
Note: Variables where Zn2+
Cr3+
and biotin and each flask represent the experimental runs and the concentration level of each
nutrient.
Therefore, 18 experiments (flask) would be sufficient to analyse the effects of each variable (at
different concentration) on the percentage cell viability. During the fermentation period, specific
gravity readings were recorded every 24 hours and the cell viability analysis was performed.
After which the optimum concentration of each nutrients was identified.

Specific Gravity Analysis
Prior to the determination of the specific gravity, samples must be at a temperature of
20 ºC. Readings were taken three times per flask and for each analysis the sanitizing solution
(5% H2SO4) was changed. This was to prevent any cross contamination of concentrations from
flask to flask.
The density of the wort sample was recorded before, during and after the fermentation process
using a 0-32% Brix Refractometer. Readings were obtained in Brix and was converted to
specific gravity. This was done not only to assess the effects of the varying concentration levels
of nutrients on the rate of fermentation, but also to compute and evaluate the apparent attenuation
and percentage alcohol produced. Additionally, it is used to indicate the end of the fermentation
process which is important for further analysis (percentage cell viability) of the beer sample.
Cell Viability Analysis
At the end of the fermentation process, each flask was continuously swirled to break up
the yeast flocs. After homogeneity was achieved, 15 mL of each sample was pipette and
transferred into sample tubes. Prior to analysis a dilution was done to allow for easy counting
of the yeast cells (counting will be difficult if the sample is too concentrated). 1000 µL of 1%
methylene blue solution was then added to the test tube. The importance of the methylene
solution was to distinguish the dead (which will absorb the stain blue) and viable cells (does not
absorbed the stain).
A cover slip was used to cover the counting areas of the hemocytometer, after which a
small amount of the sample was extracted and carefully loaded into the counting chamber. This
was done via capillary tubes. The use of capillary tubes and hemocytometer cover slips was to

prevent flooding and under loading of chambers. The hemocytometer was then placed under a
light microscope and the number of dead, living and total cells were counted and tabulated. The
percentage cell viability can then be determined which was used to assess the extent to which
yeast autolysis did occurred during the fermentation process. A detailed experimental procedure
for all method of analysis can be seen in appendix B.
Note: Time Line see appendix D

Chapter 4
Results and Data Presentations
Results
This chapter presents the results generated by the various instruments used to address the
research questions highlighted in chapter one. In order to answer the research questions the data
collected using the instruments described in chapter three were analyzed and presented using
tables, graphs and charts. The method of analysis and presentation of the results were informed
by the specific research question being addressed. However, before the four research questions
are considered, data collected concerning preliminary analysis of the wort sample were
conducted to evaluate some initial properties of the wort. The results from these analyses are
presented in Table 4.1.
Table 4.1: Preliminary Results
As shown above, the initial concentration of Zn2+
and Cr3+
ions in the wort were lower
than the experimental concentrations. As result, additional nutrients were necessary to assess and
evaluate the effects of these nutrients (in the presence and absence of biotin) on the fermentation
process and yeast performance. On the other hand, the initial pH and specific gravity readings
were within the specified range to sustain yeast growth. Hence, no adjustments were made.
Concentration of Zn2+
< 0.1 mg/L
Concentration of Cr3+
< 0.1 mg/L
pH 4.8 – 5.2
Specific Gravity 1.078 – 1.080

Research question one: What concentrations of chromium chloride (CrCl3) gives maximum
percentage viability, in the presence of biotin (vitamin B7) and zinc sulphate (ZnSO4) at their
optimum concentration?
To answer this question, five different concentrations of chromium chloride (100, 150
200 250 and 300 mg/L) were used, after which three concentrations that resulted in high
percentage viability counts were used for further investigations. Table 4.2 below presents the
experimental results.
Table 4.2: Effects of Cr3+
concentrations on yeast viability, percentage alcohol content and
attenuation at optimal concentration of Zn2+
(4mg/L) and biotin (0.6 mg/L).
Cr3+
(mg/L) Viability (%) Alcohol Content (%) Attenuation (%)
100 77.8 5.3 3.7
150 82.9 5.9 4.1
200 75 5.9 4.1
250 75.7 5.9 4.1
300 78 5.6 3.9

ions concentrations on yeast viability.
As illustrated in figure 4.1 above, the results indicate that the percentage viability
increases steadily as the Cr3+
concentration increases. After which it decreases above
concentration levels of 150 mg/L. However, at concentrations greater than 230 mg/L the
percentage viability increases. This observation led to an analysis of the percentage alcohol
produce and percentage attenuation shown in figure 4.2 below.
74
75
76
77
78
79
80
81
82
83
84
100 150 200 250 300 350
Viability(%)
Concentration (mg/L)
Graph of % Viability vs. Concentration
Viability

Figure 4.2: Effects of Cr3+ concentration on the percentage alcohol content and attenuation.
From figure 4.2 shown above, similar patterns were observed for the percentage alcohol
and attenuation with increasing concentrations of Cr3+
ions. Nevertheless, it must be noted that as
the concentration increases from 150 mg/L to about 220 mg/L (shown in figure 4.1), the
percentage viability decreases steadily. However, the percentage alcohol produced over the same
range was constant (optimum) after which it decreases at Cr3+
concentration of 250 mg/L. This
decrease accounts for the increase in percentage viability above Cr3+
concentration of 220mg/L
observed in figure 4.1. Nonetheless, the overall results indicated that the three optimal
concentrations of chromium chloride that resulted in the maximum percentage viability were 100
mg/L 150 mg/L and 300 mg/L.
0
1
2
3
4
5
6
7
100 150 200 250 300 350
ExperimentalParameters
Concentration of Cr3+ (mg/L)
Graph of Concentration vs. % Alcohol Conetnt and Attenuation
Alcoho Content (%)
Attenuation (%)

Research question 2: How does the varying concentrations of zinc sulphate (ZnSO4), and
chromium chloride (CrCl3) in the presence and absence of biotin (vitamin B7) affect yeast
viability and percentage alcohol produced?
To evaluate each nutrient in the presence and absence of biotin, the effects on the
percentage viability and percentage alcohol produced was first determined with constant
concentrations of Zn2+
and varying concentrations of Cr3+
in the presence and absence of biotin.
After which constant concentrations of Cr3+
ions and varying concentrations of Zn2+
were
analysed.
Table 4.3: Effects on percentage viability and percentage alcohol produced with constant
and varying concentration levels of Cr3+
in the presence of biotin.
Zn 2+
(mg/L) Cr3+
(mg/L) Viability (%) Alcohol Content (%)
2 100 83.5 5.7
2 150 85.5 5.7
2 300 82.6 5.5
3 100 87.8 5.3
3 150 80.6 5.7
3 300 89.7 5.7
4 100 77.6 5.7
4 150 83.1 5.6
4 300 80.4 5.7

Figure 4.3: Effects of Cr3+ on yeast viability at constant Zn2+ concentrations in the presence of
biotin.
From Figure 4.3 shown above, it was observed that concentrations of Zn2+
at 2 mg/L and
4 mg/L had comparable effects on the percentage viability. For both variables, the percentage
viability increases with increasing concentration of Cr3+
from 100 to 150 mg/L. However, above
150 mg/L, the percentage viability decreases. Conversely, concentration of Zn 2+
at 3 mg/L has a
reverse effect on the percentage viability. The graph illustrates that the percentage viability
consistently increases above 150 mg/L (Cr3+
), whereby at 220 mg/L maximum viability was
achieved compared to that of Zn2+
at 2 and 4 mg/L. The viability continued to accelerate until a
maximum at 300 mg/L.
76
78
80
82
84
86
88
90
92
100 150 200 250 300 350
Viability(%)
Zn2+ (2 mg/L)
Zn2+ (3 mg/L)
Zn2+ (4 mg/L)

Figure 4.4: Effects of Cr3+ on percentage alcohol content at constant Zn2+ concentrations in the
presence of biotin.
Initially, concentrations of Zn2+
(2 and 4 mg/L) with the addition of 100 mg/L (Cr3+
), the
alcohol content produced was 5.7%. On the other hand, Zn2+
at 3 mg/L produces a minimal of
5.3%. In addition, at this concentration, the alcohol level steadily increases with increasing
concentration of Cr3+
. It was also examined that as the concentration surpassed 150 mg/L
maximum alcohol was achieved (over 5.7%). However, a steady decline was observed at 280
mg/L until a constant quantity of 5.7%. It was evident that the trend of Zn2+
at 2 and 4 mg/L
became incomparable over 100 mg/L. At Zn2+
(2 mg/L), the alcohol level increased slowly to
150 mg/L but decreases above 150 mg/L. In contrast, Zn2+
at 4 mg/L, the alcohol level decreased
to 150 mg/L but increases above 150 mg/L.
5.25
5.3
5.35
5.4
5.45
5.5
5.55
5.6
5.65
5.7
5.75
100 150 200 250 300 350
AlcoholContent(%)
Graph of % Alcohol Content vs. Concentration
Zn2+(2 mg/L)
Zn2+(3 mg/L)
Zn2+(4mg/L)

Table 4.4: Effects on percentage viability and percentage alcohol produced with constant
and varying concentration levels of Cr3+
in the absence of biotin.
Zn 2+
(mg/L) Cr3+
(mg/L) Viability (%) Alcohol Content (%)
2 100 78.3 5.3
2 150 84.3 5.3
2 300 85.3 5.3
3 100 75.7 5.1
3 150 87.5 5.5
3 300 82.2 5.3
4 100 82.4 5.1
4 150 82.4 5.5
4 300 85.1 5.1

concentrations in the absence of
biotin.
Initially, for both concentrations of Zn2+
at 2 mg/L and 4 mg/L, the percentage viability
increases steadily as the varying concentrations of Cr3+
increases. However, concentration of
Zn2+
at 3 mg/L, the percentage viability increases to150 mg/L (Cr3+
) but gradually declines when
the concentration surpasses this point.
74
76
78
80
82
84
86
88
90
100 150 200 250 300 350
Viability(%)
Zn2+ (2 mg/L)
Zn2+ (3 mg/L)
Zn2+ (4 mg/L)

Final Major Project Report 2011

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