THIS INVENTION relates to the promotion of fungal and microbial processes. In particular, the invention relates to a nutrient additive for fungal and microbial processes, to a microbial fermentation process for producing alcohol, and to the use of a nutrient additive.

Many microbial, fungal or biosynthesis processes are known that employ one or more micro-organisms to produce a desired product or result. One of the oldest used and best- known microbial processes is the process of yeast fermentation to convert sugars to alcohol.

Fungal processes, whether macroscopic (e.g. in the case of mushrooms) or microscopic (e.g. in the case of yeasts and moulds), and other microbial processes such as processes employing bacteria, typically require a nutrient or substrate composition for the fungal organism or microbial organism used in the process. The choice of nutrient or substrate composition can have a marked effect on biosynthesis processes, and thus is important from a commercial perspective, particularly in large scale fungal, microbial or biosynthesis processes. In many fungal, microbial or biosynthesis processes a particular nutrient as raw material or feedstock material or substrate is required. Such a process can potentially be improved or enhanced by adding a nutrient additive to the raw material or feedstock material or substrate, e.g. to a yeast broth, to alter or boost the nutrient composition or substrate.

Microbial ethanol fermentation typically involves the conversion of sugars to ethanol by a yeast such as Saccharomyces cerevisiae. Ethanol fermentation by yeasts to produce alcohol for fuel is a global industry predicted to have a value of US$115.65 billion by 2025 with various factors such as increased support in Brazil and the EU driving growth. The potable ethanol industry is also currently experiencing rapid growth globally due to growth in emerging markets. It is thus clear that there will be increasing demand for ethanol. One can increase capacity by building new and/or expanding existing production facilities. This is however a capital-intensive option. Another option is by improving the efficiency of existing facilities.

The efficiency of existing ethanol production facilities can be improved by many different routes, from improving cleanliness and reducing contamination to improving yeast health through nutritional additives. In fact, a number of commercial products are marketed and purported to enhance fermentation. There is however a need for even better performing or alternative nutritional additives, particularly for a nutritional additive that is cost-effective.

The mechanisms by which yeast adapts to relatively simple environmental changes are well known and have been for some time. However, there are far more complex environmental conditions which can vary significantly from time to time in commercial fermentation systems, and the mechanisms by which yeast adapt, and can adapt are likely to be dramatically more complex than those elucidated to date in recorded research work around the globe over the past two hundred years in scientific literature.

Modern industrial molasses fermentation processes tend to use very high concentrations of molasses and hence the yeast is exposed to high osmotic stress, often high temperature, and if all goes well, high alcohol concentrations, all placing cells under stress. The strength of the yeast cell membrane and ability to tolerate high osmotic stress, and/or high ethanol concentrations is related to a healthy lipid composition. It follows that the addition of suitable lipids can allow the use of higher concentrations of molasses in fermentors, which can in turn yield higher ethanol concentrations and increase cell membrane health.

According to one aspect of the invention, there is provided a nutrient additive for a fungus or a microbe, or for use in a process employing a fungus or a microbe, the additive including

an optional diluent; and

a lipid portion or lipid composition which includes free or saponified fatty acids, free fatty alcohols, wax esters, hydrocarbons and at least one phospholipid, the free fatty alcohols making up at least 5% by mass of the lipid portion or lipid composition and the phospholipid or phospholipids making up at least 0.4% by mass of the lipid portion or lipid composition.

The free fatty alcohols may be present in the lipid portion or lipid composition in a concentration of at least about 8% by mass or at least about 10% by mass or at least about 12% by mass, e.g. about 15.6% by mass.

Typically, the free fatty alcohols are present in the lipid portion or lipid composition in a concentration of less than about 25% by mass or less than about 22% by mass or less than about 20% by mass.

The free fatty alcohols may include fatty alcohols with a carbon number ranging between 24 and 32. In other words, the fatty alcohols may include C24 - C32 fatty alcohols, i.e. saturated long chain alcohols. Typically, the fatty alcohols are even carbon numbered.

Typically, there are no free fatty alcohols of any significant concentration with a carbon number less than 24 or with a carbon number greater than 32.

The free fatty alcohols may include one or more alcohols selected from the group consisting of 1-octacosanol, 1-triacontanol, 1-tetracosanol, 1-dotriacontanol and 1-hexacosanol.

In one embodiment of the invention, the free fatty alcohols include all of 1- octacosanol, 1-triacontanol, 1-tetracosanol, 1-dotriacontanol and 1-hexacosanol. These fatty alcohols may be present in a descending concentration in the order listed hereinbefore, with 1- octacosanol thus being present in the highest concentration and 1-hexacosanol being present in the lowest concentration.

The at least one phospholipid, i.e. the one or more phospholipids, may be present in the lipid portion or lipid composition in a concentration of at least about 0.5% by mass or at least about 0.6% by mass or at least about 0.7% by mass, e.g. about 0.86% by mass. Typically, the phospholipid or phospholipids is/are present in the lipid portion in a concentration of less than about 2% by mass or less than about 1.2% by mass or less than about 1.1% by mass or less than about 1.0% by mass.

The phospholipid(s) may be in the form of one or more commercially available phospholipids, e.g. lecithin, added to a base lipid composition comprising the fatty acids, free fatty alcohols, hydrocarbons and wax esters. The lecithin may be lecithin obtained from soybeans, rapeseed, sunflower, chicken eggs, bovine milk or fish eggs, preferably from soy beans.

Advantageously, in addition to its nutrient value, the one or more phospholipids act as an emulsifier in the nutrient additive.

The fatty acids may be present in the lipid portion or lipid composition in a concentration of at least about 30% or at least about 35% or at least about 40% by mass, e.g. about 44% by mass.

Typically, the fatty acids are present in the lipid portion or lipid composition in a concentration of less than about 60% by mass or less than about 55% by mass or less than about 50% by mass.

The fatty acids may include fatty acids with a carbon number ranging between about 14 and about 28. In other words, the fatty acids may include C14 - C28 fatty acids.

Typically, there are no fatty acids of any significant concentration with a carbon number less than 14 or with a carbon number greater than 28.

The fatty acids may include palmitic acid and lignoceric acid. The palmitic acid and the lignoceric acid in combination may make up more than about 50% by mass or more than about 60% by mass or more than about 70% by mass, e.g. about 75% by mass to about 85% by mass of the fatty acids present in the lipid portion or lipid composition. The fatty acids may include additionally one or more fatty acids selected from the group consisting of tetradecanoic acid, pentadecanoic acid, stearic acid, oleic acid, vaccenic acid, linoleic acid, alpha-linolenic acid, arachidic acid, dihomo-gamma-linolenic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, behenic acid, erucic acid, cerotic acid and montanic acid.

The oleic acid may be present in the lipid portion or lipid composition in a concentration of between about 2% by mass and about 12% by mass, or between about 4% by mass and about 10% by mass, or between about 5% by mass and about 9% by mass, e.g. about 6.9% by mass.

Each of the tetradecanoic acid, pentadecanoic acid, stearic acid, vaccenic acid, linoleic acid, alpha-linolenic acid, arachidic acid, dihomo-gamma-linolenic acid, eicosatrienoic acid, eicosatetraenoic acid, eicosapentaenoic acid, behenic acid, erucic acid, cerotic acid and montanic acid, when present in the lipid portion or lipid composition, may be present in a concentration of less than about 4% by mass or less than about 3.5% by mass or less than about 3% by mass. Typically, each of these fatty acids, when present in the lipid portion or lipid composition, are present in a concentration of at least about 0.01% by mass or 0.02% by mass or 0.03% by mass.

The hydrocarbons may be present in the lipid portion or lipid composition in a concentration of at least about 8% by mass or at least about 10% by mass or at least about 12% by mass, e.g. about 14% by mass.

Typically, the hydrocarbons are present in the lipid portion or lipid composition in a concentration of less than about 20% by mass or less than about 18% by mass or less than about 16% by mass.

The hydrocarbons may include hydrocarbons with a carbon number ranging between about 25 and about 35. In other words, the hydrocarbons may include C25 - C35 hydrocarbons. Typically, there are no hydrocarbons of any significant concentration with a carbon number less than 25 or with a carbon number greater than 35.

The hydrocarbons may include one or more alkanes selected from the group consisting of 25:0, 27:0, 29:0, 31:0, 33:0 and 35:0.

The hydrocarbons may include one or more alkenes selected from the group consisting of 33:1 and 35:1.

The wax esters, i.e. esters of a fatty acid and a fatty alcohol, may be present in the lipid portion or in the lipid composition in a concentration of at least about 18% by mass or at least about 20% by mass or at least about 22% by mass, e.g. about 26% by mass.

Typically, the wax esters are present in the lipid portion or in the lipid composition in a concentration of less than about 34% by mass or less than about 32% by mass or less than about 30% by mass.

The wax esters may include wax esters with a carbon number ranging between about 34 and about 56. In other words, the wax esters may include C34 - C56 wax esters, or C36 - C54 wax esters. Typically, these wax esters are even carbon numbered, with principle alkyl esters being C40, C42, C44, C46 and C48. Some uneven numbered esters may also be present. The wax esters may predominantly be monoesters, with smaller amounts of diesters and triesters and polyesters also being present. The wax esters may also include mono, di and triglycerides.

Typically, there are no wax esters of any significant concentration with a carbon number less than 34 or with a carbon number greater than 56.

The lipid portion may include sterols and/or amino acids and/or glycerol. All of these, if present, may be present in a concentration of less than about 0.5% by mass, e.g. in the range of 0.1% - 0.5% by mass. The sterols may be attached to the fatty acids or to the free fatty alcohols. The sterols may include one or more of cholesterol, campesterol, campestanol, d5,24 stigmastadienol, d7 stigmasterol and d7 avenasterol.

The amino acids, when present, may include phenylalanine.

In the absence of a diluent, the lipid composition, and hence the nutrient additive, may be in the form of a hydrophobic powder.

The diluent may be water. When the diluent is water, the nutrient additive may be in the form of an oil-in-water emulsion.

In one embodiment of the invention, the nutrient additive is in the form of an aqueous cream. In another embodiment of the invention, the nutrient additive is in the form of a liquid.

The diluent, when water, may be present in a concentration of between about 80% and about 99% by mass, or between about 85% and about 99% by mass, or between about 90% and about 96% by mass, e.g. about 94% by mass, of the nutrient additive.

The nutrient additive may include one or more antioxidants.

In one embodiment of the invention, the antioxidant(s) include(s) potassium metabisulfite and/or ascorbic acid (vitamin C) and/or ascorbyl palmitate. Potassium metabisulfite advantageously scavenges oxygen, whereas ascorbic acid advantageously protects fats on the aqueous side of micelles with ascorbyl palmitate protecting micelles on the lipid side thereof. Typically, the minimum amount of antioxidant necessary to prevent the nutrient additive from going rancid is used. Thus, for example, the potassium metabisulfite concentration in the nutrient additive may be between about 20 parts per million and about 80 parts per million, the ascorbic acid concentration in the nutrient additive may be between about 0,04% by mass and about 0,1% by mass and the ascorbyl palmitate concentration in the nutrient additive may be between about 0,04% by mass and about 0,1% by mass. The nutrient additive may include vitamin E to inhibit rancidity. The vitamin E, when present, may be present in a concentration of between about 0,003% by mass and about 0,009% by mass.

The invention extends to the use of the nutrient additive as hereinbefore described in a process in which a fungus or microbe is used or grown, to promote yield of a desired product or to promote growth of the fungus or microbe.

The fungus may be a mushroom and the nutrient additive may thus be used to promote growth of the mushroom.

Instead, the fungus may be a yeast or a mould and the nutrient additive may thus be used to promote growth of the yeast or mould, or to promote the yield of a desired product produced by the yeast or mould.

In one embodiment of the invention, the nutrient additive is used to promote production of alcohol by a yeast.

According to another aspect of the invention, there is provided a microbial fermentation process for producing alcohol, the process including adding a lipid composition, or a nutrient additive which includes a lipid portion, to a fermentor in which a yeast is converting sugar from a sugar-containing raw material to an alcohol, the lipid composition, or the lipid portion of the nutrient additive, as the case may be, including free or saponified fatty acids, free fatty alcohols, wax esters and hydrocarbons, the free fatty alcohols making up at least 5% by mass of the lipid composition or at least 5% by mass of the lipid portion of the nutrient additive.

The nutrient additive may include a diluent. The diluent may be as hereinbefore described.

The lipid composition may be the same as, or may correspond to, the lipid portion hereinbefore described. The lipid portion may be a lipid portion as hereinbefore described.

The nutrient additive may be a nutrient additive as hereinbefore described.

The alcohol is typically ethanol.

The sugar-containing raw material may be molasses.

The yeast may be Saccharomyces cerevisiae.

The invention is now described with reference to the following examples, tables, experiments and drawings. In the drawings,

Figure 1 shows graphs of specific gravity (SG) and alcohol % volume/volume results over time for a control fermentor with no nutrient additive and a fermentor with the nutrient additive of the Example added, for Angel Thermal Tolerance Alcohol Active Dry Yeast, at 38°C;

Figure 2 shows graphs of alcohol % volume/volume results over time for a control fermentor with no nutrient additive and a fermentor with the nutrient additive of the Example added, for Angel Thermal Tolerance Alcohol Active Dry Yeast, at 35°C;

Figure 3 shows graphs of specific gravity (SG) and alcohol % volume/volume results over time for a control fermentor with no nutrient additive and for fermentors with the nutrient additive of the Example added, for Angel Super Alcohol Dry Yeast, at 35°C;

Figure 4 shows graphs of specific gravity (SG) and alcohol % volume/volume results over time for a control fermentor with no nutrient additive and fermentors with the nutrient additive of the Example added, for Angel Thermal Tolerance Alcohol Active Dry Yeast, at 35°C;

Figure 5 shows graphs of specific gravity (SG) and alcohol % volume/volume results over time for a control fermentor with no nutrient additive and fermentors with the nutrient additive of the Example added, for Angel Super Alcohol Dry Yeast, at 35°C;

Figure 6 shows graphs of specific gravity (SG) and alcohol % volume/volume results over time for a control fermentor with no nutrient additive and fermentors with the nutrient additive of the Example added, for Angel Super Alcohol Dry Yeast, at 25°C; Figure 7 shows graphs of specific gravity (SG) and alcohol % volume/volume results over time for a control fermentor with no nutrient additive and for fermentors with the nutrient additive of the Example added, for Angel Thermal Tolerance Alcohol Active Dry Yeast, at 35°C;

Figure 8 shows yeast growth results in g/l dry yeast for Angel Thermal Tolerance Alcohol Active Dry Yeast for a control fermentor with no nutrient additive and for a fermentor with the nutrient additive of the Example added;

Figure 9 shows yeast growth results in g/l dry yeast for Angel Thermal Tolerance Alcohol Active Dry Yeast for a control fermentor with no nutrient additive and for a fermentor with the nutrient additive of the Example added initially and then at a rate of O.Olg of additive/litre of broth every 18 hours;

Figure 10 shows graphs of alcohol % volume/volume results overtime for a 25-litre stirred control fermentor with no nutrient additive and a 25 litre stirred fermentor with the nutrient additive of the Example added, for Angel Thermal Tolerance Alcohol Active Dry Yeast, at 35°C; and

Figure 11 shows graphs of alcohol % volume/volume results over time for a 25 litre stirred control fermentor with no nutrient additive and a 25 litre stirred fermentor with the nutrient additive of the Example added, for Angel Thermal Tolerance Alcohol Active Dry Yeast, at 35°C, where the Angel Thermal Tolerance Alcohol Active Dry Yeast in the fermentor with the nutrient additive of the Example added was first grown for 12 hours in an incubator with aeration in the presence of the nutrient additive.

In all the drawings, "Lipid" and "Fermentanol" refer to the nutrient additive of the following Example.

Example

An exemplary nutrient additive in accordance with the invention was prepared. The nutrient additive was in the form of a liquid composition that included a base lipid portion and water as a diluent, forming a water-in-oil emulsion. The base lipid portion made up about 6.86% by mass of the nutrient additive, with the balance being predominantly water and small amounts of other ingredients or additives, including added phospholipids (equal to about 1% by mass of the base lipid portion), added potassium metabisulfite, added ascorbic acid, added ascorbyl palmitate, sterols and added vitamin E.

Table 1 provides the concentrations of all significant ingredients of the base lipid portion, including phospholipids, and hence of the nutrient additive, but for the nutrient additive Table 1 does not show the amount of water or any other added ingredients, such as added antioxidants.

Table 1

The nutrient additive of Table 1 was adjusted with the addition of antioxidants and a phospholipid in the form of lecithin, with the lecithin making up about 1.5% by mass of the final or exemplified nutrient additive. The exemplified nutrient additive was demonstrated to be stable for at least three months if stored in an airtight container. When added to water at a temperature above 35°C the exemplified nutrient additive dispersed rapidly and mixed into the water leaving no visible residues. From the analysis in Table 1 it can be seen that the exemplified nutrient additive includes a significant concentration of wax esters of fatty alcohols and fatty acids corresponding to the fatty alcohols and fatty acids listed. A smaller percentage of free fatty acids, typically in the form of potassium salts or soaps, and free fatty alcohols, as well as added phospholipids, are present and emulsify the nutrient additive to produce a unique suspension of wax esters, fatty acids, fatty alcohols, alkanes, some alkenes and lipids in general which are in a fine micellular suspension that is easily dispersed into growth media for rapid yeast absorption.

The exemplified nutrient additive was used in a number of experiments, as described hereinafter.

Fermentation experiments

Flask culture experiments were performed in triplicate in two litre Schott bottles with agitation as recommended by Azhar et al., 2017 (Azhar S.H.M., Abdulla R., Jambo S.A., Marbawi H., Gansau J.A., Faik A.A.M., Rodrigues K.F., 2017, Yeasts in sustainable bioethanol production: A review, Biochemistry and Biophysics Reports: 10, Pg 52-61).

Standard industrial molasses with a sugar content of 43% was used to adjust the specific gravity (SG) of a broth, as measured with an ANA France wine hydrometer, to a target value in each experiment. Yeast Available Nitrogen (YAN) was adjusted through the addition of 0,5 g/l diammonium phosphate (Merck). Zinc gluconate was added at a rate of 70mg/l.

Yeasts used were Angel Yeast (Angel Yeast Co., Ltd, 168 Chengdon Avenue, Yichang, Hubei, 443003, Peoples Republic of China) varieties, namely Angel Super Alcohol Dry Yeast (ASADY) (batch: 08/09/2016) and Angel Thermal Tolerance Alcohol Active Dry Yeast (ATTAADY) (batch: 08/09/2016). Angel Thermal Tolerance Alcohol Active Dry Yeast was added at a rate of 0.3g yeast/I of broth directly to fermentation flasks as per the instructions provided with the product. Angel Super Alcohol Dry Yeast was first activated by mixing yeast and water in a ratio of 1 g yeast to 20ml of 5% sugar water and incubated at 30°C for 20 minutes. The activated yeast was then dosed at a rate of 0,3g of initial dry weight yeast/I of broth.

Two concentrations of the nutrient additive were evaluated - either 0,007 grams per litre of broth or 0,014 grams per litre of broth. Fermentation experiments were conducted as follows:

Experiment 1

Angel Thermal Tolerance Alcohol Active Dry Yeast

Temp °C 38

Initial SG 1125

Duration h 48

Control No added nutrient additive

Nutrient additive concentration 0.007g/l

The results are depicted in Figure 1.

Experiment 2

Angel Thermal Tolerance Alcohol Active Dry Yeast

Temp °C 35

Initial SG 1125

Duration 48

Control No added nutrient additive

Nutrient additive concentration 0.007g/l

The results are depicted in Figure 2.

Experiment 3

Angel Super Alcohol Dry Yeast

Temp °C 35

Initial SG 1125

Duration 48

Control No added nutrient additive

Nutrient additive - fermentation 1 0.007g/l

Nutrient additive - fermentation 2 0.014g/l

The results are depicted in Figure 3. Experiment 4

Angel Super Alcohol Dry Yeast compared to Angel Thermal Tolerance Alcohol Active Dry Yeast

Temp °C 35

Initial SG 1100

Duration 48

Control for ATTAADY 0.007g/l of nutrient additive

ATTAADY fermentation 0.014g/l of nutrient additive

Control for ASADY No added nutrient additive

ASADY fermentation 1 0.007g/l of nutrient additive

ASADY fermentation 2 0.014g/l of nutrient additive

The results are depicted in Figure 4.

Experiment 5

Angel Super Alcohol Dry Yeast

Temp °C 25

Initial SG 1125

Duration hours 48

Control No added nutrient additive

Nutrient additive - fermentation 1 0.007g/l

Nutrient additive - fermentation 2 0.014g/l

The results are depicted in Figure 5. In Figure 5, the series for lipid 0,007g/l SG and the series for 0,014g/l SG have the same finish point of SG 1,038 and their graphs are hence indistinguishable; however, the alcohol percentage achieved was lower for the higher lipid addition due to excessive biomass growth. Experiment 6

Angel Thermal Tolerance Alcohol Active Dry Yeast

Duration 48

Temp °C 35

Initial SG 1100

Control No added nutrient additive

Nutrient additive - fermentation 1 0.007g/l

Nutrient additive - fermentation 2 0.014g/l

The results are depicted in Figure 6.

Fermentation was measured over the duration of fermentations using an ANA France wine hydrometer, and final alcohol determinations were performed by distillation and densitometric ethanol determination. Two concentrations of the nutrient additive were evaluated - either 0,014 grams per litre of fermentation broth or 0,007 grams per litre of fermentation broth. Initial flask experiments in Schott bottles showed that the dosage of 0,007g/l of the nutrient additive had the effect of raising ethanol concentrations in final wash upwards by a significant percentage. However, at 35°C it was noted that yeast produced very high yields of biomass when the nutrient additive was doubled to 0,014g/l, which had the effect of lowering ethanol yields compared to the control despite similar drops in specific gravity.

Yeast growth experiments

Yeast growth experiments were done as flask culture experiments performed in triplicate in two litre Schott bottles with agitation as recommended by Azhar et al., 2017, with agitation at 150rpm and incubation at 25°C in a controlled climate cabinet. Fermentors were aerated at 2 litres per minute. Sugar was added at a rate of lOg per litre fermentable sugar at 0 hrs, 24 hrs and 48 hrs. Upon completion of the growth experiments, the flasks were placed at 4°C for 12 hours to allow yeast to settle. A supernatant was withdrawn, and yeast sludge was dried at 50°C until samples attained constant weight. Samples were weighed accurately to three decimal places. Experiment 7

Angel Thermal Tolerance Alcohol Active Dry Yeast

Duration 32 h

Molasses concentration 10 g/l

Control No added nutrient additive

Nutrient additive concentration 0.014g/l

The results are depicted in Figure 7. In Figure 7, the specific gravity for the control and the specific gravity for the 0,007 g/l lipid additive (i.e. the nutrient additive) have the same finish point of SG 1,040 and their graphs are hence indistinguishable; however, these do have a significant difference in alcohol, with the control achieving an alcohol yield of 7,4% by volume and the 0,007g/l lipid additive an alcohol yield of 8,7%.

Experiment 8

Angel Thermal Tolerance Alcohol Active Dry Yeast

Duration 72h

Additions of nutrient additive of O.Olg

and fermentable sugars of 10g/l 3, at 0 hrs, 24 hrs and 48 hrs

No added nutrient additive, sugar added at 10 g/l at Control 0 hrs, 24 hrs and 48 hrs

Initial nutrient additive concentration 0.014 g/l The results are depicted in Figure 8.

The addition of the nutrient additive to the growth flasks inoculated with Angel Thermal Tolerance Alcohol Active Dry Yeast produced an increase in yeast dry weight yield of 73% over the duration of 48 hours with sequential addition of sugars and an increase of yield of 51% over 32 hours in a batch system with only one addition of sugar. Larger Scale Stirred Batch Experiment 1

25 litre carboy reactors or fermentors were filled with media prepared from standard industrial molasses with a sugar content of 47% mixed with water and adjusted to a specific gravity of 1.115, as measured with an ANA France wine hydrometer. Yeast Available Nitrogen (YAN) was adjusted through the addition of 0,5 g/l diammonium phosphate (Merck). Zinc gluconate was added at a rate of 70mg/l. Reactors were agitated as recommended by Azhar et al., 2017, with circulating pumps.

Yeasts tested were Angel Thermal Tolerance Alcohol Active Dry Yeast (ATTAADY) (batch: 08/09/2016) as this yeast is the most commonly used in the Southern African region. Angel Thermal Tolerance Alcohol Active Dry Yeast was added at a rate of O.Bg of dry yeast/I of broth directly to the carboy reactors as per the instructions provided with the product.

A control with no nutrient additive was compared to a trial with 0,014 grams of the exemplified nutrient additive per litre of media or broth.

Experiments were conducted in a controlled environment cabinet at 35°C.

The larger 25 litre glass carboy experiment allowed accurate measurements of alcohol via distillation over the entire duration of the experiment. The results are depicted in Figure 10.

From Figure 10 it is evident that the nutrient additive had the effect of raising alcohol percentages achieved, compared to a control which did not have the benefit of the nutrient additive. More significantly, and very importantly, however, is the fact that the fermentor with the nutrient additive achieved a higher percentage ethanol than the control 14 hours earlier.

Larger Scale Stirred Batch Experiment 2

20-litre reactors or fermentors were filled with media prepared from standard industrial molasses with a sugar content of 47% mixed with water and adjusted to a specific gravity of 1.115, as measured with an ANA France wine hydrometer. Yeast Available Nitrogen (YAN) was adjusted through the addition of 0,5 g/l diammonium phosphate (Merck). Zinc gluconate was added at a rate of 70mg/l. Reactors were agitated as recommended by Azhar et al., 2017, with circulating pumps.

Yeasts tested were Angel Thermal Tolerance Alcohol Active Dry Yeast (ATTAADY) (batch: 08/09/2016) as this yeast is the most commonly used in the Southern African region.

Experiments were conducted in a controlled environment cabinet at 35°C.

Two yeast samples were prepared for inoculation - one as per standard manufacturer's instructions, and one as per standard manufacturer's instructions with the addition of the exemplified nutrient additive (0,014 grams of nutrient additive per litre of media). This stage was a 12-hour growth stage with aeration in an incubator.

The yeast grown without the nutrient additive (i.e. a control) was pitched into a bioreactor (20-litre reactor) with standard media and no nutrient additive (i.e. a control).

The yeast grown with the nutrient additive was pitched into a bioreactor (20-litre reactor) with nutrient additive added (0,014 grams of nutrient additive per litre of media or broth).

As can be seen from Figure 11, the effect of growing the yeast in the presence of the nutrient additive, and then fermenting a broth also in the presence of the nutrient additive, was to bring the growth and fermentation cycle of the reactor with the nutrient additive back significantly, resulting in higher alcohol concentrations quickly. This protects against bacterial growth and increases the final alcohol levels achieved significantly over the control.

From a commercial perspective this is very important as it gives a higher yield of alcohol, faster, and most likely cleaner as well. The nutrient additive delivered an alcohol level equivalent to the control (at 48 hours) at just 32 hours. This 16-hour saving is very significant when one takes into consideration how much harder it allows one to work the significant capital tied up in an industrial scale fermentor tank infrastructure. In summary, the addition of the nutrient additive in the yeast propagation cycle, and subsequent dosing into a main fermentor allows a more rapid, more time and resource efficient fermentation that gives a higher yield per ton of molasses.

The data obtained generally shows that the nutrient additive, as exemplified, has the effect of increasing yeast growth, yeast fermentation and alcohol tolerance, which resulted in higher sugar utilisation, better ethanol yields, and higher biomass yields in growth experiments.

If one considers the fact that, in Experiment 8, the addition of 0,044g/l of the nutrient additive over time had the effect of allowing O.Bg/l inoculated dry mass of yeast to multiply to 3.115g/l of yeast dry mass in the control versus the 5.405g/l dry mass with the nutrient additive added, it is evident that the increase in mass is clearly linked to the addition of the nutrient additive. Yamada et al 2005 (Yamada EA, Sgarbieri VC. 2005, Yeast (Saccharomyces cerevisiae) protein concentrate: preparation, chemical composition, and nutritional and functional properties. J Agric Food Chem. May 18 53(10):3931-6 p.3932 table 1), list the lipid composition of dry fermentation yeast at approximately 0,5%. In Experiment 8 the addition of lipid preparation was about 0,77% of the mass of yeast produced. Hence it is safe to conclude that the nutrient additive is added in surplus to the actual requirements of the yeast, meaning that yeast may be able to store surplus lipid and thus attain greater health during fermentation cycles.

From the perspective of yeast growth, numerous papers note that the addition of lipids (fatty acids) can reduce/eliminate the need for aeration in yeast growth, allowing yeast to multiply in mass even during initial stages of fermentation (e.g. Duan et al. 2015 (Duan L.L., Shi Y., Jiang R., Yang Q., Want Y.Q., Liu P.T., Duna C.Q., Yan G.L., 2015, Effects of Adding Unsaturated Fatty Acids on Fatty Acid Composition of Saccharomyces cerevisiae and Major Volatile Compounds in Wine, SA Journal of Viticulture and Oenology, Vol 36, No.2) and Moonjal et al. 2002 (Moonjal N., Verstrepen K.J., Delvaux F.R., Derdelinckx G., Verachtert H., 2002, The Effects of Linoleic Acid Supplementation of Cropped Yeast on its Subsequent Fermentation Performance and Acetate Ester Synthesis, Journal of the Institute of Brewing, Vol 108(2) pg 227-235)). This provides for a more rapid fermentation and improves the economics of the operation as well without needing to have aeration of fermentors and all the costs and risks associated with this common practice.

The difference in ethanol concentration in experiments with the nutrient additive added versus the controls can be attributed to factors such as that lipid additives improve membrane integrity. The higher membrane health of yeast allowed them to produce and tolerate higher ethanol concentrations both by being able to tolerate the higher ethanol, and through their improved tolerance initially of the higher osmotic stresses in the cell.

From an industrial perspective, there are a number of major considerations that an ethanol plant operations team must bear in mind. Generally, the amount of alcohol produced per ton of molasses, the quality or the alcohol, and the speed with which this can be achieved are critically important. Propagator reactors produce yeast for inoculation and are aerated. These are a common source of contamination, as bacteria which contaminate the propagated yeast then enter the industrial fermentor and can produce off flavors, contaminant alcohols and organic acids which reduce ethanol fermentation speeds, yields and quality. Fermentor vessels tend to be large and expensive and hence the shorter the duration of fermentation, the more the plants operation can be optimized.

The nutrient additive, as exemplified, is unusual in that it includes free fatty alcohols, in combination with fatty acids and hydrocarbons. The nutrient additive, as exemplified, has the potential effect of reducing or eliminating the need for aeration in propagation, thus reducing the chances of contamination with ethanol quality and quantity reducing bacteria. The nutrient additive, as exemplified also has the effect of increasing biomass yield, and when added to the fermentor, it has the effect of enhancing the speed of fermentation. From a plant management perspective, the benefits are multiple. The nutrient additive, as exemplified when used in propagation reduces or potentially eliminates the need for aeration, and reduces the risk of contamination. This lowers the complexity of this stage as complicated air blower supply systems are not needed. In fermentation, higher ethanol yields are achieved. This means that distillation systems operate more efficiently and can process more volume of product. The batch fermentation time is reduced, which means the turnaround time on batch fermentations can be reduced, so that the increased efficiency of the distillation coincides with greater availability of product to distill. The reduced presence of contaminants in the distillation means that the quality of alcohol improves, and the speed with which it can be produced also are improved as complicated re-distillation to remove contaminants is avoided. Contaminant alcohols and congeners are a logistical nightmare to dispose of as well, hence the less of them the better.