catalyst to be deposited on the wires allowing for vertically aligned carbon nanotubes to be formed.

1. The carbon source, acetylene, was introduced into the reactor using argon as a carrier gas. The flowrate of acetylene was 406.3 cm ³ /min and argon was 332.1 cm ³ /min. The gas mixture was fed into the reactor for 15 min. the reactor was then purged of reactants for 15 min using argon at a flowrate 647.3 cm ³ /min.

2. The furnace and heating plate were then turned off, and the reactor was allowed to cool slowly. The wires were removed from the reactor the next day.

Scanning electron microscope (SEM) analysis of the coating showed that there were carbon microfibers and carbon nanotube (CNT) bundles present on the surface of the coating. These were removed by washing the wires in distilled water in a rotary shaker at 100 rpm for 4 hours. Figure 1 shows that after washing, the majority of the microfibers on the surface of the coating were removed however the coating itself remains intact except for some fissures and voids.

Figure 1. shows SEM image of carbon nanotube coating after washing, A; surface of coating and void (bar denotes100 m), B; vertically aligned carbon nanotube coating (bar denotes 10 pm).

10 cm sections of the CNT coated wires were then placed in a culture medium with fermenting yeast cells. The culture medium was a wort analogue at 10.7°P composed of malt extract which was sterilized and filtered to remove solids. 150 ml of culture medium was placed in 250 ml conical flasks and placed in a rotary shaker at 100 rpm and 23°C. While yeast cells were found on the surface of the coating as soon as 3 hours into the fermentation process, the amount was negligible and the cells were not well adhered to the surface. The earliest evidence of yeast cell immobilization in significant amounts was found on wires which had been exposed to the fermenting medium for 53 hours as -10-

sho n in Figure 2. In this case cells were found to be immobilized in a fissure that ranged from 40-70 m wide but was more than 200 μιτι long. The cell density in the fissure was very high as yeast cells were attached not only to the surface of the wire at the bottom of the fissure, but were also attached to the vertically aligned carbon nanotubes that formed the walls of the fissure. The morphology of the cells was not altered significantly however the cells in the fissure seemed to have a great affinity for other yeast cells and formed large clusters of cells adhering to the exposed surface of the stainless steel in between the hills of CNTs.

Figure 2. shows yeast cells immobilized in fissure. A; yeast cells in fissure showing lack of yeast cells on the surface of the coating (bar denotes 10 μητι), B; yeast cells in fissure showing interactions between yeast cells (bar denotes 1 pm).

1.5 Cell immobilization

Yeast cell immobilization was performed with CNTs while a control experiment was setup with yeast cells in the absence of CNTs. A loopful of inoculum was added to an Erlenmeyer flask containing 100 ml of sterilized yeast extract medium and incubated in a shaker at 1.10 rpm, 30 °C for 24 hours without adding more oxygen into the broth (rubber cork used to seal the flask to prevent air uptake during the study). After 24 hours the yeast cells were used for immobilization studies. 30 ml (7.04x10 ⁶ CFU/ml) of yeast broth was added to 250 ml of yeast extract medium. The broth was incubated in a shaking incubator at 30 °C [13]. Several factors that have an effect on immobilization were investigated and these included incubator agitation speed, pH of broth, immobilization temperature, CNTs concentration, calcium ions concentration and presence of glucose. The CNTs provided a surface for yeast cells to sorb onto through either the process of adsorption and/or absorption.

1.6 Analytical methods -11-

Yeast flocculation was analyzed using two methods: a qualitative process to determine the quality of the floes produced and a quantitative process to measure the flocculation weight. These two methods enabled the optimization of the parameters that affected the flocculation. The first method (qualitative in nature) involved estimating flocculation with the naked eye. This process was used to measure the quality of the floes produced. This involved looking at the sides and at the bottom of the Erlenmeyer flask and expressing flocculation qualitatively as: (-) no flocculation; (+) yeast slightly flocculent (poor); (++) yeast flocculent; and (+++) yeast very flocculent [14,15,16]. The second method involved using a centrifuge to concentrate the floes, recover them and then drying them at 40 °C for 24 hours to determine their dry weight, a quantitative process. The flocculated cells were recovered by a freeze dryer (VirTis, SP Industries) and immobilization was confirmed by JEOL JSM 840A Scanning Electron Microscopy (SEM). The floe weight was then plotted against the variable under investigation to determine the effect of the variable.

2 Results and Discussions

2.1 Immobilization of Yeast Cells

The biocatalysts were viewed under SEM as shown below (Figure 3).

Figure 3 shows SEM micrographs showing brewers' yeast flocculated by carbon nanotubes; (a) x1600, (b) x3300 and (c) x5000.

The micrographs show that the immobilized cells aligned themselves along the CNT length. This phenomenon was observed on all studies with CNTs. In contrast, free cells showed a planar structure (Figure 4).

Figure 4 shows SEM micrographs showing brewers' yeast which flocculated without carbon nanotubes; (a) x1000 and (b) x1700.

Some yeast cells were growing on top of the flocculated cells planar structure (Figure 4b). CNTs increased the flocculation rate of brewers' yeast and the -12-

flocs were more stable than floes produced by free cells. This was observed when recovering the floes for freeze drying.

The observation that CNTs could increase the flocculation rate of yeast cells was thought to be explained by the Bridging Mechanism Theory. CNTs could be considered to be long chain particles which have large surface spikes and these would enable neutralization of surface charge of brewers' yeast cells when there is contact made between the cells and the nanotubes. This would allow the cells to adsorb onto the tubes such that an individual chain can become attached to 2 or more cells thus "bridging" them together. Spike structures accumulate tip-charge, but the energy required to push a spike tip through a repulsion field would be considerably less than that for cell-cell wall contact. The spike may contain a positive tip charge (as in the case with CNTs) to most easily penetrate the negative charge repulsion of the yeast cells [17]. This mechanism is schematically depicted in Figure 5. Also the presence of CNTs seemed to have increased the water contact angle leading to an increase in CSH which in turn initiated flocculation. It was demonstrated that a relation existed between cell division arrest, the increase of Cell Surface Hydrophobicity (CSH) and initiation of flocculence during fermentation [18,19]. A high level of CSH may facilitate cell-cell contact in an aqueous medium resulting in more specific lectin-carbohydrate interactions [17].

Figure 5 shows the use of spike-structures to overcome electrostatic repulsion and ionic displacement.

Factors that affect flocculation of yeast cells in the presence of CNTs were investigated to see their effect.

2.1.1 Effect of Agitation Speed

An analysis of the effect of agitation speed on the immobilization of brewers' yeast was carried out by changing the speed from 0 to 200 revolutions per - 1 3-

minute (rpm). There was poor flocculation (.+) observed after 4 days for 0, 50, 150 and 200 rpm whilst at 110 rpm good flocculation (++) was observed (Table 1).

Table 1 : Analysis of flocculation for different agitation speeds

Agitation speed (rpm) 0 50 110 150 200

Floe quality + + ++ + +

The absence of flocculation at higher agitation speeds of 150 and 200 rpm maybe due to the disintegration of the floes as they are formed and this is attributed to surface damage or disruption of individual cells. While an increase in collisions may help to grow the floes there is a limiting agitation speed beyond which surface erosion or floe fracture sets in, which limits the stable floe to a certain optimum size [21] (Figure 6).

Figure 6 shows the effect of agitation speed on initial rate of flocculation of Saccharomyces cerevisiae S646-1B (from Stratford and Wilson, 1990).

An increase in agitation intensity should lead to a decrease in floe size with gentle agitation giving large floes while vigorous agitation should give smaller, denser floes that settle more slowly giving a more compact sediment [4]. However, this trend was not observed during the studies as there were no floes observed at lower or higher agitation speeds. From Figure 6 it can be seen that flocculation is observed between 65 and 115 rpm and this may help to explain the absence of flocculation at speeds of 50 rpm and below or 150 rpm and above.

2.12 Effect of pH

The second factor to be investigated was pH. Fermentation occurs in the pH range 3.80 - 5.60 [16], but the study was conducted in the range 1.30 - 6.50. The aim was to analyze the effect of pH on the flocculation of brewers' yeast cells. The results are summarized below (Table 2 and Figure 7). -14-

Table 2: Analysis of flocculation for different pH.

PH <4.00 4.60 5.60 6.00 6.25 6.50 6.65

Floe + ++ +++ + + + quality

Figure 7 shows the effect of changing pH on flocculation

deviations for the graphs were 0.045 for a and 0.026 for b.

Statistical analysis was conducted to see if there was a significant difference between the biocatalysts and free cells graphs as pH was increased. The p value was 0.1163 which was considered not significant as variation among the graphs was not significantly greater than expected by chance. From Figure 7 it is observed that the optimum pH when using CNTs for yeast flocculation is between 5.00 and 5.80 as there is the highest floe weight as compared with the flocculation without using CNTs. The pH range obtained is within the brewing pH range of 3.80 - 5.60 as reported in literature [16,21 ,22,23,24,25]. Yeast flocculation in the absence of CNTs was best in the pH range between 5.90 and 6.10. After pH 6.10 dry floe weight decreased rapidly up to pH 6.25 before increasing further again. The focus was mainly in the brewing pH as the application for the process was in the fermentation pH range.

Using CNTs to flocculate yeast cells had a negative effect beyond pH 5.80. This could be due to the fact that yeast cells reverse their charge above pH 5.80. In aqueous suspensions at the pH values of worts and beers (3.80-5.60), brewers' yeasts migrate to the anode in electrophoresis experiments, thus behaving as negatively charged colloids. At more acid pH values, reversal of the charge may take place [16] and this may help to explain the decrease in flocculation weight at pH below 4.60 and above 5.80 observed during the study. The fact that CNTs were able to flocculate yeast cells within the mentioned pH range showed that the CNTs are positively charged and these -15-

tend to repel the cells when the cells have a positive charge due to a change in pH. From literature, yeast cells should flocculate anywhere between pH 2.00 and 8.00, depending on strains with optimum values between pH of 3.00-6.00. The study showed the same results with flocculation observed between 5.00 and 5.80. At low pH values (2.90 - 4.00) the cells might have been denatured and this resulted in poor flocculation of cells [21 ,26].

2.1.3 Effect of Immobilization Temperature

There is an apparent contradiction in the literature about the effect of temperature on flocculation, some authors noted deflocculation with increasing temperature while others noted an increase in flocculation with increasing temperature. This discrepancy maybe attributed to differences in the response of ale and lager strains [22,23,27] found that flocculation of a lager yeast strain varied between 24.1 % at 5 °C to 66.8% at 25 °C showing an increase in flocculation as temperature was increased. However, there is little or no effect of temperature on flocculation of brewing yeast within the physiological temperature range of 15 - 32 °C [21].

Most brewing strains have an optimum temperature for growth between 30 and 34 °C [21 ,28,29] with viability losses at 30 °C for 3 days during flocculation considered negligible [30]. Yeast autolysis normally occurs at elevated temperatures of between 40 and 60 °C [30,31 , 32,33]. Studies were conducted at two different temperatures (25 and 30 °C) to investigate the effect of temperature on flocculation.

The change in temperature was analyzed to determine its effect on flocculation and the data is presented in Table 3.

Table 3: Analysis of flocculation at 25 and 30 °C. -16-

Temperature a

°C Floe Floe Standard Floe Floe Standard quality weight deviation quality weight deviation

(g) (g)

25 + 0.043 0.013 + 0.124 0.010 30 ++ 0.143 0.007 ++ 0.123 0.005

From the two temperatures investigated, the ideal temperature should be close to 30 °C to get the best results when using CNTs to aid in flocculation; 0.143±0.007g at 30 °C against 0.043±0.013 g at 25 °C with a difference of 0.100 g. In the absence of flocculation (b), flocculation rates are almost the same 0.124±0.010g at 25 °C against 0.123±0.005 g at 30 °C a difference of 0.001 g. Oztop et al. [26] found the optimum temperature for immobilization to be 25 °C when they immobilized yeast cells on a chitosan film (Figure 8).

Figure 8 shows the effect of temperature on immobilization (from Oztop et al., 2002).

Hsu et al. [34] observed an increase in flocculation with an increase in temperature from 5 - 45 °C. This was in line with the observations in the study showing an increase in flocculation with an increase in temperature. Jin et al.

[22,23] found that flocculation of a lager yeast strain varied between 24.1 % at 5 °C to 66.8% at 25 °C further supporting the observations of this study.

2.1.4 Effect of CNTs concentration

Different concentrations of CNTs were added to the broth containing yeast cells and culturing media to investigate their effect. The concentration was varied from 0 - 72 μg ml ^"1 . Ghafari et al. [35] investigated bacteria ciliated protozoa and used concentration between 0 - 17.2 μg ml ^"1 .

Changing the concentration of CNTs did have an effect on flocculation of brewers' yeast in the range investigated. A table and graph of floe weight -17-

observed against CNT concentration are presented below (Table 4 and Figure 9).

Table 4: Analysis of flocculation for different concentrations of CNTs.

Study a b c d e f g h CNT] O00 17.86 26.79 35.71 44.64 53.57 62.50 71.83

(Mg/ml)

Flocculation + ++ ++ ++ +++ +++ +++ ++ quality

Figure 9 shows the effect of CNTs concentration on brewers' yeast flocculation. The standard deviations for the graphs were 0.008 for a, 0.007 for b, 0.004 for c, 0.005 for d, 0.012 for e, 0.008 for f, 0.008 for g and 0.005 for h.

There was a general increase in floe weight with an increase in CNT concentration and the graph peaked at 53.57 μg/ml. Additional CNTs to increase their concentration above 53.57 μg/ml had negative effects on flocculation as the floe weight observed started to decrease. From 0 to 35.71 μg/ml there was a negligible gain in floe weight observed, +0.013g gained. Increasing the CNT concentration to 53.57 μg/ml gave a gain of +0.040 g as compared without using CNTs.

Studies were also conducted with CNTs in the form of bulky paper or sheet-like form resulting in poor flocculation (ranked -) as compared to CNTs in powder form. There were no floes which were recovered. These results showed the importance of surface area to volume ratio of CNTs when used for aiding flocculation of yeast cells.

2.1.5 Calcium ions concentration

Calcium ions are important in yeast cell flocculation and as such their effect on flocculation were studied [21 ,36,37]. Calcium ion concentration was varied - 18-

from 0 - 9.55 mM and introduced into the broth as anhydrous calcium chloride

(CaCI ₂ -2H ₂ 0). The calcium chloride weight was varied in steps of 0.05 grams to yield 7 experiments (a - h). Studies were done at these 7 conditions and the results are presented in Table 5 and Figure 10. The floes observed in the presence of calcium ions were powdery in nature.

Table 5: Effect of calcium ion concentration on flocculation.

Flask a b c d e f g h

Ca ²⁺ ions cone. (mM) 0.00 1.62 2.93 4.11 5.49 6.85 8.25 9.55

Flocculation quality + + + ++ +++ + + +++

Figure 10 shows the effect of Ca ions on brewers' yeast flocculation. The standard deviations for the graphs were 0.017 for a, 0.010 for b, 0.027 for c,

0.053 for d, 0.071 for e, 0.036 for f, 0.043 for g and 0.072 for h.

The best flocculation quality was observed in (d), (e) and (h) but when considering floe weight it was observed in (e) and (h). Figure 10 showed that

5.49 mM of Ca ^z+ ions (experiment e) gave the optimum conditions for flocculation when considering floe weight. Increasing the concentration to 9.55 mM yielded almost the same floe weight as at 5.49 mM. This data enables the optimisation of the concentration of calcium ions required. The presence of

Ca ²⁺ ions reduced flocculation than results obtained in the absence of the Ca ²⁺ ions.

In the study it was observed that Ca ²⁺ ions had a negative effect on flocculation in the presence of CNTs. This may be due to the repulsive forces between the

Ca ²⁺ ions and the positively charged CNTs existing in the same broth solution.

Since flocculation was observed the results are in agreement with literature

[21 ,36]. Taylor and Orton [37] concluded that the presence of calcium ions is required at a very low concentration in order to induce flocculation. For low salt -19-

concentrations (cations other than Ca ions e.g. Mg ²⁺ , Mn ²⁺ ), there was an observed flocculation enhancement, while at high concentrations inhibition of flocculation by the salt is observed [4] which might have been the case during the study.

2.1.6 Presence of glucose

The effect of glucose on flocculation of brewer's yeast cells was the last parameter to be investigated. Generally it was found that maltose and mannose were the most effective inhibitors of flocculation whereas sucrose and glucose were less effective [21]. The study was aimed at observing the effect of the presence of glucose on the flocculation process.

The presence of glucose promoted yeast cell growth and delayed the stationary phase for yeast cells thereby delaying the onset of flocculation. Ethanol was produced from the effect of yeast cells on glucose which decreases the pH of the broth resulting in the delay of flocculation. Studies were conducted to investigate the effect of glucose on flocculation using the optimised parameters. Glucose added is usually between 3 and 5 times the yeast extract weight according to literature [11 ,15,38,39] and the glucose concentration used was 18 mg/ml. The results for the study were plotted as pH and zeta potential against time (Figure 11). Figure 11 shows the effect of the presence of glucose on pH and zeta potential. The standard deviations for the graphs were 1.30 for pH and 6.50 for zeta potential.

The results showed a decrease in pH from 5.53 to 3.84 within a day and a progressive increase thereafter. pH 5.60 was reached after 3.95 days (~ 4 days) where optimum pH for flocculation onset was observed in the previous studies and this showed a delay of 4 days for yeast cell flocculation to begin. The study was repeated with the inclusion of 5.49 mM calcium ion concentration to observe their effect on the zeta potential and the flocculation process (Figure 12). -20-

Figure 12 shows the effect of the presence of glucose and calcium ions on pH and zeta potential. The standard deviations for the graphs were 1.13 for pH and 4.86 for zeta potential.

Figure 12 shows that there was a decrease in pH from 5.59 to 3.76 within a day and a progressive increase. pH 5.60 was reached after 4.90 days (~ 5 days) showing a delay of 5 days for flocculation to be observed.

These results were in agreement with literature which states that glucose inhibits flocculation [4,21,40]. Several authors have indeed found that flocculation is triggered by carbon and/or nitrogen starvation and that addition of these compounds to the growth medium delays flocculation [24,25,41].

2.2 Fermentation Studies

The biocatalysts synthesised in the previous study were assessed for their fermentation capabilities. Two fermentation studies were conducted at 15 °C [42,43,44,45] and 30 °C [43,45,46,47,48,49] with the ethanol content compared with those in literature. The experiments were stopped after 9 days for the study at 15 °C and 3.5 days for the study conducted at 30 °C. The Original Gravity and Final Gravity were measured and reported in literature.

Bekatorou et al. [42] conducted their fermentation studies on hopped, filtered and sterilised wort at 15 °C for 5.7 days using a freeze dried S. cerevisiae strain immobilised on gluten pellets. Inconomopoulou et al. [43] conducted their fermentation studies on glucose at 15 °C for 4.3 days using a freeze dried baker's yeast on DC material. Kopsahelis et al. [44] conducted their fermentation studies on pausterised wort at 15 °C for at least 1 day using an S. cerevisiae strain immobilised on brewer's spent grains. Plessas et al. [45] conducted their fermentation studies on glucose at 15 °C for at least 1 day -21-

using an S. cerevisiae strain immobilised in a starch-gluten-milk matrix usable for food.

Batistote et al. [46] conducted their fermentation studies on maltose or glucose at 30 °C for 2.5 days using 4 yeast strains; brewers' ale strain LBCC A3 and lager strain LBCC L52 and wine strains VIN7 and VIN13 as free cells. Bekatorou et al. [47] conducted their fermentation studies on glucose at 30 °C for at most 2.5 days using an S. cerevisiae strain immobilised on dried figs. Bekers et al. [48] conducted their fermentation studies on a mentioned medium at 30 °C for 2 days using an S. cerevisiae strain immobilised on modified stainless steel wire. Inconomopoulou et al. [43] conducted their fermentation studies on glucose at 30 °C for 3.5 days using a freeze dried baker's yeast on DC material. Plessas et al. [45] conducted their fermentation studies on glucose at 30 °C for at most 2.5 days using an S. cerevisiae strain immobilised in a starch-gluten-milk matrix usable for food. Speers et al. [49] conducted their fermentation studies on industrial wort at 30 °C between 3 and 5 days using an ale brewing yeast strain.

For each study, 2 experiments were conducted; one with the biocatalysts and the other with free cells. Studies were done at least twice with the mean and standard deviation included in the results. Results for the pH, residual sugar and ethanol content were presented in tabular form as mean±standard deviation or as a graph showing standard deviation bars. The free amino nitrogen or the esters were not measured in these studies. The two sugars analysed during the study were maltose and glucose and these were both broken down to ethanol and carbon dioxide according to the following balanced stoichiometric equations.

Maltose

C12H22O11 + H ₂ 0→ 4C ₂ H ₅ OH + AC0 ₂ Equation 1

Glucose

C ₆ H ₁₂ 0 ₆ → 2C ₂ H ₅ OH + 2C0 ₂ Equation 2 -22-

Only 3 parameters considered key in the study were measured in the study. These were (a) pH of the broth (b) the sugar content (glucose and maltose), and (c) alcohol content.

An increase in fermentation temperature reduces the time taken to attenuate the wort. Fermentation rates will increase with temperature by increasing the rate of yeast metabolism giving higher specific fermentation rates [50]. From this analysis the fermentation studies were conducted at the two different temperatures to see if there is an increase in ethanol content as temperature is varied.

2.2.1 pH

The pH of lager brews changes from around 5.70 initially to about 4.40 after 10 days (Figure 13). The pH falls as organic acids are produced and buffering compounds (basic amino acids and primary phosphates) are consumed. The pH reaches a minimum of 3.8 - 4.4 before rising slightly toward the end of fermentation. The lowered pH inhibits bacterial spoilage during fermentation [50].

Figure 13 shows the time course of fermentation for lager beers (from Briggs et a/., 2004).

Ross and Harrison [16] highlighted that during fermentation pH will change within the range 3.80-5.60. The pH for the studies were monitored during the fermentation processes and analysed. The results are presented in Figures 14 and 15.

Figure 14 shows the change in pH over time during fermentation at 15 °C. The standard deviations for the graphs were 0.58 for biocatalysts and 0.60 for free cells. -23-

Figure 15 shows the change in pH over time during fermentation at 30 °C. The standard deviations for the graphs were 0.83 for biocatalysts and 0.74 for free cells.

The change in pH for the two studies was almost similar. The pH change for the study at 15 °C changed from 5.09±0.01 to 3.24±0.07 for the biocatalysts (a difference of 1.85) and 3.20±0.03 for the free cells (a difference of 1.89). The pH change for the study at 30 °C changed from 5.39±0.01 to 3.39±0.09 for the biocatalysts (a difference of 2.00) and 3.59±0.10 for the free cells (a difference of 1.80). The final pH values for all experiments were outside the fermentation range of 3.80 - 5.60. Generally the pH of the biocatalysts was below that of free cells for both studies. Statistical analysis was conducted to see if there was a significant difference between the immobilised cells and non- immobilised cells graphs. The p value for Figure 14 was 0.4900 which was considered not significant as variation among the graphs was not significantly greater than expected by chance. The p value for Figure 15 was 0.4884 which was considered not significant as variation among the graphs was not significantly greater than expected by chance.

2.2.2 Sugar concentration

Hornsey [51] highlighted that from the sugars present in the malt wort; some are taken up passively by the cells in an intact form (e.g. glucose and fructose), some are hydrolysed outside the cell and the breakdown products are absorbed (sucrose), whilst others are actively transported across the cell membrane and hydrolysed in the cytosol of the cells (maltose and maltotriose). Dextrins, comprising maltotetraose and larger starch breakdown products, are not metabolised. The general pattern of disappearance of fermentable sugars from wort during fermentation is sucrose→glucose→fructose→maltose→maltotriose, although there are -24-

differences between yeast strains [46]. The sugars analysed during the study were maltose and glucose.

(A) Maltose

The results for change in maltose during the studies are presented in Figures 16 and 17.

Figure 16 shows the change in maltose over time during fermentation at 15 °C. The standard deviations for the graphs were 12.07 for biocatalysts and 13.90 for free cells.

Figure 17 shows the change in maltose over time during fermentation at 30 °C. The standard deviations for the graphs were 4.07 for biocatalysts and 3.86 for free cells.

At the end of the study, free cells utilised maltose more than the biocatalysts from both studies (Figures 16 and 17). There was a small difference in the residual maltose between the biocatalysts and free cells at the end of the experiment at 30 °C (4.84 mg/ml) than at 15 °C (5.25 mg/ml). The initial maltose concentrations were 48.23 mg/ml at 15 °C and 45.32 mg/ml for 30 °C. The final maltose concentrations are presented below (Table 6) with calculated utilisation in brackets. Statistical analysis was conducted to see if there was a significant difference between the immobilised cells and non-immobilised cells graphs. The p value for Figure 16 was 0.3511 which was considered not significant as variation among the graphs was not significantly greater than expected by chance. The p value for Figure 17 was 0.0172 which was considered significant as variation among the graphs was significantly greater than expected by chance. -25-

Table 6: Final concentration of maltose during fermentation studies, utilisation is shown in brackets.

Temperature Biocatalysts Free cells

15 °C 14.24 mg/ml (70.5 %) 8.99 mg/ml (81.4 %)

30 °C 40.77 mg/ml (10.0 %) 35.93 mg/ml (20.7 %)

The highest utilisation of maltose was from the studies at 15 °C with free cells utilising 81% of maltose and biocatalysts utilising 71%. The cells at 30 °C had more residual maltose. This could mean that the experiment was still in progress. Only 10 % maltose was utilised by the biocatalysts and 20 % by the free cells, the free cells utilised twice the amount of maltose as compared to biocatalysts. The maltose utilised during the study at 30 °C was very low than that at 15 °C showing that the metabolism of the cells was being affected. The consumption rates of maltose were calculated and are presented in Table 7. The rates showed that maltose metabolism was low at 30 °C than at 15 °C.

Table 7: Maltose consumption during fermentation studies.

Temperature 15 °C 30 °C

Biocatalysts Free Biocatalysts Free cells cells

Consumption rate 3.78 4.36 1.30 2.68 (mg/ml ^* d)

(B) Glucose

The results for change in glucose during the studies are presented in Figures 18 and 19. -26-

Figure 18 shows the change in glucose over time during fermentation at 15 °C. The standard deviations for the graphs were 3.04 for biocatalysts and 2.93 for free cells.

Figure 19 shows the change in glucose over time during fermentation at 30 °C. The standard deviations for the graphs were 2.61 for biocatalysts and 2.93 for free cells.

Utilisation of glucose was almost similar for all the studies showing that the rates of conversion of glucose were the same (Figures 18 and 19). The difference in the residual glucose between the free cells and biocatalysts at the end of the experiment at 15 °C was 0.07 mg/ml and at 30 °C it was 0.34 mg/ml. The initial glucose concentrations were 8.57 mg/ml at 15 °C and 7.08 mg/ml for 30 °C. The final glucose concentrations are presented below (Table 8) with calculated utilisation in brackets. Statistical analysis was conducted to see if there was a significant difference between the immobilised cells and non- immobilised cells graphs. The p value for Figure 18 was 0.4259 which was considered not significant as variation among the graphs was not significantly greater than expected by chance. The p value for Figure 19 was 0.3698 which was considered not significant as variation among the graphs was not significantly greater than expected by chance.

Table 8: Final concentration of glucose during fermentation studies, utilisation is provided in brackets.

Temperature Biocatalysts Free cells

15 °C 0.30 mg/ml (96.5 %) 0.22 mg/ml (97.4 %)

30 °C 2.02 mg/ml (71.5 %) 2.36 mg/ml (66.7 %)

Utilisation of glucose was above 95% for the study at 15 °C and between 66 and 72 % for the study at 30 °C. The results also showed that studies at 30 °C -27-

were stopped before all the sugars were utilised resulting in the low utilisations observed. These results were expected because the glucose was utilised first before maltose by the yeast cells. The consumption rates of glucose were calculated and are presented in Table 9.

Table 9: Glucose consumption during fermentation studies.

Temperature 15 °C 30 °C

Biocatalysts Free Biocatalysts Free cells cells

Consumption rate 0.92 0.93 1.45 1.35 (mg/ml*d)

Table 9 showed that glucose metabolism was high at 30 °C than at 15 °C. From the rates of sugar metabolism (maltose and glucose), more glucose was utilised at 30 °C and more maltose was utilised at 15 °C. The trend highlighted the change in yeast metabolism as fermentation temperature was changed.

(C) Summary

The utilisation of the sugars was compared with literature. As mentioned earlier, glucose and fructose are consumed first (Figure 20) and as the glucose concentration diminishes, the enzyme systems required for assimilating maltose are synthesised and the yeast begins to utilise maltose and maltotriose. The production of ethanol and other fusel alcohols generally follows the consumption of carbohydrates [50].

Figure 20 shows carbohydrate assimilation profiles (from Priest and Stewart, -28-

Maltose and maltotriose generally are not assimilated in appreciable quantities until most of the glucose is assimilated. The profiles of the sugar assimilation from both studies look similar to the normal lager brew (Figure 20). Glucose however, was utilised up to 7 days for the study at 15 °C and 3.5 days for the study at 30 °C as compared to 2 days from the general profile whilst less maltose was utilised at 30 °C. The overall sugar utilisation was calculated and is presented in Table 10.

Table 10: Sugar consumption during fermentation studies.

Temperature 15 °C 30 °C

Biocatalysts Free Biocatalysts Free cells cells

Consumption rate 4.70 5.29 2.75 4.03

(mg/ml ^* d)

The highest consumption rate was observed from free cells at 15 °C, followed by the biocatalysts at 15 °C. At 30 °C, the highest consumption rate was from free cells followed by the biocatalysts. The low consumption rates observed for biocatalysts maybe attributed to the low cell concentrations observed when viability tests were conducted before fermentation studies (5.22x10 ³ CFU/ml of free cells and 4.51 x10 ³ CFU/ml of biocatalyst).

2.2.3 Alcohol Content

The alcohol concentration was monitored during the two studies. The original gravity and final gravity were measured and the fermentability percentage was calculated. The results for the alcohol content and fermentability percentage are presented in Table 11 , Figures 21 and 22.

Table 11: Gravities during fermentation studies at 15 and 30 °C -29-

Temperature Type of cells Original Final Fermentability

°C Gravity Gravity (%)

Biocatalysts 1038 1024 36.8

15

Free cells 1038 1020 47.4

Biocatalysts 1041 1037 9.8

30

Free cells 1041 1034 17.1

Figure 21 shows change in ethanol content over time during fermentation at 15 °C. The standard deviations for the graphs were 4.39 for biocatalysts and 7.55 for free cells.

Figure 22 shows: the change in ethanol content over time during fermentation at 30 °C. The standard deviations for the graphs were 1.36 for biocatalysts and 1.86 for free cells.

From Figures 21 and 22 it can be seen that the free cells produced more ethanol than the biocatalysts at the two temperatures investigated. The final ethanol concentration for the study at 15 °C was 1.56 % (v/v) for biocatalysts and 2.49 % (v/v) for free cells. For the study at 30 °C, the ethanol concentration was 0.39 % (v/v) for biocatalysts and 0.52 % (v/v) for free cells. The observed ethanol content was compared with literature at 15 and 30 °C (Tables 12 and 13). Statistical analysis was conducted to see if there was a significant difference between the biocatalysts and free cells graphs. The p value for Figure 21 was 0.0474 which was considered significant as variation among the graphs was significantly greater than expected by chance. The p value for Figure 22 was 0.0030 which was considered significant as variation among the graphs was significantly greater than expected by chance. -30-

Table 12: Comparison of fermentation rates with literature for freeze-dried cells at 15 °C.

Ethanol Ethanol Residual Sugar (g/L) % (v/v) sugar utilisation

(g/L) (%)

Free cells 21.2 2.49 3.24 83.7

This study

Biocatalysts 13.3 1.56 14.5 74.4

Gluten pellets Free cells 44.4 5.50 0.90

[42] Biocatalysts 41.1 5.20 0.70

Delignified Free cells 9.5 15.1 93.4 cellulosic Biocatalysts 8.9 20.1 91.2 material [43]

From Table 12, free cells produced more ethanol than the biocatalysts. Bekatorou et al. [42] observed an alcohol concentration of 5.50 % from free cells using gluten pellets and 5.20 % from biocatalysts. The ethanol concentration observed during the study was 2.27 % (almost ^ than that observed by Bekatorou et al. [42] when using free cells and 1.15 % (almost ^ than that observed by Bekatorou et al. [42]) when using biocatalysts. Inconomopoulou et al. [43] observed less alcohol content using a baker's yeast on delignified cellulosic material. The authors from the two papers observed less alcohol produced using the biocatalysts than using free cells, the same trend observed in the study. This showed that the immobilisation process tend to affect the metabolic process for the cells.

-31-

Table 13: Comparison of fermentation rates with literature for freeze-dried cells at 30 °C.

Ethanol Ethanol Residual Sugar

(g/L) % (v/v) sugar utilisation

(g/L) (%)

Free cells 4.42 0.52 38.28 26.94

This study

Biocatalysts 3.31 0.39 42.79 18.34

Delignified Free cells 9.50 12.2 94.7 cellulose [43] Biocatalysts 9.60 18.3 92.0 Dried figs [47] Biocatalysts 40.6 5.1 2.3

Starch-gluten- Biocatalysts 50.4 6.4 25.2 81.7 milk matrix

[45]

From Table 13, free cells produced more ethanol than biocatalysts. Bekatorou et al. [47] observed an alcohol concentration of 6.20 % from biocatalysts immobilised on dried figs. The difference in the alcohol concentration was very huge. Iconomopoulou et al. [43] observed an alcohol concentration of 9.50 g/L from free cells using delignified cellulose and 9.60 g/L from biocatalysts. The ethanol concentration observed during the study was 0.39 % (almost ^ than that observed by Bekatorou et al. [47] when using biocatalysts. Iconomopoulou et al. [42] also observed more alcohol content than the study for both the free cells and biocatalysts. Plessas et al. [45] had the highest alcohol observed.

The low alcohol concentrations observed in both studies may be explained by the fact that the viability of the yeast cells reduced by a magnitude of 10 ⁴ when they were recovered by a freeze dryer. The yeast cells concentrations were on average 65.75x10 ⁶ CFU/ml after growth and 62.63x10 ⁶ CFU/ml on average before flocculation studies. The cells were freeze dried for analysis and fermentation studies, and viability tests showed that there were; on average -32-

5.22x10 ³ CFU/ml for free cells and 4.51x10 ³ CFU/ml for biocatalysts. This showed a decrease in magnitude of 10 The reduction in the viability of the cells and the residual maltose observed may help to explain the low alcohol content.

The low alcohol content observed suggest that the biocatalysts are best suited to produce low-alcohol and non-alcohol beers. Non-alcohol beer refers to a product containing less than 0.1% (v/v), whilst low-alcohol beers have high alcohol content, normally between 0.5 and 1.5% v/v [50,51].

This invention has demonstrated the potential use of CNTs to improve flocculation of brewers' yeast. The optimum agitation was 110 rpm and this was comparable with literature [17]. pH required for best flocculation rates was between 5.00 and 5.80 [16,21 ,22,23,24,25]. The temperature required for better flocculation should be between 25 and 30 °C [26]. CNT concentration which gave best results was between 44 and 54 μg/ml and for better results the nanotubes should be in powder form. Addition of calcium ions and glucose had negative effects on flocculation rate and the onset of flocculation. This may be due to the repulsive forces between the Ca ²⁺ ions and the positively charged CNTs existing in the same broth solution. Since flocculation was observed the results are in agreement with literature [21 ,36]. The negative effect due to glucose is in agreement with literature which states that glucose inhibits flocculation [4,21 ,40]. Several authors have indeed found that flocculation is triggered by carbon and/or nitrogen starvation and that addition of these compounds to the growth medium delays flocculation [24,25,41].

Commonly, flocculation occurs only when the sources of fermentable sugars are exhausted. It has been suggested that under such starvation conditions the ability to form floes may represent a stress response. Thus floes provide a sheltered environment where the chance of survival of the population is enhanced. Disaggregation of floes occurs if the cells are again exposed to a -33-

source of fermentable sugars. In this case, the re-adsorption of a single cell mode affords unimpeded opportunity to utilize the supply of sugar [28].

Live yeast cells have an intracellular negative charge because of the presence of a transmembrane potential and they can be attracted to cations or positively charged substances. However, dead cells, which have leaky membranes and cannot build a membrane potential, cannot be attracted [52]. This suggests that during flocculation using positively charged CNTs, dead yeast cells were not attracted to CNTs and could not be flocculated, by the nanotubes.

Analysis of the alcohol produced during the fermentation studies resulted in low alcohol content. Free cells produced higher alcohol content from fermentation than the biocatalysts for the two studies conducted but the alcohol content for both studies were below that reported in literature. The flocculation process can be applied in the ethanol production industry to remove the suspended yeast cells after the fermentation process to reduce the turnaround time for the process. The low alcohol content observed strongly suggest the use of the biocatalysts in production of non-alcohol and low- alcohol beers.

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