WO2011027335A1

WO2011027335A1 - Yeast cell immobilization

Info

Publication number: WO2011027335A1
Application number: PCT/IB2010/054025
Authority: WO
Inventors: Tirivaviri Augustine Mamvura; Sunny Esayegbemu Iyuke; Vusumuzi Sibanda; Yah Clarence Yah
Original assignee: University Of The Witwatersrand, Johannesburg
Priority date: 2009-09-07
Filing date: 2010-09-07
Publication date: 2011-03-10
Also published as: ZA201006438B; WO2011027335A9

Abstract

The invention describes a method for the immobilization of yeast cells in a fermentation reaction wherein carbon nanostructures are added to the reaction solution facilitating flocculation and the consequent immobilization of the yeast cells.

Description

YEAST CELL !MMOBtUZATtON

RSLD OF TH INVENTION

This invention relates to a method for the immobilization of yeast, particularly to a method for the immobilization of yeast in an ethsnol production process, more particularly to a method for the immobilization of yeast in brewing.

DEFINITIONS

"carbon nanosiruciures" a e allotropes of carbon having a bonding structure or^" sp² hybridized crblta!s.

"carbon nanotube^* a type of carbon nanostructure typciaily a fullerene-fike structure and is also known as a buckytube and includes singie-wai!ed carbon nanotubes, multi-walled carbon nanotubes, torus shaped carbon nanotubes, nanobuds and cup stacked carbon nanotu es.

BACKGROUND Of 1Ή5 INVENTION

The commercial demand for ethanol has increased considerably in recent times owing to Its use as a renewable energy source providing a potential alternative to conventional oi! based fueis.

Ethanol is typical.*/ produced by fermentation of sugars by yeast cells. The primary feedstock utilized in commercial and/or industrial ethanol production is com and sugar cane. Fermentation can be described as the process of obtaining energy from the oxidation of organic compounds, preferably carbohydrates, more preferably corn and sugar cane, while utilizing an endogenous electron acceptor. Usually the catabolic pathway produces an intermediate such as pyruvate that acts as the electron acceptor.

Toward the end of the fermentation process fiocculaiion occurs, which is of great importance in the production of ethanol. Floccuiation is defined as the phenomenon wherein free yeast ceils clump together as a resuit of the random collisions of Browntan movement and sediment rapidly from the medium in which they are suspended (1 ]. Essentially, floccuiation is the process whereby colloids come out of a suspension. It can occur naturally, or it can be artificially mduce6 by different agents. Roccuiafion is typically used to measure the progress of brewing yeast at the end of a brew.

At the end of the fermentation the yeast cells flocculate and either dump together and sink to the bottom, or rise to the surface attached to gaseous carbon dioxide bubbles. The costs of removing the yeast celis from the end product of the fermentation process in a clarification stage are high due to equipment costs and there is need to find cheaper and/or more efficient alternatives to complete this step once the fermentation is complete. Immobilization techniques have been proposed as a possible solution in finding a cheaper and more efficient way to perform the clarification step mentioned above, These techniques can be divided into four major categories based on the physical mechanism employed, nameiy attachment or adsorption on s carrier surfaces, entrapment within a porous matrix, seif aggregation by Hoccuistion {natural) or with cross-!inking agents {artificially induced by agents}, and cell containment behind barriers |3J.

The disadvantageous of the currant art includes that the immobilization techniques are expensive and Inefficient thereby negatively effecting profitability of ethanol production.

Consequently, there is a need for fioccu!ation techniques to be developed which will provide cheaper and economically profitable methods to produce ethanoi,

OBJECT OF THE INVENTION

The present invention, a method of immobilization of yeast ceils, aims to at least partly aileviate the disadvantages discussed above.

SUMMARY OF TH£ INVENTION

According to the invention there is provided a method for immobilization of yeast cells in a reaction taking place in a reaction vessel, the method comprising adding carbon nanostructures to the reaction vessel, the carbon nanostructures providing, in use, a surface to which the yeast cells can sorb facilitating fioccuiation and consequent immobilization of yeast cells. The method of immobilization of yeast ceils wherein the carbon nanostructures are added ai s time after the commencement of the reaction.

The method of immobilization of yeast ceiis wherein in the yeast ceiis sorb onto the carbon nanostrucfure through adsorption and/or absorption.

The method for immobilization of yeast cells wherein the carbon nanostructures are carbon nanotutoes.

The method for immobilization of yeast ceils wherein the reaction is an at least ethanol producing reaction,

The method for immobilization of yeast ceiis wherein the at !east ethanoi producing reaction is a fermentation reaction.

The method for immobilization of yeast ceils wherein the fermentation reaction is a saution fermentation reaction.

The method for immobilization of yeast cells wherein the reaction is agitated.

The method for immobilization of yeast ceils wherein agitation occurs at about 65 to 110 rpm.

The method for immobilisation of yeast cells wherein agitation is more than about 110 rpm.

The method for immobilization of yeast cells wherein the reaction has a pH of about 5 to 6.

The method for immobilization of yeast cells wherein the reaction has a pH of about 5.6 to 5.8. The method for immobilization of yeast cei!s wherein the reaction occurs at a temperature of about 25 to 30^CC.

The method for immobilization of yeast ceils wherein the carbon nanostructures are in powder form.

The method for immobilization of yeast cells wherein the carbon nanostructures are in sheet form.

The method for immobilization o? yeast cei!s wherein the carbon nanostructures present in a concentration of about 0 to 55 § mi^'1.

The method for immobilization of yeast cei!s wherein the carbon nanostructures are attached to a substrate.

The method for immobilization of yeast cells wherein the carbon nanostructures are attached to the substrate by coating the carbon nanostructures onto to substrate

The method for immobilization of yeast cells wherein the substrate is an inorganic substance.

The msthod for immobilization of yeast cells wherein the substrate is metallic.

The method for immobilization of yeast cells wherein the substrate is stainless steel.

The method for immobilization of yeast cel!s wherein the substrate is an organic substance. The method for immobilisation of yeast cells wherein the coated substrate comprises vertically aligned carbon nanostrudures.

DESCRIPTION OF PREFER ED EMBODIMENTS

Floccu!atson in fermentation reactions can be induced by ar artificial agent and this may increase process efficiency and/or lower the overall process cost. Carbon nanotubes (CNTs), which are increasingly recognized as promising materials for catalysis, either as catalysts themselves, as catalyst additives or as a catalyst support £5}, were employed in the immobilisation of brewers' yeast using a floccu!ation method for fermentation studies. Mu!tiwalied carbon nanotubes (MWCNTs) are relatively affordable materials making them an attractive option as artificial flocculation agents as the cost will be negligible {5}

Although no supporting materials are required for the effective immobilization the technique was further developed to provide for the use of CNTs onto a support, particularly a support being stainless steel wiring Yeast ceiis were consequently Immobilized using carbon nanotubes by coating stainless steei wire with a film of vertically aligned carbon nanotubes 50-100 um thick, Fissures in the film provided sites where cells were protected from shear forces of the fluid in the reaction vessel and cells could thus be immobilised via physical adsorption, The size of the fissures were found to be important as the sites must be large enough to aliow cells to enter the pores, however if the fissures are too wide then the cells will not be adequately protected from shear forces and will be washed out.

1 , Materials and Sfethods

1.1 microorganisms

The cultured S. cerevisiae strain NRRL Y2084 (a dry brewers yeast which was obtained from National Food Products. Emmarentia, Johannesburg) was used throughout this study and it was maintained on ma!t extract agar (UEA) slants. The cells were grown without adding more oxygen into the solution (rubber cork was used to cover the mouth of the flask throughout the experiment) at 30±0.5 °C in a 500 ml flask held on a rotary shaking incubator working at 110±2 revolutions per minute (rpm) for 24 hours and used during immobilization stages |7,8j.

Peinado et al. (9] conducted their studies at 28 °C and 150 rpm for 7 days and had successful immobilization to produce yeast biocapsuies while Sakurai et a!. [10] used 30 °C and 160 rpm during their immobilization studies of yeast ce!ls on porous cellulose carriers. Oztop et a/. [11] immobilized S, cerevisiae onto acrylamide-sodium acrylate hydrogels at 30 °C for 72 hours. Growth curves were investigated for the stationary phase and showed that the yeast ceils concentrations were on average 65.75x10^s CFU/ml after growth and 62.63x10^s CFU/mi on average before flocculation studies. The ceils were freeze dried for analysis and fermentation studies, and viability tests showed that there were; on average 5.22x10³ CFU/mi for free cells and 4.51x10³ CFU/mi for fermentation cataiyst. This showed a decrease in magnitude of 10⁴. For fermentation studies the samples were used within 3 - 4 days.

1.2 Medium

Yeast extract powder (Merck, South Africa) was dissolved in distilled water to make the medium for cell nutrients. Five grams of yeast extract powder was dissolved in one litre of distilled water and sterilized at 121 °C and 1 ,1 bars in an autoclave for 20 minutes. The pH of the medium was 6.90 and it was adjusted by an acid to within fermentation pH range of 4 to 6 before starting immobilization studies.

1.3 Carbon nanotubes (CNTs) These were produced and confirmed by JEOL JEM 100S Transmission Electron Microscopy (TEM) [12]. in a specific embodiment of the invention yeast ceils were immo ifized using carbon nanotubes by coating stainless steel wire with a film of vertically aligned carbon nanotubes 50-100 pm thick. Fissures in the fsim provided sites where ceils were protected from shear forces of the fluid in the reaction vessel and cells could thus be immobilized via physical adsorption as mentioned above.

1.4 Production of the carbon nanotube coating

The vertically aligned carbon nanotube coating was formed in a vertical chemical vapour deposition reactor. The reactor is composed of a 5 cm diameter quartz reactor. 100 cm in length placed vertically in a furnace. Gasses are fed into the reactor via a vaporization chamber at the bottom of the reactor which is heated by a heating piate. Gasses exit the system through pipes at the top of the reactor.

The vertically aligned carbon nanotube coating was produced as follows:

1. 10 cm lengths of 0.9 mm diameter type 304 stainless steel wire and 5 g of ferrocene powder were placed in the reactor tube, 30 cm from the bottom of the tube, supported by quartz wool.

2. The reactor was flushed with argon at 647.3 cnr/min for 15 min to remove oxygen from the system,

3. The furnace was heated to a temperature of S50°C, and the heating piate was heated to 350°C. The heating of the reactor causes the ferrocene catalyst to sublime to the gaseous phase, causing the ferrocene gas to descend into the vaporization chamber.

4. When the reactor reached the desired temperature, argon was fed into the system at a flowrate of 647.3 cm min for 5 min to transport the ferrocene catalyst back into the reactor. This allows the ferrocene catalyst to be deposited on the wires allowing for vertically aligned carbon nanotubes to be formed.

5. The carbon source, acetylene, was introduced into the reactor using argon as a carrier gas. The fiowraie of acetylene was 406.3 cm³/min and argon was 332.1 cm³/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 ffowrate 647.3 cm³/min.

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

Scanning electron microscope (SE ) 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 SEM image of carbon nanotube coating after washing, A: surface of coating and void (bar denotes 00 pm), 8; vertically aligned carbon nanotube coating (bar denotes 10 ym). 10 cm sections of the CNT coated wires were then placed in a culture medium with fermenting yeast ceils, The culture medium was a wort analogue at 10.7°P composed of mait extract which was sterilized and filtered to remove solids. 150 mi of culture medium was placed in 250 mi conical flasks and placed in a rotary shaker at 100 rpm and 23°C. rtiie 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 weii adhered to the surface. The earliest evidence of yeast ceil immobilization in significant amounts was found on wires which had been exposed to the fermenting medium for 53 hours as shown in Figure 2. In this case cells were found to be immobilized in a fissure that ranged from 40-70 pm wide but was more than 200 μη\ long. The ce!l 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 wails of the fissure. The morphology of the ceils 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 ceils adhering to the exposed surface of the stainless steel in between the hilis of CNTs.

Figure 2. Yeast cells immobilized in fissure. A; yeast cells in fissure showing lack of yeast cells on the surface of the coating (bar denotes 10 p.m), B; yeast cells In fissure showing interactions between yeast ceils (bar denotes um). 1.5 Cell immobilization

Yeast cell immobilization was performed with CNTs while a control experiment was setup with yeast ceiis in the absence of CNTs. A !oopfu! of inoculum was added to an Erienmeyer flask containing 100 ml of sterilized yeast extract medium and incubated in a shaker at 110 rpm, 30 °C for 24 hours without adding more oxygen into the broth (rubber cork used to sea! the flask to prevent air uptake during the study). After 24 hours the yeast cells were used for immobilization studies. 30 ml (7.04x10^sCFU/ml) of yeast broth was added to 250 ml of yeast extract medium. The broth was incubated in a shaking incubator at 30 °C [ 3]. 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 ceils to sorb onto through either the process of adsorption and/or absorption.

1.6 Analytical methods

Yeast fioccuiation was analyzed using two methods: a qualitative process to determine the quality of the floes produced and a quantitative process to measure the fioccuiation weight. These two methods enabled the optimization of the parameters that affected the fioccuiation. The first method (qualitative in nature) involved estimating fioccuiation 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 Erienmeyer flask and expressing fioccuiation qualitatively as: {-) no fioccuiation; (+) yeast slightly flocculent (poor); (++} yeast flocculent; and {+++} yeast very flocculent {14,15,16]. The second method involved using a centrifuge to concentrate the fiocs, 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 JS 840A Scanning Electron Microscopy (SEWS). The Hoc weight was then plotted against the variable nder investigation to determine the effect of the variabie.

2 Results and Discussions

2,1 Immobilization of Yeast Cells

The biocataiysts were viewed under SEM as shown beiow (Figure 3).

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

The micrographs show that the immobilized cells aligned themselves aiong the CNT length. This phenomenon was observed on aii studies with CNTs. in contrast, free ceiis showed a p!anar structure (Figure 4).

Figure 4: S£ micrographs showing brewers' yeast which flocculated without carbon nanotubes: (a) x1000 and (b) x1700.

Some yeast cel!s were growing on top of the flocculated ceiis pianar structure (Figure 4b). CNTs increased the fiocculation rate of brewers' yeast and the fiocs were more stabie than floes produced by free cells. This was observed when recovering the fiocs for freeze drying.

The observation thai CNTs couid increase the fiocculation 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 iarge surface spikes and these would enabie neutralization of surface charge of brewers' yeast ceils when there is contact made between the ceils and the nanotu es. This would ailow the ceils 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 f eld would be considerably less than that for cell-ceil 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 ceiis (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 floccuiation. it was demonstrated that a relation existed between ceil division arrest, the increase of Ceil Surface Hydrophobic^ (CSH) and initiation of fioccuience during fermentation [18,19]. A high ievei of CSH may facilitate cel!-ce!i contact in an aqueous medium resulting in more specific iectin-carbohydrate interactions [17],

Factors that affect floccuiation 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 minute {rpm). There was poor floccuiation (+) observed after 4 days for 0, 50, 150 and 200 rpm whilst at 110 rpm good floccuiation (^■*^■+} was observed (Table

1).

The absence of floccuiation 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 heip to grow the fiocs there is a limiting agitation speed beyond which surface erosion or fioc fracture sets in, which limits the stable floe to a certain optimum size [21] (Figure 6).

Figure 6: Effect of agitation speed on initial rate of floccuiation of Saccharomyces cerevisiae S6 6-1B (from Stratford and Wilson, 1S90).

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

2.1.2 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 asm was to analyze the effect of pH on the floccuiation of brewers' yeast ceils. The results are summarized below (Table 2 and Figure 7).

Figure 7: Effect of changing pH on ficcculation. The standard 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 biocataiysts and free ceiis 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 8.25 before increasing further again. The focus was mainiy 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 ceils 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 S.80 observed during the study. The fact that CNTs were able to flocculate yeast ceils within the mentioned pH range showed that the CNTs are positively charged and these tend to repei the cells when the cells have a positive charge due to a change in pH. From literature, yeast ceils 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 deftoccuiation with Increasing temperature wh!ie 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 iittle 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 fioccuiation considered negligible [30], Yeast auto!ysis 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 fioccuiation.

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

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 fioccuiation; 0.1 3±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 fioccuiation (b), fioccuiation 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 si. [26] found the optimum temperature for immobilization to be 25 °C when they immobilized yeast cefis on a chitosan film (Figure 8).

p

Figure 8: Effect of temperature on immobilization (from Oztop et al., 2002},

Hsu ei a/. [34] observed an increase in floccuiation with an increase in temperature from 5 - 45 °C. This was in line with the observations in the study showing an increase in floccuiation with an increase in temperature. Jin ef a/.

[22,23] found that floccuiation of a iager 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 ce!is and cu!tunng media to investigate their effect. The concentration was varied from 0 - 72 .ug mi^"1. G sfari et al. [35] investigated bacteria ciliated protozoa and used concentration between 0 - 17.2 ig ml^{' 1}.

Changing the concentration of CNTs did have an effect on floccuiation of brewers' yeast in the range investigated. A table and graph of floe weight observed against CNT concentration are presented beiow (Table 4 and Figure

Figure 9: Effect of CNTs concentration on brewers' yesst f!occuiatton. The standar0 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 9/ΓηΙ. Additional CNTs to increase their concentration above 53.57 g/ml had negative effects on ffocculation as the floe weight observed started to decrease. From 0 to 35.71 μ$Ιπι\ there was a negligible gain in floe weight observed, +0,0 3g gained. Increasing the CNT concentration to 53.57 ng/mi gave a gain of +0.040 g as compared without using CNTs.

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

2. 5 Calcium tons concentration

Caicium ions are important in yeast cell floccuiation and as such their effect on floccuiation were studied [21,36,37]. Calcium ion concentration was varied from 0 - 9.55 m and introduced into the broth as anhydrous calcium chloride (CaCi₂2H₂0). The caicium ch!oride 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 f!ocs observed in the presence of caicium ions were powdery in nature.

Figure 10: Effect of Ca ions on brewers' yeast fioccuiation. 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 fioccuiation 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 m of Ca^2* ions (experiment e) gave the optimum conditions for fioccuiation when considering fioc weight, increasing the concentration to 9.55 mM yielded almost the same f oe weight as at 5.49 mM. This data enabfes the optimisation of the concentration of calcium ions required. The presence of Ca^2* ions reduced fioccuiation than results obtained in the absence of the Ca^J* ions. in the study it was observed that Ca^2* ions had a negative effect on fioccuiation in the presence of CNTs. This may be due to the repuisive forces between the Ca^2* sons and the positively charged CNTs existing in the same broth solution. Since fioccuiation was observed the results are in agreement with literature [2 ,36], Taylor and Orton [37] concluded that the presence of calcium ions is required at a very icw concentration in order to induce fioccuiation. For iow salt concentrations (cations other than Ca^2* ions e.g. g²⁺, Mn²⁺), there was an observed fioccu!ation enhancement, while at high concentrations inhibition of fioccuiation 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 fioccuiation of brewer's yeast cells was the last parameter to be investigated. Generally it was found that maitose 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 fioccuiation process.

The presence of glucose promoted yeast cell growth and delayed the stationary phase for yeast cells thereby delaying the onset of fioccuiation. Ethanol was produced from the effect of yeast celis on glucose which decreases the pH of the broth resulting in the delay of fioccuiation. Studies were conducted to investigate the effect of giucose on fioccuiation 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/m!. The results for the study were plotted as pH and zeta potential against time (Figure 11).

Figure 11 : 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 ffocculation onset was observed in the previous studies and this showed a delay of 4 days for yeast cell floccutation 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 fioccuiation process (Figure 12).

Figure 12: Eifect 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 4.88 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,

Sekatorou 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 pefiets. Inconomopou!ou ef 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 ef a/. [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. Pfessas e a/. [45] conducted their fermentation studies on giucose at 15 °C for at least 1 day using an S. cerevisiae strain immobilised in a starch-giuten-milk matrix usabie 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 152 and wine strains VIN7 and VIM 13 as free ce!is. Bekatorou et al. [47] conducted their fermentation studies on giucose at 30 °C for at most 2,5 days using an S, cerevisiae strain immobilised on dried figs. Bekers et a!, [48] conducted their fermentation studies on a mentioned medium at 30 °C for 2 days using an S. cerevisiae strain immobilised on modified stainiess steel wire. Inconomopouiou ef al. [43] conducted their fermentation studies on giucose 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-giuten-mi!k matrix usabie for food. S peers 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 ceils. Studies were done at least twice with the mean and standard deviation included in the results. Results for the pH, residua! 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 ethanoi and carbon dioxide according to the following balanced stoichiometric equations.

Maltose

iz#22 ii + J¾0 ^→ 4C_ZH_S0H + CQ₂ Equation 1

Glucose

C₆H ₂0₆→ 2C_zH_sO + 2C0₂ Equation 2

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.

2.2.7 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

Ross and Harrison [16] highlighted that during fermentation pH wiii 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: Change in pH over time during fermentation at 15 °C. The standard deviations for the graphs were 0.58 for biocataiysts and 0.60 for free ce!is.

Figure 15: Change in pH over time during fermentation at 30 ^CC. The standard deviations for the graphs were 0.83 for biocataiysts and 0.74 for free ceils.

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 biocataiysts {a difference of 1.85) and 3.2Q±0.03 for the free ceils (a difference of 1.89). The pH change for the study at 30 °C changed from 5.39±0.01 to 3.39±0.0S for the biocataiysts (a difference of 2.00) and 3.59±0.10 for the free ceiSs (a difference of 1.80). The final pH vaiues for ail experiments were outside the fermentation range of 3.80 - 5.60. Generally the pH of the biocataiysts was below that of free ceils for both studies, Statistics! analysis was conducted to see if there was a significant difference between the immobiiised ceiis and non- immobilised ceils 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

Homsey [51] highlighted thai 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 ceil and the breakdown products are absorbed (sucrose), whilst others are actively transported across the cell membrane and hydroiysed in the cytosoi of the ceils (maltose and maltotriose). Dextrins, comprising maitotetraose and larger starch breakdown products, are not metabolised. The general pattern of disappearance of fermentable sugars from wort during fermentation Is sucrose- g!ucose->fructose->maitose→ma!to riose. although there are differences between yeast strains [461. 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: 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: Change in maltose over time during fermentation at 30 °C. The standard deviations for the graphs were 4.07 for biocatai sts and 3.86 for free cells.

At the end of the study, free ceits utilised maitose more than the biocataiysts from both studies (Figures 16 and 17). There was a smaii difference in the residual maltose between the biocataiysts and free cells at the end of the experiment at 30 °C (4.84 mg/m!) than at 15 °C (5.25 rng/ml). The initial maitose concentrations were 48.23 mg/mi at 15 °C and 45.32 mg/ml for 30 °C. The final maitose concentrations are presented beiow (Table 6) with calculated utilisation in brackets. Statisticai 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 0.351 1 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. Tabie 6: Final concentration of maltose during fermentation studies, utilisation is shown in brackets.

The highest utilisation of maltose was from the studies at 15 "C with free ceils utilising 81% of maltose and biocatai sts utilising 71%, The cells at 30 °C hat- 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 maitose as compared to biocataiysts. The maltose utilised during the study at 30 °C was very low than that at 15 °C showing that the metabolism of the ceils 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.

(8) Glucose

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

Figure 18: Change in glucose over time during fermentation at 15 °C. The standard deviations for the graphs were 3.04 for biocataiysts and 2.93 for free ce!is.

Figure 19: Change in glucose over time during fermentation at 30 °C. The standard deviations for the graphs were 2.61 for biocataiysts and 2.93 for free celts. 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 ceils and biocata!ysts at the end of the experiment at 15 °C was 0.07 mg/mi 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/m! 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 celis and non- immobilised cells graphs. The p vaiue 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 vaiue 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.

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 were stopped before ail 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 celis. The consumption rates of glucose were calculated and are presented in Tabie 9.

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, gfucose 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 maitotriose. The production of ethanoi and other fusel alcohols generally follows the consumption of carbohydrates [50],

Figure 20: Carbohydrate assimilation profiles {from Priest and Stewart, 2006). Maltose and maitotriose generally are not assimilated in appreciable quantities until most of the glucose is assimilated. The profiles of tie 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.

The highest consumption rate was observed from free cells at 15 °C, followed by the biocataiysts at 15 °C. At 30 °C, the highest consumption rate was from free cells followed by the biocataiysts, The low consumption rates observed for biocataiysts maybe attributed to the low ceil concentrations observed when viability tests were conducted before fermentation studies (5.22x10³ CFU/mi of free cells and 4.51x10^s CFU/mi 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 fermentabt!ity percentage was calculated. The results for the alcohol content and fermentability percentage are presented in Table 11, Figures 21 and 22.

Tab!e 11: Gravities during fermentation studies at 15 and 30 °C

Figure 21 : Change in ethano! content over time during fermentation at 15 °C. The standard deviations for the graphs were 4.39 for biocatalysts and 7.55 for free ceils.

Figure 22: Change in ethanoi content over time during fermentation at 30 °C. The standard deviations for the graphs were 1.36 for biocata!ysts and 1.36 for free cells.

From Figures 21 and 22 it can be seen that the free cells produced more ethanoi than the biocataiysts at the two temperatures investigated. The final ethanoi concentration for the study at 15 °C was 1.56 % (v/v) for biocataiysts and 2.49 % (v/v) for free cells. For the study at 30 °C, the ethanoi concentration was 0.39 % (v/v) for biocataiysts and 0.52 % (v v) for free cei!s. The observed ethanoi content was compared with literature at S and 30 °C (Tables 12 and 13). Statistical analysis was conducted to see if there was a significant difference between the biocataiysts 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.

From Table 12, free cells produced more ethanol than the biocatalysts. Bekatorou et a . [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 a!. [42] when using free cells and 1.15 % (aimost ~ than that observed by Bekatorou et at. [42]) when using biocatalysts. inconomopouiou et at. [43] observed less alcohol content using a baker's yeast on deiignlfied ceiiulosic materia!. The authors from the two papers observed less alcohol produced using the biocatalysts than using free celis. the same trend observed in the study. This showed that the immobilisation process tend to affect the metabolic process for the cells.

From Table 3, free cefls produced more ethanoi than biocatalysts. Sekatorou et al. [47] observed an alcohol concentration of 6.20 % from biocatalysts immobfitsed on dried figs. The difference in the alcohol concentration was very huge, !conomopouiou et al. [43] observed an aicoho! concentration of 9.50 g/L from free cells using deiignified cellulose and 9.60 g/L from biocatalysts. The ethanoi concentration observed during the study was 0.39 % (almost— than that observed by Bekatorou ef a/. [47] when using biocatalysts. !conomopou!ou ei a . [42] also observed more alcohol content than the study for both the free cells and biocatalysts. Plessas ei 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* when they were recovered by a freeze dryer The yeast cells concentrations were on average 65.75x10^® CFU/ml after growth and 62.63x10^s CFU/m! on average before floccufation studies. The ceils were freeze dried for analysis and fermentation studies, and viability tests showed that there were; on average 5.22x10³ CFU/ml for free ceils and 4.51x1Q³ CFU/mi for biocatalysts. This showed a decrease in magnitude of 10⁴. The reduction In the viability of the cells and the residua! maltose observed may heip to explain the low aicohoi content.

The low aicohoi 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 iess than 0.1% (v v), whilst iow-aicohol beers have high aicohoi content norma!!y between 0.5 and 1.5% v/v [50,51 J.

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

Commonly, fioccuiation occurs oniy when the sources of fermentable sugars are exhausted, it has been suggested that under such starvation conditions the ability to form fiocs 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 ceils are again exposed to a source of fermentable sugars, in this case, the re-adsorption of a single ceil mode affords unimpeded opportunity to utilize the supply of sugar [28].

Live yeast ceiis 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 ceils, which have !eaky membranes and cannot build a membrane potential, cannot be attracted [52], This suggests that during floccu!ation 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 ceils 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 floccuiatton 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-alcohoi and iow- alcohoi beers.

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Claims

1. A method for Immobilization of yeast cells in a reaction taking piace in a reaction vessel, the method comprising adding carbon nanostructures to the reaction vessel, the carbon nanostructures providing, in use, a surface to which the yeast ceils can sorb facilitating flocculation and consequent immobilization of yeast ceils.

2. The method of Immobilization of yeast cells according to ciaim 1, wherein the carbon nanostructures are added to the reaction vessel at a time after commencement of the reaction

3. The method of immobilization of yeast ceils according to ciaims 1 and 2, wherein in the yeast ceils sorb onto the carbon nanostructures through adsorption and/or absorption,

4. The method for immobilization of yeast ceiis according to any one of ciaims 1 to 3, wherein the carbon nanostructures are carbon nanotubes.

5. The method for immobilization of yeast ceils according to any one of claims 1 to 4, wherein the reaction is an at least ethanol producing reaction.

6. The method for immobilization of yeast cells according to ciaim 5, wherein the at ieast ethanol producing reaction is a fermentation reaction.

7. The method for immobilization of yeast cells according to claim 6, wherein the fermentation reaction is a solution fermentation reaction. The method for immobilization of yeast ceils according to any one of claims 1 to 7, wherein the reaction is agitated. The method for immobilization of yeast ceils according to claim 8, wherein agitation occurs at about 65 to 110 rpm. The method for immobilization of yeast cells according to claim 8, wherein agitation is more than about 110 rpm. The method for immobilization of yeast ceils according to any one of claims 1 to 10, wherein the reaction has a pH of about 5 to 6. The method for immobilization of yeast ceils according to any one of claims 1 to 11, wherein the reaction has a pH of about 5.6 to 5.8. The method for immobilization of yeast cells according to any one of claims 1 to 12, wherein the reaction occurs at a temperature of about 25 to 30'C. The method for immobilization of yeast ceils according to any one of claims 1 to 13, wherein the carbon nanostrucfures are in powder form, The method for immobilization of yeast ceils according to any one of claims 1 to 14, wherein the carbon nanostrucfures are in sheet form. The method for immobilization of yeast ceils according to any one of claims 1 to 15, wherein the carbon structures are present in a concentration of about 0 to 55 9 mi^'1. The method for Immobilization of yeast cells according to any one of claims 1 to 16, wherein the carbon structures are attached to a substrate. The method for immobilization of yeast ceils according to claim 17, wherein the carbon nanostructures are attached to the substrate by coating the carbon nanostructures onto to substrate, The method for immobilization of yeast cells according to claim 17, wherein the substrate is an inorganic substance. The method for immobilization of yeast cells according to claim 17. wherein the substrate is metallic. The method for immobilization of yeast ceils according to claim 19 or 20, wherein the substrate is stainless steel. The method for immobilization of yeast cells according to claim 17, wherein the substrate is an organic substance. The method for immobilization of yeast cells according to any one of claims 17 to 22, wherein the coated substrate comprises vertically aligned carbon nanostructures. The method for immobilization of yeast cells substantially as in any one embodiment herein described, illustrated and exemplified.