Fermentation Kinetics of Different Sugars - Essay Example

Fermentation Kinetics of Different Sugars Abstract The process of fermentation has been in used for centuries. As the importance of fermentation increases day by day in many industries one must understand the factors which may increase or decrease the rate of the process. As fermentation uses many molecules mostly carbohydrates as the principal source, this paper investigates the role of different carbohydrates namely glucose, sucrose, maltose and lactose on the rate of fermentation. Furthermore the paper explores the use of cofactor in different process highlighting the role of magnesium ion in glycolysis which is believed to be the precursor process to fermentation. The experiment thus also includes the effect of adding magnesium chloride and sodium fluoride separately to two different fermentation mixtures. The process was carried out under controlled environment with the fermentation being carried out in a 42oc incubator and the volume of carbon dioxide evolved measured to the rate of fermentation. The findings achieved through the experiment showed an increase rate of fermentation in tubes with glucose and sucrose as the substrate while lactose showed a massively decreased rate of fermentation. The addition of sodium fluoride also caused a decreased rate of fermentation. Analysis of the complete data suggested that the carbohydrates used by Saccharomyces cerevisiae for fermentation plays a great role in the final rate of fermentation. Keywords: Saccharomyces cerevisiae, fermentation, carbohydrates, magnesium Fermentation Cells and tissues irrespective of belonging to animal or plant have a minimum requirement of energy. Different processes such as synthesis of molecules, transportation, DNA replication and cell repairs have varying requirements of energy. To successfully complete these processes cells undertake many metabolic processes to achieve their supply of energy. Glucose being the most important carbohydrate and the end product of almost all food sources is the beginning point of these metabolic processes. Energy conversion starts from the process of glycolysis. As explained by Agrimi et al., (2011) glycolysis begins with the entry of a single glucose molecule and terminates with the production of two pyruvate molecules. The process immediately yields four ATP molecules. However with the consumption of two ATP molecules at two different steps in the cycle the net production via substrate level phosphorylation turns out to be two. Although the process itself is not affected by the presence or absence of oxygen, the final production of the ATPs is hugely affected under hypoxic conditions as only 2 ATP molecules per glucose are produced instead of 36 ATP molecules per every glucose molecule. Depending on the availability of oxygen the pyruvates produced at the end of glycolysis are either shuttled into either cellular respiration / Krebs cycle or they are used in the process of fermentation. Fermentation has been derived from a Latin word ‘fevere’ meaning to ferment. Since the 19th century, humans have been taking advantage of the fermentative action of different yeasts in the production of bread and alcohol. It was around that time that observations were made about Saccharomyces cerevisiae; also known as baker’s yeast. Nowadays fermentation is one of the most important processes involved in the production of many products including wine, dairy products, fuels, single cell proteins and antibiotics. Saccharomyces cerevisiae are classified as unicellular organisms belonging to the kingdom of fungi. It has a very short generation time and can easily be cultured, one of its very important positive characteristics as it allows multiple cell lines to be maintained at low cost. They are grouped among facultative anaerobes, which mean that they have the ability to participate in both cellular respiration and fermentation according to the presence or absence of oxygen. Anaerobic conditions in the muscles of human body results in the production of lactic acid while the same conditions causes the yeast to produce ethanol (alcohol). This example shows that different cells have different oxygen requirements and every cell will have different type of end product if it undergoes fermentation due to hypoxic conditions. Regardless of the end product of fermentation, every cell which will participate in the fermentation process uses two pyruvate molecules as the starting ingredient. Below the two equations shows the two fermentations taking place in muscle cell and yeast In muscle cells: C6H12O6 2CH3CHOHCOOH In yeast cells: C6H12O6 2C2H5OH + 2CO2 Under anaerobic conditions the yeast reduces the NADH back to NAD in a two step process. Firstly pyruvate is decarboxylated to acetaldehyde by pyruvate decarboxylase and secondly this acetaldehyde is also reduced into ethanol by alcohol dehydrogenase. Both the end products in yeast fermentation have been used in the industry. Ethanol produced is used in wine industry while carbon dioxide has been the factor which is used to produce the airiness of a loaf of bread Understanding the importance of this process we must analyze the different factors which can increase or decrease the fermentation rate. As these processes have different enzymes catalyzing phosphorylation reactions, Mg2+ is involved as the cofactors in six of these reactions. The experiment was conducted in order to study the effect of two variables; effect of different food sources and the effect of different cofactors. Glucose (structure shown in figure – 1) was used as the only monosaccharide while three disaccharides, maltose, sucrose and lactose (structure shown in figure – 1) were used. Magnesium chloride and sodium fluoride were used to study the effect of cofactor. Different research articles have commented on this topic. Magnesium chloride is hypothesized to convert into magnesium and chloride ion with magnesium ion having a synergistic action on the different enzymes involved in glycolysis, including hexokinase, phosphofructokinase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase, enolase and pyruvate kinase. Coming towards the effect of different carbohydrates on the rate of fermentation, Domingues, Guimaraes & Oliveira, (2010) state that lactose will yield the least volume of ethanol and carbon dioxide. The volume of carbon dioxide evolved during the experiment and the rate at which it evolved was used as the method for the measurement of the rate of fermentation. Glucose Sucrose Lactose Maltose Figure – 1: Structure of different carbohydrates used in the fermentation process Materials and Methods Materials The following materials have been used in the experiment. Their respective quantity along with their concentration for a single tube is also mentioned. Baker’s Yeast (Saccharomyces cerevisiae) = 0.3g 10% Glucose = 2ml 10% Sucrose = 2ml 10% Maltose = 2ml 10% Lactose = 2ml 0.1 M MgCl2 = 0.5ml 0.1 M NaF = 0.5ml Water = to make up 20ml Six beaker and six fermentation gas tubes were used for the experiment. The solution for every tube was prepared according to the table presented below. 1/16 of a teaspoon was taken equal to 0.3g for the measurement of yeast. The fermentation tubes were tipped in order to completely fill the gas collection tubes. The top of every tube was sealed off with a piece of parafilm. Before any other step it is compulsory to confirm the absence of any air bubbles present before the start of fermentation. An incubator set at 42oc was used and the tubes were set in it. To check the rate of fermentation rate the production of CO2 was measured. The volume of CO2 produce can be estimated by measuring the depth of the layer of bubbles trapped in foam on top of the yeast solution. On observation the balloons will get bigger as they catch the CO2 produced by the yeast. Intervals of 10 min were chosen as the optimum time to measure volume of CO2. For accurate measurement and to minimize the chances of errors the CO2 gas line is leveled if the gas collector is not at 90o to the table. Every group data was repeated five times after which the mean was calculated. Safety precautions taken like wearing goggles and abstaining from tasting any reagents are similar to any other experiment. Tube No. Yeast (g) 10% Glucose 10% Sucrose 10% Maltose 10% Lactose 0.1 M MgCl2 0.1 M NaF pH 4 H2O 1 0.3 2ml 18ml 2 0.3 2ml 18ml 3 0.3 2ml 18ml 4 0.3 2ml 18ml 5 0.3 2ml 0.5ml 17.5ml 6 0.3 2ml 0.5ml 17.5ml Table 1: Fermentation solution mixing chart Results The rate of fermentation of different carbohydrates and in presence of different cofactors was assessed through the production of carbon dioxide. The volume of the gas produced per every interval of 10 min will give an idea as to which carbohydrate fermented faster. The fermentation gas tube containing glucose was deemed to be the control tube. The mean values along with the standard deviation of five different sets of reading at every interval from all six fermentation mixture are tabulated below. The table displays a clear trend in the production of carbon dioxide. The volume of gas collected increases in all the tubes as the time progresses. Lactose and Sodium fluoride have produced the least volume of CO2 in 1 hour with 0.0264 ml and 0.106 ml being measured. Greatest evolution of CO2 is reported in tube containing glucose with the volume of gas measured at 0.974ml. Sucrose, maltose and tube containing magnesium chloride have all produced around 0.860, 0.640 and 0.562ml of carbon dioxide. Time (min) Mean Values (ml) Glucose Sucrose Maltose Lactose Magnesium Chloride Sodium Fluoride 10 0.028 ± 0.018 0.018 ± 0.019 0.002 ± 0.004 0.0062 ± 0.01 0.012 ± 0.022 0.006 ± 0.013 20 0.190 ± 0.151 0.114 ± 0.216 0.030 ± 0.014 0.0122 ± 0.01 0.050 ± 0.048 0.018 ± 0.022 30 0.390 ± 0.336 0.232 ± 0.339 0.178 ± 0.163 0.0164 ± 0.01 0.226 ± 0.212 0.048 ± 0.048 40 0.510 ± 0.397 0.360 ± 0.378 0.420 ± 0.356 0.0204 ± 0.02 0.292 ± 0.248 0.062 ± 0.061 50 0.630 ± 0.476 0.460 ± 0.456 0.490 ± 0.381 0.0244 ± 0.02 0.422 ± 0.367 0.086 ± 0.077 60 0.974 ± 0.629 0.860 ± 0.796 0.640 ± 0.358 0.0264 ± 0.03 0.562 ± 0.539 0.106 ± 0.093 Table 2: Class data of CO2 evolution Graph 1: Volume of CO2 produced by different carbohydrates at every interval Graph 2: Volume of CO2 produced from two different cofactors at every interval The graphs above depict the trend in the values of the volume measured. The R2 value being approximately equal to 1 confirms that the readings are accurate and give the right picture and understanding of the experiment. The equation obtained by using the trend line (best fit line) gives the gradient of the curve. The gradient in this scenario is interpreted as the rate of fermentation. Here also lactose shows the lowest rate of 0.0040 ml/min while glucose shows the quickest rate of 0.1763 ml/min. The addition of magnesium chloride instead of producing any marked increase in the rate of fermentation has slowed the rate from 0.1763 ml/min to 0.1123 ml/min. However the addition of sodium fluoride has resulted in a slow rate of fermentation of 0.0205 ml/min. Fermentation rate (ml/min) Glucose Sucrose Maltose Lactose Glucose + MgCl2 Glucose + NaF Saccharomyces cerevisiae 0.1763 0.1536 0.1375 0.0040 0.1123 0.0205 Table 3: Class data of Saccharomyces cerevisiae fermentation rate Discussion This lab research paper discusses two of the major factors which may affect the rate of fermentation. The advancement in the technology has also shown the importance of fermentative process. Firstly the experiment was conducted in order to determine the role of different carbohydrates on the rate of fermentation. The experiment confirms the initial hypotheses that lactose is the least likely of the disaccharides to be fermented by Saccharomyces cerevisiae. Glucose has been described as the primary molecule which is used for energy by all living organisms either it be human beings, plants or yeast. It is because of this reason that glucose has yielded the greatest amount and volume of carbon dioxide. Glucose is utilized faster than any other monosaccharide by Saccharomyces cerevisiae. As glycolysis originates from a single molecule of glucose, this allows yeast to immediately process the glucose which has been taken in. As a result the fastest rate of fermentation is seen among the four carbohydrates used. Sucrose is a combination of two monosaccharide glucose and fructose. Saccharomyces cerevisiae utilizes both fructose and glucose though the rate at which glucose is used is much higher than that of fructose (Wang et al., 2004). Saccharomyces cerevisiae is therefore also classified as glucophilic with some strains acting as exceptions. Sucrose undergoes extracellular hydrolysis. The enzyme invertase is present in greater quantities outside. As a result the initial rate is much faster compared to other disaccharides. As shown in the results above sucrose achieves the second greatest volume of carbon dioxide and a better fermentation throughout the whole process as compared to other disaccharides. Although maltose is composed of two glucose molecules it should yield the highest volume of carbon dioxide among all the disaccharides. However due to the lack of an extracellular enzyme the hydrolysis only takes place intracellularly resulting in a decreased rate of fermentation and also producing low volume of carbon dioxide. Maltose is metabolized by an intracellular enzyme known as maltase (Sutton & Lampen, 2002). Nevertheless the volume of gas evolved is much greater than that of lactose which is explained below. Lactose is composed of glucose and galactose molecule. In yeasts it is metabolized by the enzyme β-galactosidase. This enzyme is possessed by Kluyveromyces and is not present both intracellularly and extracellularly in Saccharomyces cerevisiae. This results in an inability to ferment lactose and as a result the rate of fermentation drastically decreases and the production of ethanol and carbon dioxide is also a lot less than what is achieved through the other disaccharides. This is also shown by the mean values obtained in the results above. Cofactors are described as very important molecules needed for enzymes to work. Magnesium ion has a very important role in glycolysis as it acts as a cofactor for a host of enzymes. However the role of cofactor is only to an extent. Cofactors are only needed as a starting ignition for the enzymes to work. They have no effect on the rate at which the enzyme work or on the amount of product formed. This is further explained by the experimental values achieved during the fermentation. The addition of magnesium chloride which was believed to dissociate into magnesium ion and produce an increase in rate of fermentation had no positive effect. Although it did result in a decrease in the final volume of carbon dioxide but that wasn’t significant. However Sodium fluoride produced very interesting effects on the rate of fermentation. Like lactose earlier it caused very little evolution of carbon dioxide and also showed very slow rate of fermentation. The reason behind this interesting finding is hidden in the characteristics of sodium ion. Sodium ion is an inhibitor of the enzyme enolase which plays a part in the process of glycolysis by forming phosphoenolpyruvate. As glycolysis is the starting point of the whole fermentation process a decrease in its end product or any kind of decrease in the rate of glycolysis will ultimately affect the rate of fermentation as seen in the results above. The need to know the perfect combination for fermentation is very important. Any mistakes in this can cause massive industrial losses. It can lead to microbial instability and can result in an off-taste of the final product due to the presence of residual sugars. The change in sugar and its concentration has a massive affect on the volume of the final products as well as the rate of the process. This underlies the need for further experiments and practical investigations into the mode of sugar use and its uptake as well as to study the kinetic behavior of fermentation in Saccharomyces cerevisiae and other yeasts. However the use of magnesium compounds to enhance the production rate of fermentation products is not profitable as no apparent increase is seen or reported in this experiment. References Agrimi, G., Brambilla, L., Frascotti, G., Pisano, I., Vai, M. & Palmieri, L. (2011). Deletion or overexpression of mitochondrial NAD+ carriers in Saccharomyces cerevisiae alters cellular NAD and ATP contents and affects mitochondrial metabolism and the rate of glycolysis. Applied and Environmental Microbiology, 77(7), 2239-2246. Domingues, L., Guimaraes, P. M. R. & Oliveira, C. (2010). Metabolic engineering of Saccharomyces cerevisiaefor lactose/whey fermentation. Bioengineered Bugs, 1(3), 164-171. Sutton, D. D. & Lampen, J. O., (2002). Localization of sucrose and maltose fermenting systems in Saccharomyces cerevisiae. Biochimica et Biophysica Acta, 56, 303-312. Wang, D., Xy, Y., Hu, J. & Zhao, G., (2004). Fermentation kinetics of different sugars by apple wine yeast Saccharomyces cerevisiae, Journal of the Institute of Brewing, 110(4), 340-346. Read More