Fermentation Technology BTK 4301

Name Matrix Number Group Title

: : : :

MOHD UZAIR BIN JAAFAR 151516 2E Growth of microorganism: Respond to nutrient requirement (different carbon source)

Submission Date

22/2/2011

Title Growth of microorganism: Respond to nutrient requirement (different carbon source) Introduction Every organism must find in its environment all of the substances required for energy generation and cellular biosynthesis. The chemicals and elements of this environment that are utilized for bacterial growth are referred to as nutrients or nutritional requirements. Many bacteria can be grown the laboratory in culture media which are designed to provide all the essential nutrients in solution for bacterial growth. Energy for growth comes from either the oxidation of medium components or from light. Most of industrial microorganisms are chemoheterotrophs, therefore the commonest source of energy will be the carbon source such as carbohydrates, lipid or proteins. Microbes, especially bacteria can grow on variable growth substrates and hence possess versatile metabolic activities. Nutritional versatility makes bacteria adaptable to different types of habitats and environments. Microbes produce adaptive enzymes in response to the presence of substrates that are not usually preferred by other organisms. Metabolic versatility is hence the function of enzymes; hydrolases especially, which confer anextraordinary ability to grow on unusual substrates. Because of this versatility, in industrial fermentation, the suitable carbon source can be chose depending on the desire product to be produce. However the utilization of different carbon source may result differently such as effect on growth of bacteria and production of product. In this experiment, three type of carbon source is used to investigate the growth of microorganism, E.Coli, that is maltose, glucose and xylose. Maltose or malt sugar, is a disaccharide formed from two units of glucose joined with an (14)bond. Maltose is the second member of an important biochemical series of glucose chains. Maltose can be broken down into two glucose molecules by hydrolysis. Glucose is a simple sugar (monosaccharide) and an important carbohydrate in biology. Cells use it as a source of energy and a metabolic intermediate as it is readlly carbon souce. Xylose is a sugar first isolated from wood, and named for it. Xylose is classified as a monosaccharide of the aldopentose type, which means that it contains five carbon atoms and includes an aldehyde functional group. It is the precursor to hemicellulose, one of the main constituents of biomass. Like most sugars, it can adopt several structures depending on conditions. With its free carbonyl group, it is a reducing sugar.

Objective To investigate growth of microorganism using different carbon source as a source of energy Material Escherichia Coli fresh culture LB broth Maltose Glucose Xylose Conical flask Incubator shaker

Method

1. A single colony of E.Coli was inoculated in 100ml LB broth and incubated at 200 rpm, 37C until the OD of the culture reach approximately 600. 2. 10 ml of the culture was added to 80ml of LB broth with 10g/l of maltose in a conical flask. 3. Step 2 was repeated by replacing 10g/l of maltose with 10g/l of glucose and 10g/l of xylose with different conical flask. 4. All the culture was incubated at 200 rpm, 37C. 5. Each culture was sampling for 2ml every 1.5 hour.

A) Sampling for bacteria growth 1. 1ml of each sampling (glucose medium, maltose medium, xylose medium) was centrifuge at 10 00 rpm for 10 minute. 2. The supernatant was removed and 1 ml of NaCl was added. 3. The sample was resuspend and read the absorbance at 600nm (if the reading >1.0, dilute the sample before taking the reading).

B) Sampling for determination of glucose concentration by using DNS method 1. 1ml of sampling was added with 1ml of DNS 2. 2 drops of 0.1 NaOH was added and mix by using vortex. 3. The sample was boil for 5 minute and let it cool by running it with tap water. 4. 10 ml of dH2O was added. 5. The absorbance was read at 540nm after 20 minutes.

Result a) Sampling for bacteria growth Table 1 : cell density (OD) against cultivation time OD reading at 600nm with dilution time (hours) 0 1.5 3 4.5 6 7.5 18 19.5 21 22.5 24 maltose 0.296 0.232 0.962 1.317 0.368 0.284 0.315 0.95 0.423 0.282 0.147 glucose 0.312 0.225 1.07 1.18 0.193 0.212 0.234 0.035 0.246 0.235 0.109 xylose 0.278 0.191 0.877 1.165 0.156 0.268 0.238 0.238 0.417 0.24 0.101 Dilution factor 1 1 1 1 10 10 10 10 10 10 10 maltose 0.296 0.232 0.962 1.317 3.680 2.840 3.150 9.500 4.230 2.820 1.470 Actual OD glucose 0.312 0.225 1.070 1.180 1.930 2.120 2.340 0.350 2.460 2.350 1.090 xylose 0.278 0.191 0.877 1.165 1.560 2.680 2.380 2.380 4.170 2.400 1.010

Cell density (OD) versus cultivation time of different carbon source

10.000 OD reading at 600nm 8.000 6.000 OD maltose 4.000 2.000 0.000 0 5 10 time (hours) 15 20 OD glucose OD xylose

Figure 1: Cell density (OD) versus cultivation time of different carbon source

b) Sampling for determination of glucose concentration by using DNS method Table 2: concentration of maltose against time Absorbance Dilution factor reading at 540nm with dilution actual reading 20 0.392 7.840 20 0.320 6.400 20 0.282 5.640 20 0.264 5.280 20 0.160 3.200 20 0.167 3.340 20 0.101 2.020 1 2.071 2.071 1 2.170 2.170 1 2.075 2.075 1 2.110 2.110

Time (hours) 0 1.5 3 4.5 6 7.5 18 19.5 21 22.5 24

Concentration of maltose (g/l) 11.136 9.091 8.011 7.500 4.545 4.744 2.869 2.942 3.082 2.947 2.997

Time (hours) 0 1.5 3 4.5 6 7.5 18 19.5 21 22.5 24

Table 3: concentration of glucose against time Absorbance Dilution factor reading at 540nm with dilution actual reading 20 0.405 8.100 20 0.398 7.960 20 0.346 6.920 20 0.348 6.960 20 0.314 6.280 20 0.321 6.420 20 0.257 5.140 1 1.843 2.038 1 2.038 1.970 1 1.712 1.843 1 1.970 1.712

Concentration of glucose g/l 11.506 11.307 9.830 9.886 8.920 9.119 7.301 2.895 2.798 2.618 2.432

Time (hours) 0 1.5 3 4.5 6 7.5 18 19.5 21 22.5 24

Table 4: concentration of xylose against time Absorbance Dilution factor reading at 540nm Concentration of xylose with dilution actual reading g/l 20 0.241 4.820 6.847 20 0.688 13.760 19.545 20 0.639 12.780 18.153 20 0.644 12.880 18.295 20 0.510 10.200 14.489 20 0.516 10.320 14.659 20 0.641 12.820 18.210 1 2.085 2.085 2.962 1 2.296 2.296 3.261 1 1.951 1.951 2.771 1 2.024 2.024 2.875

Cell density (OD) and concentration of maltose vesus cultivation time

10.000 9.000 OD reading at 600nm 8.000 7.000 6.000 5.000 4.000 3.000 2.000 1.000 0.000 0 5 10 time (hours) 15 20 0.000 4.000 2.000 6.000 10.000 8.000 12.000 concentration of maltose g/l

OD [maltose]

Figure 2: cell density (OD) and concentration of maltose versus cultivation time

Cell density (OD) and concentration of glucose versus cultivation time

3.000 OD reading at 600nm 2.500 2.000 1.500 6.000 1.000 0.500 0.000 0 5 10 time (hours) 15 20 4.000 2.000 0.000 14.000 12.000 10.000 8.000 concentration of glucose g/l

OD [glucose]

Figure 3: cell density (OD) and concentration of glucose versus cultivation time

Cell density (OD) and concentration of xylose versus cultivation time

4.500 4.000 OD reading at 600nm 3.500 3.000 2.500 2.000 1.500 1.000 0.500 0.000 0 5 10 time (hours) 15 20 0.000 20.000 15.000 10.000 5.000 25.000 concentration of xylose g/l

OD [xylose]

Figure 4: cell density (OD) and concentration of xylose versus cultivation time

Discussion Because of microbes produce adaptive enzymes in response to the presence of substrates that are not usually preferred by other organisms, they can accept different carbon source but will undergo different pathway of utilizing it. Glucose is the most preferable and readily form of carbon source to be used by microorganism. In utilization of maltose, it is initially transformed to polysaccharide and glucose. This reaction followed by the phosphorolytic decomposition of the polysaccaharide and the usual sequence of glycolytic steps. Thus the final product will become glucose as a readily carbon source to be used by the microorganism. In metabolism of xylose, there is three pathway that are common in prokaryote that is isomerase pathway, Weimberg pathway and Dahms pathway. The isomerase pathway the enzyme xylose isomerase is responsible for the conversion of D-xylose into D-xylulose. D-xylulose is then phosphorylated to D-xylulose-5-phosphate as in the oxireductive pathway. The Weimberg pathway is an oxidative pathway where the D-xylose is oxidized to D-xylono-lactone by a D-xylose dehydrogenase followed by a lactonase to hydrolyze the lactone to D-xylonic acid. A xylonate dehydratase is splitting off a water molecule resulting in 2-keto 3-deoxy-xylonate. A second dehydratase forms the 2-keto glutarate semialdehyde which is subsequently oxidised to 2-ketoglutarate.The Dahms pathwaystarts as the Weimberg pathway but the 2-keto-3 deoxy-xylonate is split by an aldolase to pyruvate and glycoladehyde.

Based on the graph of cell density (OD) versus cultivation time of different carbon source it show the microbial growth kinetics consist of lag phase, log phase and stationary phase. During lag phase, the growth of microorganism are almost the same in each carbon source. There is only a small increase in numbers as they are still adapting the growth and adjusting the new condition. The growth start to enter the log phase on the forth hours in each carbon source. It show that maltose as carbon source has higher growth limit after 5th hour followed by xylose and glucose. This because maltose contain more carbon compare to glucose and xylose. maltose has higher number of carbon and many of the maltose has been broken down to a simple sugar and readily consumed by the bacteria for rapid growth of cell as maltose has higher number of carbon that can support large number of bacterial requirement for carbon. In glucose as carbon source, the growth limit is low because glucose is simplest type of sugar that is easily consumed by the E.coli for the cell growth, so it is faster utilize by the microorganism and faster depleted compare to other like maltose and xylose that must undergo several pathway before it can be used.

In comparing the growth rate in this log phase, it show maltose as carbon source has greatest rate. However, glucose as carbon source has greater rate compare to xylose only until 6th hours, then entering stationary phase, while xylose still increasing until approximately 7th hours before entering stationary phase. Stationary phase is where the growth rate slows and the production of new cells equals the rate of cell death. It involves the establishment of an equilibrium in population numbers and a slowing of the metabolic activities of individual cells. The stationary phase reflects a change in growing condition for example, a lack of nutrients and/or the accumulation of waste products. Suppose that after stationary it enter death phase. Death phase is when the rate of cell deaths exceeds the number of new cells formed, the population equilibrium shifts to a net reduction in numbers and the population enters the death phase, or logarithmic decline phase. The population may diminish until only a few cells remain, or the population may die out entirely. Based on the gowth curve, suppose that the cell density decrease between 8th to 18th hours. As the result is not taken during that time, the deacreasing might not be taken. The cell decrease may due to accumulation of waste product. As only few cell still survive, it become stable back and start to increase the cell density but not to high. However in maltose and xylose the increasing is higher because the carbon contain is higher. The cell density will eventually decreasing back as the nutrient is no longer sufficient to support the gowth.

In the second part of the experiment, the DNS method, 3,5-Dinitrosalicylic acid (DNS) is an aromatic compound that reacts with reducing sugars and other reducing molecules to form 3-amino-5nitrosalicylic acid, which can absorb light by spectrophotometer. Thus, as the colour of the reacted DNS darken, the absorbance reading of the sample is increased. Since the DNS reacted with the reducing sugar, the concentration of glucose, maltose and xylose can be determined respectively. In the result obtained, the absorbance reading for concentration of maltose, glucose and xylose is decreasing gradually as the cultivation time increases and the number of cell is increases also. This means that the amount of cell increases as the cells consumes the substrate to grow in the culture.

Based on figure 2, it show that glucose is rapidly decreasing. This is because, it is the simplest form of sugar and easily consumed by the cells. For xylose, the graph in figure 4, the early reading of the xylose concentration is lower and suddenly increase may due to error in collecting reading. After the 3rd hours the xylose concentraion was unevenly distributed throughout the inoculation time. This maybe because xylose is hardly degraded by bacteria since it is bigger and more complex metabolism compare to glucose or maltose. In figure 2, the maltose concentration decrasing rapidly.

This is because, in metabolism of maltose in E.coli, the maltose is initially transformed into polysaccharide and glucose by enzyme called amylomaltose. This reaction is followed by the phosphorolytic decomposition of the polysaccharide and the usual sequence of glycolytic steps. The DNS cannot detect the polysaccharide.

In precaution step, when taking the absorbance reading of the sample, always try to keep below 1. Because spectrometer, percentage of transmittance is related logarithmically with concentration, it means that from 1-99% transmittance it can detect approximately 2 order of magnitude analyte concentration. Any order of magnitude greater than that will be detected in the range of 0-1% of transmittance. In order to maximize accuracy, the solution must be diluted if have to do so that the transmittance reading is not maxed out in that region.

Conclusion The usage of different carbon source has different effect on growth rate and growth limit of microorganism on certain growth phase. Thus this variable can be used to control the growth rate in industrial microorganism.

Reference 1. Hughes, D., Wimpeny, J. and Lloyd, D. in Methods in Microbiology Academic Press, London, 1971 2. Direct utilization of maltose by escherichia coli; michael doudoroff, w. 2. Hassid, e. W.Putman, and a. L. Potter ;Universily of Wisconsin ; December 29, 1948) 3. http://www.textbookofbacteriology.net/index.html