Microbial Growth and Yield Coefficient Determination by Turbidimetry

Experiment 2 Determination of growth and yield co-efficient by turbidimetry Date: Growth is an orderly increase in cellular components. It may be considered as the increase in cell size and/or number, marked by an increase in net or gross weight of microbes. The rate of growth is influenced by both internal and external environmental factors. The internal factors may be the genetic constitution of the microorganisms, the age of the inoculum used. External environmental factors represent the incubation temperature, pH, light and the nutritional factors. Growth rate represents the number of cells produced by cell division or by budding per unit time. When bacteria are grown in the liquid medium, the nature of the growth of the bacterial population can be studied by plotting the cell number versus incubation time and analyzing the resulting growth curve. In microorganisms reproducing by binary fission, the logarithm of cell number is plotted on semi-logarithmic graph paper. The curve has four distinct phases. The rate of growth and death of the organism has been divided into a number of phases. The general growth phases of bacteria are: Lag phase: In this phase, the inoculum remains temporarily constant, but the individual cells are physically active. They get adjusted to the new environment and increase in cell size but do not multiply. Log phase: It is the logarithmic or exponential phase. Here the cells divide steadily, and the maximum rate of increase in population is obtained. Stationary phase: Due to the exhaustion of nutrients, the metabolic activity will be in a reduced state. Here the rate of increase in population and death of cells is balanced. Decline phase: The rate of bacterial death is more than the rate of reproduction, and hence there is a steady decline in the population. AGM 312 Microbial Biotechnology (1+1) Page 6 Yeast is a unicellular eukaryote, whereas lactic acid bacteria are a prokaryotic probiotic. Both of them differ in their nutritional requirements. The nutrient source for maximum growth of yeast and lactic acid bacteria is assessed by their growth pattern in various carbon and nitrogen sources. Yeast multiplies by budding while lactic acid bacteria by binary fission. When growing exponentially by binary fission, the increase in bacterial population is by geometric progression. The generation time is the time interval required for the cells to divide. The time needed for the population to double is termed as doubling time. No of generations (n) = 𝐿𝑜𝑔 𝑐𝑒𝑙𝑙𝑠 𝑎𝑡 𝑡𝑕𝑒 𝑒𝑛𝑑 𝑜𝑓 𝑖𝑛𝑐𝑢𝑏𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒– 𝐿𝑜𝑔 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑎𝑡 𝑖𝑛𝑖𝑡𝑖𝑎𝑙 0.301 Generation time = a) Direct methods 𝑡 𝑛 It includes:     Filtration Plate count Direct Microscopic count Most Probable number b) Indirect Methods of Measurement 1. Turbidimetry: Turbidimetry refers to the determination of cell density by optical measurements. It is one of the simplest methods used to analyze trends in growth because it uses a spectrophotometer to track changes in the optical density (OD) over time. The turbidity or optical density of a suspension of cells is directly related to cell mass or cell number. The method is simple and non-destructive. But the sensitivity is limited to about 107 cells/ml for most bacteria. 2. Metabolic Activity: As the microbe grows in the culture media, they produce certain metabolic products such as carbon dioxide and acids. Quantification of these metabolic products provides details about microbial growth. In addition to this, cellular protein can also be estimated and correlated with turbidity and cell count. Disadvantage: Measuring the metabolic products is expensive AGM 312 Microbial Biotechnology (1+1) Page 7 3.Dry Weight/Biomass: The microbial growth is determined on a weight basis. Bacteria or fungi in liquid media are centrifuged, and the resulting cell pellet is weighed. Disadvantage: Doesn‟t distinguish live and dead cells. Aim: To determine the growth and yield coefficiency of yeast and lactic acid bacteria by turbidity measurement. Materials Required: YPD broth ( Yeast) Peptic digest of animal tissue Yeast extract Dextrose Distilled water pH : : : : : MRS broth composition (Lactobacillus) Glucose : Peptone : Beef extract : Yeast extract : K2 HPO4 : Sodium acetate : Triammonium citrate : Magnesium sulfate : Manganese sulfate : Tween 80 : Distilled water : pH : AGM 312 Microbial Biotechnology (1+1) 20 g 10 g 20 g 1000 ml 6.5 20 g 10 g 10 g 5g 2g 5g 2g 0.2g 0.05g 1ml 1000 ml 6.0 Page 8 C sources: glucose, sucrose, fructose, maltose, mannitol N sources: Peptone, Ammonium chloride Procedure: a. Growth pattern of Yeast         Prepare YPD broth with given C and N sources Inoculate fresh culture of yeast (logarithmic phase) @ 2% Incubate at 30±2 ºC for 24- 48 h Harvest the cells by centrifugation at 10,000 rpm for 5 min Transfer the cell pellet into a pre-weighed filter paper and observe the fresh weight Dry the pellet in a hot air oven at 105ºC until a constant weight Observe the dry weight Express the biomass of yeast in terms of mg/l or mg/g of C source. Note: Alternatively, the cells can be filtered through a sterile pre-weighed filter paper. The fresh and dry weight is measured, as mentioned above. b. Growth pattern of Lactic acid bacteria       Inoculate the given lactic acid bacteria (2% inoculum) into MRS broth prepared with different C and N sources Incubate at 30±2 ºC for 24- 48 h Observe the growth by measuring the OD at 600 nm Plot the absorbance values against time in a semi-log graph paper Plate count the corresponding population in the respective medium at every 2 h Calculate the number of generations (n) and generation time (t). Activity:       Prepare YPD and MRS broth with the given C and N source Measure the absorbance at regular interval Determine the fresh and dry weight Plot graph based on OD value and cell population Work out the generation time Observe the most preferred C and N source for maximum growth AGM 312 Microbial Biotechnology (1+1) Page 9 Results: Review Questions: 1. What are the different phases of bacterial growth? 2. What happens when a microbial population is transferred from rich medium to poor or minimal medium? 3. What is actually assessed in the turbidimetric method? AGM 312 Microbial Biotechnology (1+1) Page 10 4. Differentiate autotrophs and heterotrophs? 5. What are the factors required for the successful cultivation of microbes? 6. If you are an entrepreneur, planning for yeast-based single-cell production, what are the criteria for you to consider for enhancing yeast biomass production? Reference JoVE Science Education Database. Environmental Microbiology. Bacterial Growth Curve Analysis and its Environmental Applications. JoVE, Cambridge, MA (2020). AGM 312 Microbial Biotechnology (1+1) Page 11