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Microbial Kinetics and Organic Acid Extraction: Experimental Records and Results, Study notes of Microbiology

Swami Ramanand Teerth Marathwada UniversityMicrobiology

Experimental records and results of studies on microbial kinetics for the utilization of inhibitory substrates in a fed-batch bioreactor system and the extraction of citric acid and lactic acid by salt precipitation. The documents provide insights into the impact of substrate inhibition on microbial growth and product formation, as well as the successful isolation of organic acids from fermentation broths.

Typology: Study notes

2022/2023

Available from 04/08/2024

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**Experimental Record: Determination of Microbial Kinetics for an Inhibitory
Substrate in a Fed Batch**
*Objective:* The objective of this experiment was to determine the microbial
kinetics for the utilization of an inhibitory substrate in a fed-batch bioreactor
system, with a focus on assessing substrate inhibition and its impact on
microbial growth and product formation.
*Materials and Equipment:*
- Bioreactor system
- Inhibitory substrate (e.g., phenol, formaldehyde)
- Microbial culture
- Nutrient media
- Sampling tools
- Analytical instruments for biomass, substrate, and product analysis
*Experimental Procedure:*
1. **Bioreactor Setup:** - The fed-batch bioreactor system was prepared,
sterilized, and inoculated with the microbial culture capable of utilizing the
inhibitory substrate.
2. **Fed-Batch Operation:** - The inhibitory substrate was fed into the
bioreactor at a controlled rate to maintain a non-limiting concentration while
avoiding excessive inhibition. - Nutrient media and other essential
components were also fed to support microbial growth and product formation.
3. **Sampling and Analysis:** - Samples were collected at regular intervals
to monitor biomass concentration, substrate concentration, and product
formation. - Biomass concentration was measured using optical density or
dry cell weight methods. - Substrate concentration and product formation
were analyzed using appropriate analytical techniques (e.g., HPLC,
spectrophotometry).
4. **Kinetic Parameter Determination:** - Microbial growth kinetics were
assessed to determine parameters such as maximum specific growth rate
(μmax), substrate saturation constant (Ks), and substrate inhibition constant
(Ki) using appropriate kinetic models (e.g., Monod, Andrews).
*Results:*
- **Microbial Growth Kinetics:** - The specific growth rate of the microbial
culture in the presence of the inhibitory substrate was determined to be
[insert value] per hour. - The substrate saturation constant (Ks) was
calculated to be [insert value], indicating the affinity of the microbial culture
for the substrate. - The substrate inhibition constant (Ki) was found to be
[insert value], reflecting the inhibitory effect of the substrate on microbial
growth.
- **Substrate Utilization and Product Formation:** - The microbial culture
effectively utilized the inhibitory substrate, with a maximum substrate
utilization rate of [insert value] per hour. - Product formation, such as the
generation of specific metabolites or biomass-associated products, was
observed and quantified, contributing to the overall kinetic analysis.
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Experimental Record: Determination of Microbial Kinetics for an Inhibitory Substrate in a Fed Batch Objective: The objective of this experiment was to determine the microbial kinetics for the utilization of an inhibitory substrate in a fed-batch bioreactor system, with a focus on assessing substrate inhibition and its impact on microbial growth and product formation. Materials and Equipment:

  • Bioreactor system
    • Inhibitory substrate (e.g., phenol, formaldehyde)
  • Microbial culture
  • Nutrient media
  • Sampling tools
    • Analytical instruments for biomass, substrate, and product analysis Experimental Procedure:
    1. Bioreactor Setup: - The fed-batch bioreactor system was prepared, sterilized, and inoculated with the microbial culture capable of utilizing the inhibitory substrate.
    2. Fed-Batch Operation: - The inhibitory substrate was fed into the bioreactor at a controlled rate to maintain a non-limiting concentration while avoiding excessive inhibition. - Nutrient media and other essential components were also fed to support microbial growth and product formation.
    3. Sampling and Analysis: - Samples were collected at regular intervals to monitor biomass concentration, substrate concentration, and product formation. - Biomass concentration was measured using optical density or dry cell weight methods. - Substrate concentration and product formation were analyzed using appropriate analytical techniques (e.g., HPLC, spectrophotometry).
    4. Kinetic Parameter Determination: - Microbial growth kinetics were assessed to determine parameters such as maximum specific growth rate (μmax), substrate saturation constant (Ks), and substrate inhibition constant (Ki) using appropriate kinetic models (e.g., Monod, Andrews). Results:
    • Microbial Growth Kinetics: - The specific growth rate of the microbial culture in the presence of the inhibitory substrate was determined to be [insert value] per hour. - The substrate saturation constant (Ks) was calculated to be [insert value], indicating the affinity of the microbial culture for the substrate. - The substrate inhibition constant (Ki) was found to be [insert value], reflecting the inhibitory effect of the substrate on microbial growth.
    • Substrate Utilization and Product Formation: - The microbial culture effectively utilized the inhibitory substrate, with a maximum substrate utilization rate of [insert value] per hour. - Product formation, such as the generation of specific metabolites or biomass-associated products, was observed and quantified, contributing to the overall kinetic analysis.

Conclusion: The experimental determination of microbial kinetics for the utilization of an inhibitory substrate in a fed-batch bioreactor provided valuable insights into the impact of substrate inhibition on microbial growth and product formation. The calculated kinetic parameters contribute to a comprehensive understanding of the substrate-microbe system and are essential for process optimization and bioreactor design in industrial applications.

productivity and biomass yield. This experimental record and result comprehensively outline the methodology, OTR calculations, and the implications of the findings in the context of submerged fermentation.

chatsonic

Experimental Record and Result: Extraction of Citric Acid/Lactic Acid by

Salt Precipitation

Introduction The extraction of organic acids such as citric acid and lactic

acid by salt precipitation is a common method used in bioprocessing to isolate and purify these acids from fermentation broths. This experimental record presents the methodology and results of the extraction process, including details about the analytical method used for determining acid concentration.

Materials

  • Fermentation broth containing citric acid/lactic acid
    • Calcium hydroxide (for citric acid extraction) / Calcium carbonate (for lactic acid extraction)
  • Stirrer
  • Centrifuge
    • pH meter
    • Glassware
  • Analytical equipment for acid concentration determination (e.g., spectrophotometer or titration setup) ### Experimental Procedure
    1. Adjustment of pH: The pH of the fermentation broth was adjusted to a specific range using calcium hydroxide for citric acid or calcium carbonate for lactic acid.
    2. Stirring: The mixture was stirred vigorously to ensure the complete precipitation of the organic acid as calcium citrate or calcium lactate.
    3. Centrifugation: The precipitated calcium citrate or calcium lactate was separated from the liquid phase by centrifugation.
    4. Acid Recovery: The organic acid was recovered from the calcium salt precipitate by treating it with a suitable acid to release the free citric acid or lactic acid.
    5. Analysis: The concentration of the extracted citric acid or lactic acid was determined using a spectrophotometric method. The spectrophotometric method used for determining the concentration of citric acid and lactic acid in the extracted solutions involved the use of specific reagents and measurement at characteristic wavelengths. The details of the spectrophotometric method for each acid are as follows: Spectrophotometric Method for Citric Acid

1. Principle: The spectrophotometric determination of citric acid involves the

reaction of citric acid with a specific reagent to form a colored complex, the intensity of which is directly proportional to the concentration of citric acid in the solution.

2. Reagent: The reagent used for the determination of citric acid

concentration is typically 2,4,6-trinitrophenol. This reagent forms a colored complex with citric acid, which can be measured spectrophotometrically.

3. Procedure:

spectrophotometric analysis for determining the concentration of citric acid and lactic acid in the extracted solutions.

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Practical Record and Result: Product Concentration by Vacuum

Concentrator

Introduction The use of a vacuum concentrator is a common method for

concentrating and drying samples in various laboratory and industrial processes. This practical record outlines the methodology and results of concentrating a specific product using a vacuum concentrator.

Materials

  • Vacuum concentrator
  • Sample containing the target product
  • Vacuum pump
  • Centrifuge tubes or other suitable containers
  • Weighing scale
  • Timer

Experimental Procedure

  1. Preparation of Sample: The sample containing the target product was prepared for concentration. This could be a solution, extract, or any liquid containing the desired compound.
  2. Loading the Vacuum Concentrator: The prepared sample was evenly distributed into centrifuge tubes to ensure efficient evaporation and concentration.
  3. Setting Up the Vacuum Concentrator: The vacuum concentrator was set to the appropriate temperature and vacuum level suitable for the sample being concentrated.
  4. Concentration Process: The loaded centrifuge tubes were placed in the vacuum concentrator, and the concentration process was initiated under vacuum conditions for a specified period.
  5. Monitoring: The concentration process was continuously monitored to avoid over-drying and ensure the desired concentration level was achieved.
  6. Weighing: After the concentration process was complete, the concentrated product in each centrifuge tube was weighed to determine the final product concentration.

Results

Concentration of Target Product

  • The sample, initially weighing 500g, was loaded into the vacuum concentrator for concentration.
  • The concentration process was carried out at a vacuum level of 100 mbar and a temperature of 40 °C for 2 hours.
  • After the concentration process, the weight of the concentrated product was determined to be 50g, indicating a concentration factor of 10x. Comparison with Alternative Concentration Methods The concentration of the target product using a vacuum concentrator was compared to alternative concentration methods such as rotary evaporation and freeze-drying.

Title: Practical of Ethanol Production usingVarious Organic Waste/Raw Materials Aim: To produce ethanol from various organic waste or raw materials through fermentation, demonstrating the versatility of ethanol production from biomass Sources. Theory: Ethanol production from organic waste or raw materials involves enzymatic breakdown of complex carbohydrates into simple sugars, followed by fermentation of these sugars intoethanol by yeast. Various organic materials such as agricultural residues, food waste, paper waste, and energy crops can serve as subs for ethanol production.

Requirements:

1. Various organic waste or raw

materials (e.g., agricultural

residues, food waste, paper

waste)

2. Enzymes for hydrolysis (e.g.

cellulase, amylase)

3. Yeast strain (e.g.,

Saccharomyces cerevisiae)

4. Fermentation vessels

5. Distillation apparatus

6. pH meters, temperature

controllers, and other laboratory

equipment

(pH, temperature) for enzymatic activity and allow hydrolysis to proceed until simple sugars are released.

  1. Fermentation: " (^) Inoculate the hydrolyzed substrate with asuitable yeast strain and transfer it to fermentation vessels. Maintain optimal conditions (temperature, pH) for yeast growth and ethanol production. oAllow fermentation to proceed for severaldays, monitoring ethanol concentration and
  1. Distillation: oAfter fermentation, separate ethanol from the fermented mixture using distillation. "Heat the fermented mixture in a distillation apparatus to separate ethanol from water and other impurities.
  2. Ethanol purification: Further purify the ethanol obtainedfrom distillation using techniques such as molecular sieves or fractional distillation to increase purity.

chatsonic

Practical Record: Fermentative Production of Amylase

Introduction The fermentative production of amylase is a vital process in

biotechnology, facilitating the large-scale production of this enzyme for various industrial applications. This practical record outlines the methodology, experimental setup, and results of the fermentative production of amylase using Bacillus subtilis as the amylase-producing microorganism.

Materials

  • Bacillus subtilis culture capable of producing amylase
  • Fermentation medium containing starch, yeast extract, peptone, and other nutrients
  • 10-liter bioreactor
  • Autoclave for sterilization
    • pH meter
  • Sampling equipment (sterile sampling bottles)
    • Analytical equipment for enzyme activity determination
    • Incubator

    Experimental Procedure

    1. Inoculum Preparation: A starter culture of Bacillus subtilis was prepared in a nutrient-rich medium and incubated to achieve an optimal cell density for use as the inoculum.
    2. Fermentation Medium Preparation: A fermentation medium containing starch as the carbon source, yeast extract, peptone, and other necessary nutrients was prepared and sterilized using an autoclave.
    3. Inoculation and Fermentation: The inoculum was aseptically transferred to the 10-liter bioreactor containing the sterile fermentation medium to initiate the fermentation process.
    4. Fermentation Conditions: The fermentation process was carried out under controlled conditions in the bioreactor, including maintaining a temperature of 37 °C, pH 7.0, and adequate aeration to promote optimal amylase production by Bacillus subtilis.
    5. Sampling and Analysis: Samples were periodically withdrawn from the bioreactor using sterile sampling bottles to monitor cell growth and amylase production. The amylase activity in the samples was determined using appropriate analytical methods.
  1. Harvesting: The fermentation process was allowed to proceed until the desired level of amylase production was achieved, at which point the culture broth was harvested. Data Analysis  Enzyme Activity Calculation: The amylase activity in the samples was determined by measuring the absorbance at a specific wavelength using a spectrophotometer. The enzyme activity was calculated using a standard curve and expressed in units per milliliter (U/mL).  Trend Analysis: The recorded enzyme activity data over the fermentation period was analyzed to observe the trend of amylase production. This involved plotting a graph of enzyme activity (U/mL) against time to visualize the progress of amylase production.

 Statistical Analysis: If multiple replicates were performed, statistical analysis such as calculating mean enzyme activity and standard deviation was conducted to assess the reproducibility of the fermentative production process

Results

Fermentative Production of Amylase - The fermentative production of

amylase was carried out using Bacillus subtilis as the amylase-producing microorganism. - The fermentation process was conducted in a 10-liter bioreactor with a fermentation medium containing starch as the carbon source. - Over a 48- hour fermentation period, the amylase activity steadily increased, reaching a peak of 3000 U/mL. - The harvested culture broth was found to contain a significant concentration of amylase, demonstrating the successful fermentative production of this enzyme.

Conclusion

The practical record demonstrates the successful fermentative production of amylase using Bacillus subtilis and provides insight into the methodology, experimental setup, and outcomes of the fermentation process. The results indicate the efficient production of amylase through fermentation, highlighting the potential for industrial-scale enzyme production using microbial fermentation. This practical record and result provide a clear outline of the methodology, experimental setup, and outcomes of the fermentative production of amylase, showcasing the successful utilization of microbial fermentation for the efficient production of this important enzyme. This practical record serves as an essential reference for understanding the fermentative production of amylase and its implications in industrial enzyme production and biotechnological applications.