7.3: Microbial Cultivation and Manipulation

Biotech Focus

Are bacteria the next "hot" thing in fashion? The clothes people wear require a massive amount of energy and resources to produce. Only to have those clothes thrown away when next year's fashion trends appear. So, when it comes to sustainability, the fashion industry is pretty unfashionable. The solution? Bacteria, or rather, bacterial cellulose (BC). BC is a network of carbohydrate molecules that can be excreted by some species of bacteria. BC is an exciting idea in the fashion industry because it can be crafted into everything from handbags to jackets and because BC is biodegradable, it is an attractive solution to an unattractive problem. But how is BC transformed into "fabulous fashion”? Microbiologists grow the bacteria in large bioreactors under carefully controlled conditions that promotes their growth and production of BC. The resulting BC fibers are extracted from the bioreactor, washed, and dried. Depending how the bacteria are cultured in the bioreactor, the BC can be similar to leather, thread, or paper-like. Despite how exciting BC is, it is still in the prototype phase and has only been shown at a few fashion shows. But it may only be a matter of time before BC-containing fashions become mainstream in the fashion world and out on the streets.

To read more about BC and the fashion industry, check out the article Are Bacteria the Next Big Thing in Fashion?

Introduction

The cultivation and manipulation of microbes, like bacteria and yeast, are foundational aspects of microbiology and biotechnology. The ability to grow, control, and genetically manipulate bacteria and yeast has revolutionized industries from medicine to agriculture, environmental management, and even energy production. This page explores the principles and techniques involved in microbial cultivation, with an emphasis on bacteria cultivation principles like aseptic techinque, and outlines some of the the challenges that arise in manipulating these organisms.

Learning Objectives

Microbiology and biotechnology rely upon the cultivation and manipulation of microbes like bacteria and yeast. At the end of this section, you will be able to:

• Describe the physical conditions necessary for microbial growth
• Describe the types of media used in growing microbes
• Explain the four phases of a microbial growth curve
• Describe microbial aseptic technique
• Describe bioreactors and fermenters and how they are used in microbial cultivation
• Describe how microbes can be manipulated genetically and metabolically
• Explain a microbial consortia and co-cultivation

Microbial Cultivation

Microbial cultivation in microbiology and biotechnology refers to the process of growing microbes, like bacteria or yeast, under controlled laboratory conditions in order to study their physiology, biochemistry, genetics, or for industrial production of valuable compounds. The ultimate goal of cultivation is to provide an environment that mimics the organism's natural habitat as closely as possible, allowing for growth and replication. In order to provide the optimal growth conditions, it is important to consider the species, the type of medium and the physical conditions for growth.

Physical Conditions for Growth

Microbes, like bacteria and yeast, require specific environmental conditions in order to optimize their growth. The primary factors that need to be controlled during cultivation include:

1. Temperature
2. Ph
3. Oxygen levels
4. Nutrients

Temperature

When culturing microbes, it’s essential to consider the incubation temperature specific to each organism, as they have preferred growth temperatures. For instance, E. coli, commonly found in the human intestine, thrive at 37°C, aligning with body temperature. Similarly, the yeast Saccharomyces cerevisiae, used in brewing and baking, grows optimally between 28°C and 32°C. Based on their ideal growth temperatures, microbes can be classified into three main groups: psychrophiles (cold-loving, optimal growth between -20°C and 20°C), mesophiles (moderate temperature-loving, best growth between 20°C and 45°C), and thermophiles (heat-loving, growing above 50°C).

pH

The pH level of an environment, indicating its acidity or alkalinity, significantly influences enzyme activity and cellular processes. Like temperature, it is important to consider the pH when culturing microbes, as each type has an optimal pH for growth. Acidophiles thrive in acidic conditions, neutrophiles prefer a neutral pH, and alkaliphiles grow best in alkaline environments.

Oxygen Availability

Microbes have varying requirements for and responses to oxygen, so oxygen levels must be carefully controlled in culture conditions. Oxygen is essential for aerobic respiration as it provides an efficient way to generate ATP. However, oxygen can also produce harmful reactive oxygen species (ROS), highly reactive molecules that can damage lipids, proteins, and DNA. ROS molecules are produced as normal by-products of cellular metabolism, but are also generated by environmental stresses. Some cells have enzymes that neutralize these toxic byproducts, while others lack this capability and therefore must be cultured in anaerobic conditions. Certain microbes require oxygen to grow, while others can switch to less efficient pathways, like fermentation, in its absence.

Microbes exhibit a spectrum of oxygen needs, including:

• obligate aerobes: cells that require oxygen to grow; use cellular respiration to generate ATP
• facultative anaerobes: cells that prefer to grow in the presence of oxygen but can survive in its absence; use aerobic respiration, fermentation and other anaerobic mechanisms to generate ATP
• aerotolerant anaerobes: cells that do not use oxygen but can survive in its presence; use only fermentation to generate ATP
• obligate anaerobes: cells that cannot survive in the presence of oxygen; use anaerobic metabolism like fermentation to generate ATP

Nutrient Availability

The availability of nutrients in microbial cultures is crucial to optimizing cellular growth rate and behavior. These nutrients serve as the building blocks for cell growth, development and reproduction, and provide essential elements for cellular processes like energy production and biosynthesis. Microbes need a carbon source (e.g., glucose, lactose), nitrogen, phosphorus, sulfur, trace elements, and vitamins for growth. Carbon sources, like glucose, provide essential energy, while nitrogen supports protein and nucleotide synthesis. Phosphorus is critical for nucleic acids and energy molecules like ATP, while sulfur is necessary for specific amino acids and vitamins. Minerals and trace elements, including potassium, magnesium, and iron, are crucial for enzyme function and cellular stability. Many microbes also need growth factors or vitamins as coenzymes for metabolic pathways, especially fastidious species requiring enriched media. By optimizing these nutrient sources, microbial growth and function can be effectively supported in culture. Therefore, specific types of media have been designed based on the metabolic profile of the microbe, in order to ensure that all nutrients are present in the correct forms and concentrations to support optimal microbial activity and research outcomes.

Types of Culture Media

Essential nutrients are supplied through a culture medium (plural = media). Different types of culture media serve different purposes. For additional information, go to Chapter 7.2 Microbial Identification.

• Solid Medium: Often containing agar, a gelatinous substance derived from seaweed, solid media supports surface growth, allowing for the formation of colonies. It is useful for isolating pure cultures and observing colony morphology. Solid media can be formed into plates, slants and stabs.
• Liquid Medium: Also known as broth, a liquid medium is used for growing large numbers of cells in a homogeneous suspension. Broth culture is ideal for studying growth kinetics and conducting biochemical tests.
• Semi-solid Medium: Has a lower concentration of agar, allowing it to be used for motility studies and the propagation of organisms that require low-levels of oxygen to grow (i.e., microaerophilic).
• Selective Medium: Contains compounds that suppress the growth of certain microorganisms while promoting the growth of others. For example, MacConkey agar selects for Gram-negative bacteria.
• Differential Medium: Designed to differentiate between cell types based on metabolic activity. Differential medium contains indicators that reveal specific biochemical reactions, such as the ability to ferment carbohydrates.
• Enriched Medium: Provides additional nutrients to support the growth of fastidious organisms that require specific growth factors.

Cultivation Growth Phases

When microbes, like bacteria and yeast, are grown in batch culture, they typically pass through four distinct phases of growth (Figure \(\PageIndex{1}\)):

1. Lag Phase: Cells are adapting to the new environment, synthesizing enzymes, and preparing for division. There is little to no cell division during this phase.
2. Log Phase (Exponential Phase): Cells divide at a constant rate, and growth is exponential. This phase is ideal for studying metabolism and for industrial processes.
3. Stationary Phase: The rate of cell growth equals the rate of cell death, as nutrients become limited and waste products accumulate. The culture reaches a plateau in population size.
4. Death Phase: Nutrients in the culture medium are depleted, and waste products build up to toxic levels, causing the death of cells at an exponential rate.

Figure \(\PageIndex{1}\): Cell growth curve. The curve in this figure shows the number of bacterial cells plotted over time. The curve is divided into four distinct phases: the Lag phase with minimal increase in cell number, the Log phase marked by rapid cell multiplication, the Stationary phase where growth stabilizes, and the Death phase, reflecting a decrease in the number of live cells. (Cell Growth Curve by Kareen Martin; CC BY 4.0)

Aseptic Conditions for Microbial Cultivation

Aseptic conditions in microbial cultivation are essential to prevent contamination and the growth of unwanted microbes. Preparation of liquid and solid media, in addition to the establishment of bacterial cultures, are all done under aseptic conditions. Aseptic conditions for microbial cultivation include the use of sterile materials, the creation of a controlled environment, and the employment of specific aseptic techniques. Autoclaving to sterilize materials and equipment should be considered where appropriate. The purchase of pre-sterilized materials may also be an option. Work areas should be disinfected with 70% ethanol, a dilute bleach solution, or another suitable disinfectant. The work area should also be kept clutter free and should only contain the materials to be used. Hands should be washed thoroughly before and after working with microbes. A lab coat may be worn, along with gloves, a face mask or goggles. Wearing a face mask or goggles is not required but will depend on the specific microbe being manipulated. As with face masks and goggles, the use of gloves is not mandatory but is generally encouraged when working with any microbe.

When establishing a microbial culture, specific steps are used in microbiology to prevent contamination. These steps are collectively referred to as aseptic technique. Key practices underlying aseptic technique include:

• Disinfect work surfaces before and after use.
• Work using a flame or in a laminar flow hood.
• Always sterilize inoculating loops and needles by flaming until red-hot.
• Do not place caps or lids on the bench; hold them in hand or keep upright.
• Minimize exposure time of media and cultures to air.

The most important tool used in microbial aseptic technique is the Bunsen burner. The Bunsen burner is used to "flame" tools like inoculating loops or needles until red hot before and after use. The burner is also used to quickly flame the opening of bottles, tubes, and flasks to eliminate potential contaminants. Wearing gloves when working with a Bunsen burner should also be carefully considered, as most disposable gloves (e.g., latex, nitrile) are flammable. Therefore, hands should be kept away from open flames.

Concept in Action

Aseptic Technique in Microbiology

While Chapter 6: Mammalian Cell Culture Fundamentals also describes aseptic technique, this technique is specific to the culture of mammalian cells. However, the principles of mammalian cell aseptic technique are much the same as bacterial aseptic technique in that sterile materials, a controlled environment, and specific techniques are used. There are some differences, such as the absence of a Bunsen burner and the use of a biosafety cabinet to ensure sterility.

Bioreactors Are Used in Microbial Cultivation

A bioreactor is a controlled vessel used to grow biological organisms under controlled conditions. The purpose of a bioreactors is the continuous culture of biological organisms, like microbes, for various applications. Bioreactors are widely used in industries like biotechnology, pharmaceuticals, and food production for the large-scale synthesis of products like antibiotics, enzymes, and pharmaceuticals. For example, insulin production uses genetically-modified bacteria or yeast grown in bioreactors to create human insulin for diabetic patients. The microbial fermenter (i.e. fermenter) is a type of bioreactor that provides the ideal environment for microbes, such as bacteria or yeast, to grow and perform fermentation processes under controlled conditions. Despite their name, fermenters can also grow organisms under aerobic conditions since aerobic fermentation is more efficient and can significantly increase cell biomass and their desired products. They can be used for batch culture or continuous culture.

Fermenters can range from small-scale laboratory reactors to large industrial fermenters. A typical industrial fermenter is a large stainless steel chamber (or multiple chambers) that can hold large volumes of culture medium (Figure \(\PageIndex{2}\)). The fermenter has a sterilization system to ensure the environment is free from contaminants before the microbe is added, an inoculation port into which the desired microbe can be added, and a nutrient feed system to provide a continuous supply of optimized culture medium. The fermenter agitates the culture while it regulates key parameters such as temperature, pH, oxygen levels, and nutrient supply to optimize microbial activity and maximize yield. By-products of fermentation, like carbon dioxide, can be collected and released. The final products of fermentation can be harvested at the completion of the fermentation process.

Figure \(\PageIndex{2}\): The bioreactor. Bioreactors for bacterial fermentation for production of vaccines. (The Bioreactor by Peter grotzinger, CC BY-SA 3.0)

Microbial Manipulation

Microbial manipulation involves modifying microbes to suit specific scientific, medical, or industrial purposes. This can include genetic engineering, metabolic pathway modifications, and environmental adaptations.

Genetic Manipulation of Microorganisms

Genetic manipulation, particularly with bacteria and yeast, is a crucial tool in biotechnology and synthetic biology. There are several techniques used to manipulate microbial genomes, including:

• Recombinant DNA Technology: This involves the insertion of foreign DNA into the microbial genome. The foreign DNA encodes for the production of a protein, allowing microbess to synthesize valuable products such as insulin, growth factors, or vaccines. Bacteria and yeast are used most often in this technology. For more about recombinant DNA technology, go to Chapter 4 Genetic Engineering and Recombinant DNA Technology.
• CRISPR-Cas9 System: This technology enables precise "gene editing" by utilizing the CRISPR-Cas9 system of bacteria to target specific genes in a genome for deletion, insertion, or modification. CRISPR is widely used to modify either prokaryotic and eukaryotic cells. For more about CRISPR, go to Chapter 8.1 Applications of Animal Biotechnology.

Metabolic Engineering

Microbial metabolic engineering is the practice of optimizing the metabolic pathways within microbes to increase the production of desired compounds or degrade specific environmental pollutants. Some examples of metabolic manipulation include:

• Overproduction of Enzymes: By enhancing the expression of genes involved in specific metabolic pathways, microbes, such as bacteria, can be engineered to produce large quantities of industrial enzymes, such as proteases, amylases, and cellulases.
• Biosynthesis of Pharmaceuticals: Yeast and bacteria are engineered to produce antibiotics, vaccines, and other therapeutics. For example, genetically-modified Saccharomyces cerevisiae can produce artemisinin, an important anti-malarial drug.
• Biodegradation of Pollutants: Bacteria such as Pseudomonas species have been engineered to degrade hydrocarbons and other pollutants, contributing to bioremediation efforts.

Microbial Consortia and Co-Cultivation

In many natural environments, microbes exist in complex communities where they interact with one another. This has led to the development of a microbial consortia in laboratory settings, where multiple species are cultivated together to perform complex tasks that single organisms cannot accomplish alone. Applications of such co-cultivation techniques include:

• Biofuel Production: Co-cultivation of photosynthetic algae and fermentative bacteria can enhance the production of biofuels like ethanol and biodiesel.
• Bioremediation: Microbial consortia are often more effective than single species in breaking down pollutants, as different species can metabolize different components of complex waste.

Key Concepts

Microbial cultivation and manipulation are at the heart of modern microbiology and biotechnology, offering powerful tools to address pressing global challenges. From advancing medical therapies to environmental sustainability, the ability to grow and engineer microorganisms holds tremendous potential for innovation.

Some important concepts to remember are:

• Microbes, like bacteria and yeast, require specific environmental conditions in order to optimize their growth
• Environmental conditions for growth include temperature, pH, oxygen, and nutrients
• Nutrients are provided using a medium; medium forms include liquid, solid, selective, and differential
• Bacterial growth curves track the growth of a bacterial culture over time
• Growth curves are made up of 4 phases: lag, log, stationary, and death
• Bioreactors are used for the large-scale growth of microbes, like bacteria and yeast
• Microbial manipulation of microbes can be achieved through genetic changes, metabolic engineering, and the creation of microbial consortia

Glossary

Agar: a gelatinous substance derived from seaweed; used as a solidifying agent in microbial culture media

Aerobic: a process, environment, or organism that requires oxygen

Anaerobic: a process, environment, or organism that does not require oxygen

Batch culture: closed system of microbial growth where nutrients are provided at the beginning, and no additional nutrients are added during cultivation

Broth: a liquid medium used to grow microorganisms in suspension

Colony: a visible cluster of microorganisms, like bacteria, growing on a solid medium and originating from a single cell or a group of identical cells

Continuous culture: a system in which fresh nutrients are continuously added, and waste products are removed to maintain microbial growth over time

Culture medium: a nutrient-rich solution or gel used to support microbial growth; it can be solid, liquid, or semi-solid

Facultative anaerobe: microorganism that can grow with or without oxygen but prefers oxygen for more efficient energy production

Fermentation: a biological process where microbes (like bacteria and yeast) convert sugars into other products like alcohol, gases, or acids

Inoculation: the introduction of microorganisms into a culture medium to promote growth

Incubator: laboratory equipment used to grow and maintain microbiological cultures or cell cultures under optimal temperature, humidity, and carbon dioxide levels

Lag Phase: the initial stage of microbial growth in which cells adjust to new conditions before active division begins

Log (Exponential) Phase: a phase of rapid microbial growth and division when conditions are optimal

Obligate Aerobe: a microorganism that requires oxygen for survival and metabolism

Obligate Anaerobe: a microorganism that cannot survive in the presence of oxygen

Selective medium: a culture medium designed to inhibit the growth of certain microbes while promoting the growth of others