Microbial Nutrition, Growth, and Environmental Factors

Microbial Nutrition and Growth

Essential Nutrients

Essential nutrient: Any substance that must be provided to an organism to sustain life and growth.
Macronutrients: Required in relatively large quantities and play principal roles in cell structure and metabolism.
- Examples: Carbon (C), Hydrogen (H), and Oxygen (O).
Micronutrients (Trace elements): Present in much smaller amounts and are primarily involved in enzyme function and maintenance of protein structure.
- Examples: Manganese, Zinc, Nickel.

Categorizing Nutrients According to Their Carbon Content

Inorganic nutrients:
- An atom or simple molecule that contains a combination of atoms other than C and H.
- Found in the Earth's crust, bodies of water, and the atmosphere.
- Examples: Metals and their salts (magnesium sulfate, ferric nitrate, sodium phosphate), gases (oxygen, carbon dioxide), and water.
Organic nutrients:
- Contain carbon and hydrogen atoms and are usually products of living things.
- Examples: Simple organic molecules such as methane, and large polymers like carbohydrates, lipids, proteins, and nucleic acids.

Chemical Analysis of the Microbial Cytoplasm

Water: Approximately $70\%$ of all cellular components.
Proteins and Organic Compounds: Make up $97\%$ of the dry cell weight.
Elements CHONPS: Account for $96\%$ of the dry cell weight (Carbon, Hydrogen, Oxygen, Nitrogen, Phosphorus, Sulfur).
Most chemical elements are available to the cell as compounds, not as pure elements.
Only a few types of nutrients are needed to synthesize over $5,000$ different compounds.

Chemical Composition of E. coli

Organic Compounds (% Dry Weight):
- Proteins: $50\%$
- Nucleic Acids–RNA: $20\%$
- Nucleic Acids–DNA: $3\%$
- Carbohydrates: $10\%$
- Lipids: $10\%$
- Miscellaneous: $4\%$
Inorganic Compounds (% Dry Weight):
- Water: Not measured in dry weight.
- All others (salts, minerals): $3\%$
Elements (% Dry Weight):
- Carbon (C): $50\%$
- Oxygen (O): $20\%$
- Nitrogen (N): $14\%$
- Hydrogen (H): $8\%$
- Phosphorus (P): $3\%$
- Sulfur (S): $1\%$
- Potassium (K): $1\%$
- Sodium (Na): $1\%$
- Calcium (Ca): $0.5\%$
- Magnesium (Mg): $0.5\%$
- Chlorine (Cl): $0.5\%$
- Iron (Fe): $0.2\%$
- Trace metals: $0.3\%$

Nutritional Categories of Microbes

Heterotroph: An organism that must obtain its carbon in an organic form.
Autotroph: An organism that uses inorganic $CO2$ as its carbon source; has the capacity to convert $CO2$ into organic compounds and is not nutritionally dependent on other living things.
Phototroph: A microbe that photosynthesizes, deriving energy from sunlight.
Chemotroph: A microbe that gets its energy from chemical compounds.

Categories by Energy and Carbon Source

Photoautotroph
- Energy Source: Sunlight
- Carbon Source: $CO_2$
- Examples: Photosynthetic organisms such as algae, plants, cyanobacteria. They produce organic molecules using $CO_2$ that can be used by themselves and by heterotrophs.
Chemoautotroph
- Chemoorganic autotrophs:
  - Energy Source: Organic compounds
  - Carbon Source: $CO_2$
  - Example: Methanogens.
- Chemolithoautotrophs:
  - Energy Source: Inorganic compounds (minerals)
  - Carbon Source: $CO_2$
  - Examples: Thiobacillus, "rock-eating" bacteria. They rely totally on inorganic minerals and require neither sunlight nor organic nutrients.
Photoheterotroph
- Energy Source: Sunlight
- Carbon Source: Organic compounds
- Examples: Purple and green photosynthetic bacteria.
Chemoheterotroph
- Energy Source: Metabolic conversion of nutrients from other organisms
- Carbon Source: Organic compounds
- Examples: Protozoa, fungi, many bacteria, animals. They process organic molecules through cellular respiration or fermentation.
- Saprobe:
  - Description: Free-living organisms that feed on organic detritus from dead organisms; they are decomposers of plant litter, animal matter, and dead microbes, recycling organic nutrients.
  - Energy/Carbon Source: Metabolizing the organic matter of dead organisms, obtain organic carbon.
  - Examples: Fungi, bacteria.
- Parasite:
  - Description: Derive nutrients from the cells or tissues of a living host.
  - Energy/Carbon Source: Utilizing the tissues, fluids of a live host, obtain organic carbon.
  - Examples: Various parasites and pathogens (bacteria, fungi, protozoa, animals).
  - Pathogens: Cause damage to tissues or even death.
  - Types:
    - Ectoparasites: Live on the body.
    - Endoparasites: Live in the organs and tissues.
    - Intracellular parasites: Live within cells.
    - Obligate parasites: Unable to grow outside of a living host (e.g., Leprosy bacillus, syphilis spirochete).
Oversimplification: The terms saprobe and parasite can be oversimplified because some microbes possess attributes of both types or can switch their nutritional mode based on environmental conditions.

Detailed Essential Nutrients

Carbon: Among the common organic molecules that satisfy carbon requirements are proteins, carbohydrates, lipids, and nucleic acids. These molecules often provide several other nutrients as well.
Hydrogen: A major element in all organic and several inorganic compounds, including water ( $H2O$ ), salts (e.g., $Ca(OH)2$ ), and certain naturally occurring gases ( $H2S$ , $CH4$ ). Hydrogen helps cells maintain their pH, is useful for forming hydrogen bonds between molecules, and serves as a source of free energy in respiration.
Oxygen: A major component of organic compounds (carbohydrates, lipids, nucleic acids, proteins), playing an important role in structural and enzymatic cell functions. It is also a common component of inorganic salts (sulfates, phosphates, nitrates) and water. Free gaseous oxygen ( $O_2$ ) makes up $20\%$ of the atmosphere.
Nitrogen: The main reservoir is nitrogen gas ( $N2$ ), making up $79\%$ of the Earth's atmosphere. It is indispensable for the structure of proteins, DNA, RNA, and ATP. Heterotrophs use these compounds after degrading them into amino acids and nucleotides. Some bacteria and algae utilize inorganic nitrogenous nutrients ( $NO3^-$ , $NO2^-$ , or $NH3$ ). A small number of bacteria and archaea perform nitrogen fixation, transforming $N2$ into usable compounds. Regardless of the initial form, inorganic nitrogen must be converted to $NH3$ , the only form that can be directly combined with carbon to synthesize amino acids and other compounds.
Phosphate ( $PO4^{3-}$ ): The main inorganic source of phosphorus, derived from phosphoric acid ( $H3PO_4$ ) and found in rocks and oceanic mineral deposits. Phosphate is a key component of nucleic acids (essential to cell and virus genetics), ATP (cellular energy transfers), phospholipids in cytoplasmic membranes, and coenzymes like NAD $\text{+}$ .
Sulfur: Widely distributed in the environment in mineral form. Rocks and sediments can contain sulfate ( $SO4^{2-}$ ), sulfides (FeS), hydrogen sulfide gas ( $H2S$ ), and elemental sulfur (S). Sulfur is an essential component of some vitamins (e.g., vitamin B1) and amino acids (methionine and cysteine); cysteine helps determine protein shape and structural stability by forming disulfide bonds.

Other Important Nutrients

Potassium (K): Essential for protein synthesis and membrane function.
Sodium (Na): Important for certain types of cell transport.
Calcium (Ca): Stabilizer of bacterial cell walls and endospores.
Magnesium (Mg): Component of chlorophyll and a stabilizer of membranes and ribosomes.
Iron (Fe): Important component of the cytochrome proteins involved in cell respiration.
Zinc (Zn): Essential regulatory element for eukaryotic genetics.

How Microbes Eat: Transport Mechanisms

Transport of necessary nutrients occurs across the cytoplasmic membrane, even in organisms with cell walls.
The driving force of transport is atomic and molecular movement.

Diffusion and Osmosis

Diffusion: The phenomenon of molecular movement, in which atoms or molecules move in a gradient from an area of higher density or concentration to an area of lower density or concentration. No energy expenditure is required.
Osmosis: The diffusion of water through a selectively (or differentially) permeable membrane.
- The membrane has passageways that allow free diffusion of water but block other dissolved solute molecules.
- Water will diffuse at a faster rate from the side with more water (lower solute concentration) to the side with less water (higher solute concentration).
- This continues until the concentration of water is equalized on both sides of the membrane.

Cell Responses to Osmosis

Isotonic conditions: The environment has the same solute concentration as the cell's cytoplasm, resulting in no net movement of water. The cell maintains its normal shape and volume.
Hypotonic conditions: The environment has a lower solute concentration than the cell's cytoplasm. Water diffuses into the cell, causing it to swell and potentially burst (lysis in animal cells) or create turgor pressure against the cell wall (in bacteria, fungi, algae, plants).
Hypertonic conditions: The environment has a higher solute concentration than the cell's cytoplasm. Water diffuses out of the cell, causing it to shrink and wrinkle (crenation in animal cells) or the cytoplasmic membrane to pull away from the cell wall (plasmolysis in walled cells).

Passive vs. Active Transport

Passive Transport: Does not require cellular energy expenditure. Substances move down a concentration gradient.
- Simple diffusion: Molecules move freely across the membrane from higher to lower concentration.
- Facilitated diffusion: A molecule binds to a specific receptor or carrier protein in the membrane and is carried to the other side. This process is molecule-specific, can go in both directions, and the rate of transport is limited by the number of binding sites on transport proteins. Still moves down a gradient (from higher to lower concentration) and requires no energy.
Active Transport: Requires specific membrane proteins (permeases and pumps) and the expenditure of cellular energy (ATP or the proton motive force).
- Moves nutrients against the diffusion gradient (from lower to higher concentration) or in the same direction as the natural gradient but at a rate faster than by diffusion alone.
- Examples of actively transported substances: Monosaccharides, amino acids, organic acids, phosphates, and metal ions.

Endocytosis: Eating and Drinking by Cells

Endocytosis: A process where the cell encloses a substance in its membrane, simultaneously forming a vacuole and engulfing the substance.
- Phagocytosis: Ingestion of whole cells or large solid matter. It is accomplished by amoebas and white blood cells.
- Pinocytosis: Ingestion of liquids such as oils or molecules in solution.

Environmental Factors Affecting Microbial Growth

Cardinal Temperatures

Cardinal temperatures: The range of temperatures for the growth of a given microbial species.
- Minimum temperature: The lowest temperature that permits a microbe’s continued growth and metabolism; below this temperature, its activities stop.
- Maximum temperature: The highest temperature at which growth and metabolism can proceed before proteins are denatured (lose their function).
- Optimum temperature: An intermediate temperature between the minimum and the maximum that promotes the fastest rate of growth and metabolism.

Ecological Groups by Temperature Range

Psychrophiles:
- Optimum temperature below $15°C$ .
- Capable of growth at $0°C$ .
- Obligate with respect to cold; cannot grow above $20°C$ .
- Storage at refrigerator temperatures causes them to grow rather than inhibiting them.
- Natural habitats: Lakes, rivers, snowfields, polar ice, and the deep ocean. Rarely pathogenic.
Psychrotrophs:
- Grow slowly in the cold.
- Optimum temperature between $15°C$ and $30°C$ .
- Examples: Staphylococcus aureus and Listeria monocytogenes can grow at refrigerator temperatures and cause food-borne illness.
Mesophiles:
- Majority of medically significant microorganisms.
- Grow at intermediate temperatures between $20°C$ and $40°C$ .
- Habitats: Animals and plants, as well as soil and water in temperate, subtropical, and tropical regions.
- Human pathogens typically have optimal temperatures between $30°C$ and $40°C$ .
Thermoduric Microbes:
- Can survive short exposure to high temperatures but are normally mesophiles.
- Common contaminants of heated or pasteurized foods.
- Examples: Heat-resistant endospore formers such as Bacillus and Clostridium.
Thermophiles:
- Grow optimally at temperatures greater than $45°C$ .
- Habitats: Soil and water associated with volcanic activity, compost piles, and habitats directly exposed to the sun.
- Vary in heat requirements, with a growth range typically from $45°C$ to $80°C$ . Most eukaryotic forms cannot survive above $60°C$ .
Extreme thermophiles:
- Grow between $80°C$ and $121°C$ .

Gases

The atmospheric gases that influence microbial growth are O2 $and$ CO2.
$O_2$ has the greatest impact on microbial growth; it is an important respiratory gas and a powerful oxidizing agent.

How Microbes Process Oxygen

As oxygen enters cellular reactions, it is transformed into several toxic products:
- Singlet oxygen (O): An extremely reactive molecule that can damage and destroy a cell by the oxidation of membrane lipids.
- Superoxide ion ( $O_2^-$ ): Highly reactive.
- Hydrogen peroxide (Hg2O2): Toxic to cells and used as a disinfectant.
- Hydroxyl radical ( $OH^-$ ): Also highly reactive.

How Microbes Protect Themselves Against Damage from Oxygen By-products

Most cells have developed enzymes that scavenge and neutralize reactive oxygen by-products through a two-step process requiring two enzymes:
- Step 1: Superoxide ion is converted into hydrogen peroxide by superoxide dismutase.
 $2O2^- + 2H^+ \xrightarrow{\text{Superoxide dismutase}} H2O2 + O2$
- Step 2: Hydrogen peroxide is converted into harmless water and oxygen by catalase.
 $2H2O2 \xrightarrow{\text{Catalase}} 2H2O + O2$

Oxygen Usage and Tolerance Patterns in Microbes

Aerobes: Can use gaseous oxygen in their metabolism and possess the enzymes needed to process toxic oxygen products.
- Obligate aerobe: An organism that cannot grow without oxygen.
  - Examples: Most fungi, protozoa, and many bacteria, such as Bacillus species and Mycobacterium tuberculosis.
Microaerophiles:
- Are harmed by normal atmospheric concentrations of oxygen but require a small amount of it in metabolism.
- Examples: Helicobacter pylori, Borrelia burgdorferi.
Facultative anaerobes:
- Do not require oxygen for metabolism but use it when it is present.
- Examples: Many gram-negative intestinal bacteria, staphylococci.
Anaerobes: Lack the metabolic enzyme systems for using oxygen in respiration.
- Obligate anaerobes: Also lack the enzymes for processing toxic oxygen and die in its presence.
  - Examples: Many oral bacteria, intestinal bacteria.
Aerotolerant anaerobes:
- Do not utilize oxygen but can survive and grow to a limited extent in its presence.
- Not harmed by oxygen, mainly because they possess alternate mechanisms for breaking down peroxides and superoxide.
- Examples: Certain lactobacilli and streptococci, Clostridial species.

Carbon Dioxide

Capnophiles: Organisms that grow best at a higher $CO_2$ tension than is normally present in the atmosphere.
They are important in the initial isolation of certain clinical specimens:
- Neisseria (a genus causing gonorrhea and meningitis)
- Brucella (undulant fever)
- Streptococcus pneumoniae

pH

pH is defined as the degree of acidity or alkalinity of a solution, expressed by a scale ranging from $0$ to $14$ .
- $7.0$ is the pH of pure water.
- As pH decreases toward $0$ , acidity increases.
- As pH increases toward $14$ , alkalinity increases.
The majority of organisms live or grow in habitats with a pH between $6$ and $8$ .
- Acidophiles: Organisms that thrive in acidic environments.
  - Examples: Euglena mutabilis (grows in acid pools between pH $0$ and $1$ ), Thermoplasma (lives in coal piles at pH $1$ or $2$ ), Picrophilus (thrives at pH $0.7$ , can grow at pH $0$ ). Many molds and yeasts tolerate acid and are common spoilage agents of pickled foods.
- Alkalinophiles: Organisms that thrive in alkaline conditions.
  - Examples: Natronomonas (live in hot pools and soils up to pH $12$ ), Proteus (can create alkaline conditions to neutralize urine and colonize/infect the urinary system).

Osmotic Pressure

Osmophiles: Live in habitats with high solute concentration.
Halophiles: Prefer high concentrations of salt.
- Obligate halophiles: Examples like Halobacterium and Halococcus. They grow optimally at solutions of $25\%$ NaCl but require at least $9\%$ NaCl.
- Facultative halophiles: Remarkably resistant to salt, even though they do not normally reside in high salt environments. Example: Staphylococcus aureus can grow on NaCl media ranging from $0.1\%$ to $20\%$ .

Radiation

Phototrophs: Use visible light rays as an energy source.
Nonphotosynthetic microbes tend to be damaged by toxic oxygen products produced by contact with light.
Some microbial species produce yellow carotenoid pigments to absorb and dismantle toxic oxygen.
Ultraviolet and ionizing radiation can be used in microbial control.

Pressure

Barophiles:
- Exist under pressures that range from a few times to over $1,000$ times the pressure of the atmosphere.
- These bacteria are so strictly adapted to high pressures that they will rupture when exposed to normal atmospheric pressure.

Microbial Associations

In most instances, microbes live in shared habitats, forming associations with similar or dissimilar types of microbes, or with multicellular organisms (animals, plants).
Interactions can be beneficial, harmful, or have no particular effect, and can be obligatory or nonobligatory. They often involve nutritional interactions.

Strong Partnerships: Symbioses

Symbiosis: A general term denoting a situation in which two organisms live together in a close partnership. The members are called symbionts.
Three main types of symbiosis:
- Mutualism: Both organisms live in an obligatory but mutually beneficial relationship.
- Commensalism: One partner (the commensal) receives benefits, while its partner is neither harmed nor benefited.
- Parasitism: A relationship in which the host organism provides the parasitic microbe with nutrients and a habitat, and the host suffers from the relationship.

Associations but Not Partnerships

Antagonism: An association between free-living species that arises when members of a community compete.
- Antibiosis: Production of inhibitory compounds (such as antibiotics) into the surrounding environment that inhibit or destroy another microbe in the same habitat. The first microbe gains a competitive advantage by increasing the space and nutrients available to it. Common in soil where mixed communities compete for space and food.
Synergism:
- An interrelationship between two organisms that benefits them.
- However, it is not necessary for their survival; they can both grow independently.
- Together, the participants cooperate to produce a result that neither could do alone.
- Examples: Gum disease, dental caries, and some bloodstream infections involve mixed infections that are examples of bacteria interacting synergistically.

Biofilms: The Epitome of Synergy

Biofilms: Mixed communities of bacteria and other microbes that are attached to a surface and to each other.
Formation of a biofilm:
1. A "pioneer" colonizer initially attaches to a surface.
2. Other microbes then attach to those bacteria or to a polymeric sugar or protein substance secreted by the microbial colonizers.
3. Attached cells are stimulated to release chemicals as the cell population grows.
Characteristics: Bacteria in biofilms behave and respond very differently than planktonic (free-living) bacteria; different genes are activated, leading to altered characteristics like increased resistance to antimicrobials and host defenses.

The Study of Bacterial Growth

Binary Fission

Binary fission: The process by which one cell becomes two.
1. The parent cell enlarges.
2. It duplicates its chromosome.
3. The cell envelope starts to pull together at the center of the cell.
4. A cell wall eventually forms a complete septum, dividing the cell into two daughter cells.

Rate of Population Growth

Generation time (doubling time): The time required for a complete fission cycle, from a parent cell to two daughter cells. Each generation increases the population by a factor of two.
As long as the environment remains favorable, the doubling effect can continue at a constant rate, leading to exponential growth.
Length of Generation Time: A measure of the growth rate of an organism.
- Average generation time: $30$ to $60$ minutes.
- Shortest generation times: Can be $10$ to $12$ minutes.
- Longest generation times: Mycobacterium leprae has a generation time of $10$ to $30$ days; some environmental bacteria have generation times measured in months.
- Most pathogens have relatively short generation times.

Mathematics of Population Growth

The size of a population can be calculated by the following equation: $N_t = (N)2^n$
- $N_t$ : The total number of cells in the population at some point in time (t).
- $N$ : The starting number of cells.
- $n$ : The generation number (the number of times the population has doubled).
- $2^n$ : Represents the number of cells in that specific generation.

The Population Growth Curve

A predictable pattern of bacterial population growth in a closed system (batch culture) can be measured by inoculating a tiny number of cells into a sterile broth, incubating, sampling at regular intervals, plating each sample onto solid media, and counting the colonies.

Stages in the Normal Growth Curve

Lag phase:
- A "flat" period of growth where newly inoculated cells require a period of adjustment, enlargement, and synthesis.
- Cells are not yet multiplying at their maximum rate.
- The population of cells is often so sparse or dilute that initial sampling may miss them.
Exponential growth (logarithmic or log) phase:
- The growth curve increases geometrically.
- Cells are multiplying at their maximum rate.
- This phase will continue as long as cells have adequate nutrients and the environment remains favorable. During this phase, the population exhibits the briefest doubling time.
Stationary growth phase:
- The rate of cell birth and cell death are equal, resulting in no net increase in population size.
- The cell division rate is slowing down due to depleted nutrients and oxygen, plus the excretion of organic acids and biochemical pollutants into the growth medium.
Death phase:
- Cells begin to die at an exponential rate due to the buildup of wastes and continued nutrient depletion.
- The speed with which death occurs depends on the resistance of the species and how toxic the conditions are.
- This phase is typically slower than the exponential growth phase.
- Viable nonculturable state (VNC): Many cells in a culture in the death phase may stay alive but become dormant, meaning they will not grow on culture medium and are therefore missed in standard colony counts.

The Practical Importance of the Growth Curve

The tendency for populations to exhibit these phases of rapid growth, slow growth, and death has important implications for controlling microbes.
Microbes in the exponential growth phase are generally more vulnerable to antimicrobial agents and heat.
Cells in the rapid growth phase are more vulnerable to conditions that disrupt cell metabolism and binary fission.
In general, actively growing cells are more susceptible to growth inhibition and destruction.

Analyzing Population Size Without Culturing

Turbidity/Turbidometry:
- A clear nutrient solution becomes turbid, or cloudy, as microbes grow in it.
- The greater the turbidity (cloudiness), the larger the population size.
- Measurements are usually taken with a spectrophotometer, which measures absorbance or transmittance of light through the culture.
Counting Methods:
- Direct cell count: Measured microscopically using a calibrated counting chamber (e.g., Petroff-Hausser counting chamber).
- Coulter counter: An electronic device that scans a fluid as it passes through a tiny pipette, counting cells based on changes in electrical resistance.
- Flow cytometer: Works similarly to a Coulter counter but can also measure cell size and differentiate between live and dead cells by using fluorescent dyes.
Genetic Probing: Uses real-time PCR (Polymerase Chain Reaction) to quantify bacteria and other organisms present in environmental or tissue samples, detecting specific DNA sequences.