Microbiology and Ecology: A Comprehensive Guide

Posted on Aug 23, 2024 in Biology

This comprehensive guide explores the fascinating world of microbiology and ecology, covering topics from bacterial structure and growth to population dynamics and environmental impacts.

Bacterial Structure and Growth

Bacteria are microscopic organisms that play crucial roles in various ecosystems. Their shape is determined by their rigid cell walls, and they can be classified into different types based on their morphology:

Bacterial Shapes

• Coccus (spheres): Staphylococcus (food poisoning), Diplococci (pneumonia), Streptococci (chain, sore throats)
• Bacillus (rod): Salmonella (single, typhoid fever), Bacillus (anthrax)
• Spirillium (helical): Treponema pallidum (syphilis)

Gram Staining

Gram staining is a technique used to differentiate bacteria based on their cell wall structure. The process involves:

1. Making a film of the sample bacteria.
2. Flooding with crystal violet dye.
3. Flooding with Lugol’s iodine.
4. Decolorizing with acetone.
5. Counterstaining with safranin.

Gram-positive bacteria retain the crystal violet dye complex and appear purple, while Gram-negative bacteria are decolorized and stain red by the counterstain. This difference is due to the structure of their cell walls:

• Gram-positive: Thick layered cell wall consisting of peptidoglycan.
• Gram-negative: Thin layer of peptidoglycan covered by a layer containing lipopolysaccharides, providing some protection against antibiotics and the lysozyme enzyme, making them more difficult to kill.

Aseptic Techniques and Bacterial Growth Phases

Aseptic techniques are essential when isolating and culturing bacteria to prevent contamination from unwanted organisms and the environment. These techniques include:

• Sterilizing equipment with heat (e.g., autoclave at 121°C for 15 minutes).
• Washing hands with antibacterial soap before and after handling cultures.
• Cleaning work surfaces with disinfectant.
• Using sterile Petri dishes and agar jelly.
• Inoculating plates close to a Bunsen burner to provide an updraft that moves airborne microorganisms away.
• Keeping the lid of a culture bottle in hand away from the bench surface and flaming the mouth of the bottle to kill unwanted microbes.
• Flaming an inoculating loop to transfer bacterial culture to an agar plate.
• Opening the lid of a Petri dish just wide enough when inoculating.
• Securing plates with adhesive tape and incubating at a lower temperature of 25°C, as human pathogenic bacteria grow best at 37°C and in anaerobic conditions.

Bacterial growth follows distinct phases:

Bacterial Growth Phases

• Lag phase: A small increase in cell number as bacteria adjust to a new environment by switching on genes to produce digestive enzymes (saprophytes/extracellular). Soluble products of digestion are absorbed, and time is also required for DNA replication before cells can divide.
• Log phase: When nutrients are in plentiful supply and waste products are at low concentrations, the rate of cell division is maximum, and the population is growing at an exponential rate.
• Stationary phase: Carrying capacity is reached (maximum population the environment can support), where the number of new cells produced is equal to those dying. Limiting factors, such as nutrient depletion and waste accumulation, come into play.
• Death phase: More cells are dying than being produced due to a shortage of nutrients and waste accumulation, so the population decreases.

Optimum Growth Conditions for Bacteria

Bacteria require specific conditions for optimal growth:

• Nutrients: A source of carbon (organic molecules), nitrogen (amino acids, proteins, and nucleic acids), and phosphorus (ATP), a respiratory substrate (e.g., glucose), vitamins and minerals as coenzymes, and water as metabolic reactions occur in an aqueous solution.
• Temperature: Too low, enzyme-catalyzed reactions become too slow to sustain life. Too high, enzymes denature, leading to death. The optimum temperature is between 20-49°C (mesophiles). Above 45°C, thermophiles thrive, and below 20°C, psychrophiles are found.
• pH: Microorganisms can tolerate a higher pH range than plants and animals. For most, it’s 5-7.5, but some species can grow at 2.5 or 9.
• Oxygen:
  • Obligate aerobes: Need oxygen for growth at all times.
  • Obligate anaerobes: Find oxygen toxic as it inhibits respiration.
  • Facultative anaerobes: Grow best with oxygen but can survive without it, although population growth is low.

Measuring Bacterial Populations

Two methods are commonly used to measure bacterial populations:

Total Count

Includes both living and dead cells (by haemocytometry), but the number in the population may be an overestimate.

Viable Count

Only counts living cells. Bacteria are grown to form distinct colonies based on the assumption that a single cell gives rise to one colony. The number can be underestimated due to clumping of cells when plates are made.

Dilution Plating

Dilution plating is used to distinguish between individual colonies. The original culture is diluted down (in 10-fold steps) to produce a countable range. This involves serial dilutions:

1. 1 cm³ of the original culture is transferred to 9 cm³ of sterile medium and mixed for even distribution, resulting in a 10x dilution.
2. 1 cm³ of this mixture is transferred to a different sterile medium of 9 cm³, resulting in a 100x dilution.
3. This process is repeated to achieve dilutions of 10,000-100,000x.
4. A known volume (1-0.1 cm³) from the final dilution is added to agar and incubated.
5. The number of colonies is multiplied by the dilution factor.

Industrial Fermentation: Penicillin Production

Industrial fermentation is used to produce various products, including antibiotics like penicillin. The process involves:

Penicillin Production

• Primary metabolite: Needed for normal metabolism (e.g., enzymes). Continuous fermentation is used, and microbes are kept in the exponential phase for maximum production of the primary metabolite.
• Secondary metabolite: Produced in overcrowded conditions and shortage of nutrients, used to kill competing microbes. Batch fermentation is used so microbes can reach the stationary phase when secondary metabolites are produced.

The fungus Penicillium notatum produces penicillin as a secondary metabolite when glucose (food source) is depleted to reduce competition. The process takes place in a batch fermenter:

Batch Fermenter

• Vessels are sterilized with high-pressure steam to kill microbes that may infect the batch.
• Sterile nutrient medium is added.
• A pure culture of Penicillium notatum is added.
• Sterile air provides oxygen and prevents contamination from airborne microbes.
• The fermenter is heated to the optimum temperature for growth, maintained by a cooling jacket that removes excess heat from fungal respiration.
• Optimum pH is maintained by adding acid or alkali.
• The culture and nutrients are thoroughly mixed by sparging rings for aeration.
• Penicillin is produced at the stationary phase once glucose is no longer in excess.
• The culture fluid is filtered to remove mycelium and purified to extract penicillin.

Penicillin’s Mechanism of Action

Penicillin prevents the formation of cross-links between peptidoglycan units during cell division, weakening cell walls and making them susceptible to osmotic lysis. It is effective against Gram-positive bacteria due to their thick peptidoglycan layer but less effective against Gram-negative bacteria due to the extra lipopolysaccharide layer.

Population Ecology

Population ecology studies the dynamics of populations, including their growth, regulation, and interactions with the environment.

Population Growth

A population is a group of individuals of the same species in a particular habitat at a particular time. Population growth is determined by the following equation:

Population growth = (Births + Immigration) – (Deaths + Emigration)

The lag phase in population growth is due to a limited number of individuals at reproductive age. The log phase occurs due to little intraspecific competition and a plentiful supply of resources, leading to a maximum birth rate during the exponential phase.

Carrying Capacity

Carrying capacity is the maximum population size that an environment can sustainably support. As population growth nears carrying capacity, it slows down due to environmental resistance, which limits further population growth.

Density-Dependent Factors

Density-dependent factors affect a greater percentage of the population at higher population densities. Examples include:

• Food: As population density increases, competition for food intensifies, leading to reduced food availability and slower growth rates.
• Competition: Intraspecific competition for resources, such as food, mates, and territory, increases with population density.
• Predation: Predators often focus on areas with higher prey densities, increasing predation rates as population density rises.
• Disease: Pathogens (e.g., influenza virus) spread more easily from individual to individual at higher densities, leading to a higher percentage of infected individuals.

Most populations fluctuate around carrying capacity. The stationary phase is influenced by nutrient availability (e.g., nitrates). A rise in carrying capacity results in intraspecific competition, and the death rate will exceed the birth rate, causing the population to decrease. If the population falls below carrying capacity, competition decreases, the birth rate will exceed the death rate again, and the population increases.

Density-Independent Factors

Density-independent factors have the same effect regardless of population size, affecting the same percentage of the population. Examples include:

• Weather and climate: Sudden changes in temperature can kill a certain percentage of a population, leading to a population crash.

Pest Control

Pests are unwanted organisms that interfere directly or indirectly with human activity, such as agriculture and food production. Various methods are used to reduce pest populations to a level that doesn’t result in economic loss to farmers.

Chemical Control

Chemical control involves using poisons (pesticides) to eradicate pests.

Advantages

• Fast eradication over a specific area before the pest population can increase.
• Cost-effective and doesn’t exceed the economic gain of selling the product.
• Chemicals can be applied on a small scale.
• Doesn’t require a high level of skill.

Disadvantages

• Non-selective and can kill a wide range of beneficial organisms (e.g., pollinating bees and natural pest predators), causing a resurgence of the pest population as individuals that survive can migrate into the treated area to find ideal conditions.
• Persistent and non-biodegradable, remaining in the soil for years (e.g., DDT).
• Fat-soluble and aren’t excreted, so they accumulate in fatty tissues, increasing the level of toxicity as organisms grow (bioaccumulation). As the pesticide passes from one trophic level to the next, it becomes more concentrated in the animals’ bodies (biomagnification), so animals higher up the food chain will have the highest concentrations, which can be fatal.
• Individuals in the pest population can contain alleles with genetic resistance to the pesticide and survive. With a lack of intraspecific competition, they will reproduce and pass on their resistant alleles to offspring, leading to the evolution of resistant populations.

Biological Control

Biological control exploits natural predators, parasites, or disease-causing organisms of the target pest species. It aims to reduce the pest population below the economic damage threshold, as it would be counterproductive to eradicate the pest species, as then the control agent would die out.

Advantages

• Specific to a target species.
• The environment is not contaminated and non-toxic to other species.
• Pests are unable to develop resistance.
• Self-perpetuation when a natural population of predators vs. pest is established.
• Relatively inexpensive in the long term, although initial research costs may be high.

Disadvantages

• Slow, taking time for the control agent population to increase to an effective level.
• Requires a high skill level to implement and research.
• An exotic control agent may itself become a pest (e.g., cane toad).
• Repeated application of the control agent may be needed to achieve long-term population balance.
• Must be large-scale.

Integrated Pest Management (IPM)

IPM combines biological control agents with minimal, well-targeted applications of selective pesticides.

Nutrient Cycling

Decomposers (fungi and bacteria) break down organic molecules in dead tissue, releasing inorganic molecules into the environment, enabling nutrients to be recycled in ecosystems.

Carbon Cycle

Carbon is found in carbohydrates, lipids, proteins, and nucleic acids in living organisms. Carbon is made available from the atmosphere and oceans via photosynthesis and carbon fixation by green plants and algae, producing complex organic molecules (e.g., glucose). Carbon dioxide is produced by all organisms during respiration and released back into the atmosphere.

Human effects: Carbon is also stored in wood and fossil fuels (coal) formed by undecomposed dead matter. Coal is fossilized wood, and oil/gas is formed from soft animal tissue. Combustion of these releases more stored carbon dioxide into the atmosphere.

Nitrogen Cycle

Nitrogen leaves as indigestible matter in feces and in urea.

1. Putrefaction/Ammonification: Saprophytic bacteria and fungi metabolize organic nitrogen-containing matter, producing ammonium ions.
2. Nitrification: Under aerobic conditions, nitrifying bacteria (Nitrosomonas) convert ammonia to nitrites, and Nitrobacter converts nitrites to nitrates, which are then taken up from the soil by producers.
3. Dentrification: Dentrifying bacteria (Pseudomonas) under anaerobic conditions convert nitrates and ammonium ions back to atmospheric nitrogen, removing it from ecosystems.
4. Nitrogen fixation: Nitrogen-fixing bacteria convert atmospheric nitrogen (N₂) to ammonium ions. Rhizobium is a mutualistic bacterium found in leguminous plants (e.g., clover). The plant rapidly converts ammonium ions to nitrogen-containing organic compounds, where the bacterium benefits by gaining energy and nutrients (e.g., carbohydrates) from the plant and gains leg hemoglobin from the plant, which provides anaerobic conditions. Azotobacter is a free-living nitrogen-fixing bacterium found in the soil.

Photoperiodism

Photoperiod is the relative length of day and night, which varies with the time of year in temperate latitudes.

Photoperiodism is a plant’s response to changes in the length of the photoperiod, affecting flowering, fruit, and seed production.

Types of Photoperiodic Plants

• Long-day plants: Lettuce, clover, and cereals flower in late spring/early summer when light periods are long.
• Short-day plants: Chrysanthemums, tobacco, and strawberries flower in late summer/early spring when light periods are short.
• Day-neutral plants: Tomatoes and cucumbers, day length has no effect on flowering.

Phytochrome

Phytochrome is the photoreceptor responsible for absorbing light. It’s a blue-green pigment that absorbs light in the red region of the visible spectrum (wavelength 625-740 nm).

• Pr: Absorbs red light (660 nm).
• Pfr: Absorbs far-red light (730 nm).

When Pr absorbs red light, it rapidly converts to Pfr, and vice versa (with absorption of far-red). Sunlight has more red light than far-red, so Pr is converted to Pfr during the day, which accumulates in cells and is reverted back to Pr at night. Low levels of Pfr are associated with short plants, while high levels of Pfr are associated with long-day plants.

If high levels of Pr are found, both long-day and short-day plants will flower. During a short day, Pr is converted to Pfr, so due to the longer dark period, most Pfr reverts back to Pr. If low levels of Pfr are found, long-day plants won’t flower, while short-day plants will.

It is the length of the unbroken dark period (1940) that is critical. In normal conditions or a long dark period, most Pfr is converted to Pr. However, a brief light period during the dark period leads to a rapid conversion of Pr to Pfr, resulting in high levels of Pfr. Long-day plants will now flower, while short-day plants won’t.

Horticulturalists manipulate the flowering of plants for particular dates and allow plant breeders to create new hybrid varieties by cross-pollination.

With long-day plants, short dark periods induce flowering, but if briefly exposed to light during a long dark period, they won’t flower. Short-day plants require long dark periods to induce flowering, but brief exposure to light during a long dark period will prevent flowering.

If one leaf of a short-day plant is exposed to a daily routine of short days and long nights while the rest of the plant is kept in constant darkness, only that one leaf needs to be exposed to the appropriate photoperiod for flowering to occur. This indicates that the photoperiod is detected by leaves, and a chemical substance must be transported from the leaf to the bud to bring about flowering.

If a short-day plant is grafted onto a long-day plant, and both are exposed to a routine of short days and long nights, both plants will flower. This suggests that the chemical produced in the short-day plant must be transported into the long-day plant, having the same effect on both.

Florigen, a chemical hormone, is thought to be responsible for flowering. In short-day plants, low levels of Pfr may stimulate Florigen production, while in long-day plants, high levels of Pfr may stimulate Florigen production.

Energy Yields

– not all glucose molecule transferred to ATP due to loss of energy via heat/aerobic respiration= breakdown of glucose>co2+h2o>38ATP per glucose=40% effeciant
Anaerobic respiration- incomplete breakdown of glucose>2ATP=2% efficient as energy