Chapter 7- Microbial Nutrition, Ecology, and Microbial Growth*

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Chapter 7- Microbial Nutrition, Ecology, and Microbial Growth* *Lecture notes are to be used as a study guide only and do not represent the comprehensive information you will need to know for the exams.

7.1 Microbial Nutrition

Microbes live in various types of habitats. Environmental factors such, as nutrient and energy sources, temperature, gas content, water, salt, pH and radiation. Microbes have the ability to adapt to their environments.

Nutrients are chemical substances that allow cells to grow. Nutrients can be divided into several groups: essential nutrients, those substances that must be provided to an organism; essential nutrients can be divided into two categories: 1) macronutrients, those nutrients needed in large amounts, and 2) micronutrients, those nutrients needed in small amounts, such as trace elements.

Nutrients can also be classified as organic, contains carbon and hydrogen, and most often are derived from living things; inorganic, nutrients that contain elements other than carbon and hydrogen, such as metals, salts and carbon dioxide.

Chemical Analysis of Cell Contents

Water is the highest content in the cell, proteins are the most prevalent compound, the most common elements are CHONPS.

Forms, Sources, and Functions of Essential Nutrients

The source of nutrients is extremely varied. See Fig. 7.1 and Table 7.1.

Carbon-based Nutritional Types:

Microbes can be classified based on their carbon usage. See Table 7.2.

Heterotrophs = organisms that ingest food; i.e. gets carbon from other organisms.

Autotrophs = organisms that produce their own food; utilize inorganic CO2 as its carbon source.

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Carbon Source versus Carbon Function

No matter the source of carbon, organic or inorganic, once carbon enters the cell, it become organic.

Growth Factors: Essential Organic Nutrients

Fastidious bacteria need nutrients that they can’t synthesize. A nutrient that must be provided is called a growth factor.

7.2 Classification of Nutritional Types

General trends of microbial nutrition are listed in Table 7.2 and 7.1 MAKING CONNECTIONS.

Microbes can be organized based on their carbon and energy source. The two types of energy sources are described using the terms phototroph, which uses light as an energy source; and chemotroph, which uses organic material as an energy source.

Autotrophs and Their Energy Sources

Photoautotrophs derive their energy from sunlight and their carbon from CO2.

Chemoautotrophs use inorganic sources such as minerals and gases. For example, methanogens.

Heterotrophs and Their Energy Sources

Heterotrophs obtain carbon and energy from organic compounds in their environments. They are called chemoheterotrophs. Typically, these organisms use fermentation and/or respiration to make energy in the form of ATP. Heterotrophs = organisms that ingest food; i.e. gets carbon from other organisms.

Microbes can obtain their carbon and energy by being saprobes, they survive on decaying matter, and/or, by being symbiots, living with another organism(s).

Saprobic Microorganisms Saprobic organisms live on decaying matter and recycle nutrients back to the environment. Saprobic organisms, like bacteria and fungi, are decomposers. These microbes obtain their nutrients by breaking down large organic molecules (ex. proteins and polysaccharides) outside of the cell using exoenzymes, also called extracellular enzymes (digestive enzymes that they secrete into their environment). The bacteria then absorb small organic molecules (fig 7.3). 2

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

When a saprobe infects a host it is called a facultative parasite, and if that host is immune-compromised, the same saprobe can become an opportunistic pathogen.

Parasitic Microorganisms Parasites are organisms that survive by using living tissues as it’s nutrient source and harms the host. Parasites can be pathogens by ultimately causing diseases and sometimes death. Obligate parasites must live on a host to survive.

Note: In nature, organisms compete for nutrients. In the laboratory, the nutrients are provided in their media. Media refers to any solution or solid agar that supplies the essential nutrients the organisms need.

There are about 22 different chemical elements found in bacterial cells. Not all elements are found in every cell. It depends upon the nutritional requirements of a particular organism. There are six elements that are essential to all cells: CARBON, NITROGEN, HYDROGEN, OXYGEN, PHOSPHORUS, and SULFUR.

In addition to the essential elements needed for survival, bacteria also require trace mineral elements such as copper, zinc, iron, etc. Other growth factors that they cannot always synthesize include: vitamins and some amino acids.

*Note: We will not cover section 7.3 Transport: Movement of Substances Across the Cell Membrane

7.4 Environmental Factors That Influence Microbes

In nature, microorganisms can only grow in an environment where their nutritional and environmental requirements are met, their ecological niche. Physical or environmental requirements include: osmotic pressure, temperature, gaseous requirements and pH.

Adaptations to Temperature

Note: Growth ranges, not only temperature, can be describes by its minimum growth at the lowest tolerable point, it’s maximum growth, the highest tolerable point, and it’s optimum growth at the point where enzymes are most active.

The optimum growth temperature is the temperature that allows for most rapid growth during a short time period (12-24 hours). Growth slows near the lower limit (reduced metabolic activity) and drops sharply if it extends beyond the upper limit (enzymes and proteins are destroyed).

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Bacteria can be classified into three general categories depending on their optimum growth temperature (fig. 7.9):

a. psychrophiles - Range: -15ºC to 20ºC. Optimum = 0ºC These organisms must be incubated in the freezer, fig. 7.10.

b. mesophiles - Range: although some may grow at extremes of 10ºC up to 50ºC, they optimally grow at 20-40ºC. Most human pathogens grow between 30-40ºC because body temperature = 37ºC. Thermoduric bacteria are those which can tolerate short exposure to high temperatures (such as pasteurization), but are normally mesophilic. They are often cyst-forming, spore-forming, or thick walled. Some mesophilic pathogens (ex. Listeria monocytogenes) can still grow when refrigerated (typical temp. = ~4ºC, or ~40ºF).

c. thermophiles - Range: 45ºC to 80ºC. These bacteria typically live in hot springs or near deep sea volcanic vents. Remember, water boils at 100ºC.

Gas Requirements

Gas requirements refers here to the organism’s use of oxygen (fig. 7.11). Bacteria have a wide range of oxygen requirements and these requirements often are crucial to the pathogenicity of bacterial infections.

How Microbes Process Oxygen

 obligate aerobic bacteria – MUST have oxygen for growth.  obligate anaerobic bacteria – cannot utilize oxygen and do not grow in its presence. Some organisms can actually be killed by traces of oxygen.  facultative anaerobic bacteria do not require O2 , although they will grow faster with it because respiration yields more energy (ATP) than fermentation.  microaerophiles – organism that require less O2 (2-10%) than is found in air (20%).  capnophiles – grow best in the presence of CO2 (fig. 7.12).

Pathology Note: Despite the fact that humans breathe O2 and O2 is present throughout our tissues, there are still anaerobic pockets (microhabitats) where bacterial colonization and infection can occur. Two such sites: Dental caries (cavities) are partly due to a mixture of aerobic and anaerobic bacteria; the large intestine.

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Effects of pH

The pH scale is used to measure of the degree of acidity or alkalinity of a solution by measuring the H+ concentration in the solution. pH ranges from 1 to 14, with 1 being the most acidic and 14 being alkaline or basic. ph 7 is neutral. Bacteria grow best around 7, although they tolerate a range from 5 to 8 (optimally 6.5 to 7.5 for human1 pathogens). Fungi prefer a more acidic environment.

 neutrophiles – grow best in the pH range of 5.5 – 8.  acidophiles – grow best in the pH range of 0 – 4.  alkaliphiles – grow best in the pH range of 9-12.

Osmotic pressure

Osmotic pressure is defined as the minimum amount of pressure that must be applied to a solution to prevent the flow of water across a membrane within a solution. Most bacteria live in hypotonic environments. Osmotic pressure increases as more and more water is taken up by the cell. The cell wall keeps the cell from bursting. In cells which lack a cell wall (ex. mycoplasmas) or in marine bacteria which require high salt contents in their medium, osmotic pressure becomes critical. In environments where there is a large quantity of salt, water diffuses out of the microbe in an effort to dilute the salt in the surrounding environment (trying to establish equilibrium). The organism dehydrates as a result. This is why salt and sugar (solutes) can be used to preserve foods. High salt drains the water needed by bacteria for normal metabolic activities.

7.5 Ecological Associations Among Microorganisms

Many organisms live in symbiotic relationships, called symbioses, that advantages one of the members of the relationship (fig. 7.13).

 mutualism – all members benefit from the interrelationship.  commensalism – one member benefits, while the other is not harmed.  parasitism – the parasite benefits and the host is harmed, some parasites are obligate intracellular parasites, where they reside in the host’s cells.  syntrophy – communal feeding of organisms that share a habitat.  amensalism – one microbe disrupts the living efforts of another microbe, such as antibiosis.

1 Average human blood pH = 7.35-7.45 5

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Biofilms – A Microbial Conversation

Biofilms are common among many types of bacteria. They rely on quorum sensing, a mechanism that bacteria use to “count” themselves to initiate a genetic response to their environment. The counting molecule is called an autoinducer. Bacteria use this molecule to help establish biofilms (fig. 7.14).

7.6 The Study of Microbial Growth

The Basis of Population Growth: Binary Fission and the Bacterial Cell Cycle

Bacteria grow by a process called transverse binary fission (fig 7.15). That is, one cell divides into 2; those 2 into 4; those 4 into 8, etc. If a single cell begins dividing on a culture plate, a visible colony will appear in approximately 10-24 hours through multiple cell divisions. A visible colony represents approximately 1 billion (1 x 109) cells.

The Rate of Population Growth

The time required for one cell to divide is called the generation time. Generation times vary between different species; can be as low as 5 minutes and as long as 30 hours. Growth, for bacteria, is measured in numbers of cells, not the size of the individual cell. Growth is expressed in generation time. Theoretically, the increase in cell number is exponential (fig. 7.16).

The growth of bacteria can be described mathematically by the equation:

n Nf = Ni2 n = number of generations

Nf = the final number of bacteria

Ni = the initial number of bacteria

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Hypothetical Example:

Let’s say you’re having a picnic. You put a bowl of potato salad on the picnic table and then go off for 3 hour softball game. If there had been just one Staphylococcus aureus bacterium in the salad, and it was growing optimally (i.e. a hot Houston day), would the potato salad be safe to eat when you got back?

Assume 1 cell to start and a generation time of 10 minutes.

3 hours = 180 minutes

180 min ÷ 10 [generation time] = 18 generations

18 Nf = (1)2 = 2x2x2x2x2x2x2x2x2x2x2x2x2x2x2x2x2x2 = 262,144 bacteria in the potato salad after 3 hours.

262,144 Staphylococcus aureus bacteria are plenty to give you food poisoning.

What if the number of bacteria in the potato salad were 1000 to start with?

Determinants of Population Growth

Bacterial growth follows a predictive growth pattern called a growth curve over a time period.

The Viable Plate Count: Batch Culture Method

A flask is inoculated and over time points, samples are removed to determine the number of live cells in the culture. The colonies are counted, where one colony represents one cell, called colony-forming unit (CFU) (fig. 7.17).

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Stages in the Normal Growth Curve:

Bacteria have four well-defined phases of growth. The same test tube of liquid medium cannot support a bacteria population indefinitely: nutrients are depleted; toxins and wastes build up; overcrowding leads to competition and selection. However, from the time bacteria are inoculated into a medium, the rate of increase follows a characteristic and predictable sequence (fig. 7.18).

1. lag phase - Each individual cell is increasing its cell mass. The cell manufactures and accumulates the molecules necessary for cell division - ATP for energy, ribosomes for protein synthesis and enzyme production. Cells are dividing slower than optimum.

2. exponential or log phase - During this phase, the cells divide at their maximum rate. The size of a cell population before it stops growing depends on their genetic constitution (can it utilize available nutrients?) and environmental factors (pH, temp., etc.). This is also the phase during which cells are most susceptible to the effects of antibiotics and chemical agents.

3. stationary phase - Bacterial growth rate is essentially zero. Either the accumulation of toxic waste products or the depletion of a required nutrient are responsible for the slow down in growth. The constancy (balance) of cell numbers during this phase may be the result of: the cells have quit multiplying, but are not dying - or - a balance of cell division and cell death exists. The length of time that cells remain in stationary phase varies depending upon the species and environmental conditions.

4. Death phase - This phase is marked by a decrease in the total number of viable cells in the population. A cell is dead if it is incapable of multiplying. Viable means living, and non-viable means non-living.

BIOL 2320 J.L. Marshall, Ph.D. HCC-Stafford Campus

Other Methods of Analyzing Population Growth

As microbes grow in liquid media, the media becomes turbid (fig. 7.19a). Turbidity can be measured using a spectrophotometer, where the higher the absorbance implies the higher the bacterial cell count, but not all cells are alive (fig. 7.19b).

Quantifying and Analyzing Cultures

Direct / Total cell count uses a cytometer to count all cells, live and dead (fig. 7.20).

Flow cytometer is an automated cell counting instrument that can separate cells that are alive and dead based on fluorescent dyes. The results are plotted on a graph (fig. 7.21).