Lesson 6:

Protozoa


Objective

In this lesson we will learn the following:

 

Reading Assignment

In addition to the online lecture, read chapter 5 in Wastewater Microbiology .


Lecture

Eukaryotic Cells

Protozoa are euckaryotes, so let's first understand how they are different from bacteria, which are prokaryotes. A eucaryotic cell is larger and more complex than a procaryotic cell and found in animals, plants, algae, fungi, and even YOU!

 

 

Where prokaryotes are bacteria and Archaea, eukaryotes is literally everything else! Eukaryotes also have specialized energy producing organelles called mitochondria and plants also have chloroplasts. These vital organelles are involved in metabolism and energy conversion within the cell. Depending upon the organism, eukaryotic cells can reproduce in one of several ways, including meiosis (sexual reproduction) and mitosis (cell division producing identical daughter cells).

The most noticeable feature that differentiates these cells from prokaryotes is the presence of a nucleus; a double membrane-bound control center separating the genetic material, DNA, from the rest of the cell. The eukaryotic cell also has an endomembrane system composed of different membrane-bound organelles that transport materials around the cell. The endomembrane system includes the nuclear membrane, round and smooth endoplasmic reticulum, Golgi apparatus and different types of transport vesicles.

Let's watch a video that explains the difference between eukaryotes and prokaryotes.

 

 

 

 

Structure of the Eucaryotic Cell

Plasma Membrane and Cytoplasm

The plasma membrane (also known as the cell membrane or cytoplasmic membrane) surrounds a eucaryotic cell and serves as a barrier between the inner cell and its environment. The membrane consists of a double layer of lipids called phospholipids. As shown below, proteins are also in important component of the plasma membrane. Some of them pass all the way through the membrane, serving as channels or signal receptors, while others are simply attached at the edge. Different types of lipids, such as cholesterol, may also be found in the cell membrane and affect its fluidity.

 

 

 

In a eucaryotic microorganism, the cytoskeleton provides support and shape for cells and helps transport substances through the cell. The plasma membrane of a eucaryotic cell functions like the plasma membrane of a procaryotic cell. How easily these molecules can cross the membrane depends on their size and polarity. Some small, nonpolar molecules, such as oxygen, can pass directly through the phospholipid portion of the membrane. Other, larger, more polar substances enter and leave the cell through the cytoplasmic membrane by using simple diffusion, facilitated diffusion, osmosis, and active transport.

 

 

Transport Across the Membrane

All substances that move through the membrane do so by one of two general methods, which are categorized based on whether or not energy is required. Passive (non-energy requiring) transport is the movement of substances across the membrane without the expenditure of cellular energy. During this type of transport, materials move by simple diffusion or by facilitated diffusion through the membrane, down their concentration gradient. Water passes through the membrane in a diffusion process called osmosis.

Let's watch a video explaining the difference between passive and active transport.

 

 

Passive Transport

The two passive transport methods include diffusion and osmosis. In the process of diffusion, a substance tends to move from an area of high concentration to an area of low concentration until its concentration becomes equal throughout a space. Osmosis is a process during which substances move from an area of low concentration to an area of high concentration (the opposite of diffusion).

 

 

Let's watch a video explaining diffusion and osmosis in more detail.

 

 

Active Transport

Passive transport is a great strategy for moving molecules into or out of a cell. All the cell has to do is sit there and let the molecules diffuse in. In active transport the cell expends energy, usually in the form of ATP. If a substance must move into the cell against its concentration gradient, that is, if the concentration of the substance inside the cell must be greater than its concentration in the extracellular fluid, the cell must use energy to move the substance. Some cells are even capable of engulfing entire unicellular microorganisms.

A common mistake is that active transport is the same as facilitated diffusion. Both are active transport systems and facilitated diffusion does use proteins to assist in transport, however, active transport works against the concentration gradient, moving substances from areas of low concentration to areas of high concentration. Active transport uses carrier proteins, not channel proteins. These carrier proteins are different than the ones seen in facilitated diffusion, as they need ATP in order to change conformation.

 

 

Endocytosis is a type of active transport that moves particles, such as large molecules, parts of cells, and even whole cells, into a cell. There are different variations, but all share a common characteristic: the plasma membrane introverts, forming a pocket around the target particle. The pocket pinches off, resulting in the particle being contained in a newly created vacuole, or vesicle, that is formed from the plasma membrane. Variations of endocytosis include phagocytosis ("cell eating"), pinocytosis ("cell drinking") and receptor-mediated.

Let's watch a video explaining the different methods of bulk transport through endocytosis, phagocytosis and pinocytosis.

 

 

 

The Nucleus and Ribosomes

The nucleus houses the cell's genetic material, or DNA, and is also the site of synthesis of ribosomes, the cellular machines that assemble proteins. Inside the nucleus, chromatin (DNA wrapped around proteins, is stored in a gel-like substance called nucleoplasm. Enclosing the nucleoplasm is the nuclear envelops, which is made up of two layers of membrane: an outer membrane and an inner membrane. There's a thin space between the two layers which is directly connected to the interior of another membranous organelle, the endoplasmic reticulum. Nuclear pores, small channels that span the nuclear envelope, let substances enter and exit the nucleus.

 

 

 

Ribosomes are the molecular machines responsible for protein synthesis and is made out of RNA and proteins. Ribosomes may be either free, meaning that they are floating around in the cytoplasm, or bound, meaning that they are attached to the endoplasmic reticulum or the outside of the nuclear envelope.

 

 

Endomembrane System

The endomembrane system is a group of membranes and organelles that work together to modify, package, and transport lipids and proteins. It includes the nuclear envelope as well as the endoplasmic reticulum and Golgi apparatus. Although it's not technically inside the cell, the plasma membrane is also part of the endomembrane system. The membrane interacts with the other endomembrane organelles, and its the site where secreted proteins are exported. It is important to note that the endomembrane system does not include mitochondria, chloroplasts, or peroxisomes.

Let's watch a video outlining the endomembrane system. When he references the endoplasmic reticulum videos previously watched, don't worry, we'll watch it in the next section!

 

 

Endoplasmic Reticulum

The endoplasmic reticulum (ER) plays a key role in the modification of proteins and the synthesis of lipids. It consists of a network of membranous tubules and flattened sacs. The discs and tubules of the ER are hollow, like a straw, and the space inside is called the lumen.

 

 

Rough ER

The rough endoplasmic reticulum (rough ER) gets its name from the bumpy ribosomes attached to its cytoplasmic surface. As these ribosomes make proteins, they feed the newly forming protein chains into the open space (lumen). Some are transferred fully into the ER and float inside, while others are anchored in the membrane. Inside the ER, protein modifications occur, and if these modified proteins are not destined to stay in the ER, they will be packaged into vesicles (small sphers of membrane that are used for transport) and shipped to the Golgi apparatus. The rough ER also makes phospholipids for other cellular membranes, which are transported whent he vesicle forms. Basically, the rough ER helps modify proteins that will be secreted from the cell.

 

 

Smooth ER

The smooth endoplasmic reticulum (smooth ER) is continuous with the rough ER but has few or no ribosomes on its cytoplasmic surface. Functions of the smooth ER include the synthesis of carbohydrates, lipids and steroid hormones; detoxification of medications and poisons; and storage of calcium ions.

 

 

The Golgi Apparatus

When vesicles bud off from the ER, where do they go? Before reaching their final destination, the lipids and proteins in the transport vesicles need to be sorted, packaged, and tagged so that they wind up in the right place. This sorting, tagging, packaging, and distribution takes place in the Golgi apparatus (Golgi body), an organelle made up of flattened discs of membrane.

 

As proteins and lipids travel through the Golgi, they undergo further modifications. Short chains of sugar molecules might be added or removed, or phosphate groups attached as tags. Finally, the modified proteins are sorted (based on markers such as amino acid sequences and chemical tags) and packaged into vesicles that bud from the face of the Golgi. Some of these vesicles deliver their contents to other parts of the cell where they will be used, while others fuse with the plasma membrane, delivering proteins there and releasing proteins outside the cell.

Let's watch a video that goes into detail about the ER and Golgi bodies.

 

 

Lysosomes

The lysosome is an organelle that contains digestive enzymes and acts as the organelle-recycling facility of an animal cell. It breaks down old and unnecessary structures so their molecules can be reused. Lysosomes are part of the endomembrane system, and some vesicles that leave the Golgi are bound for the lysosome. They can also digest foreign particles that are brought into the cell from outside.

 

 

Mitochondria and Chloroplasts

Two important organelles that are responsible for turning stored food energy into something that can be used are the mitochondria and chloroplasts. Mitochondria are the powerhouses of the cell, breaking down fuel molecules and capturing energy in cellular respiration. Chloroplasts are found in plants and algae and are responsible for capturing light energy to make sugars in photosynthesis.

Mitochondria are often called the powerhouses or energy factories of the cell. Their job is to make a steady supply of adenosine triphosphate (ATP), the cell's main energy-carrying molecule. The process of making ATP using chemical energy from fuels such as sugars is called cellular respiration, and many of its steps occur inside the mitochondria.

Chloroplasts are found only in plants and photosynthetic algae. Their job is to carry out photosynthesis, where light energy is collected and used to build sugars from carbon dioxide. The sugars produced in photosynthesis may be used by the plant cell, or may be consumed by animals that eat the plant, such as humans. The energy contained in these sugars is harvested through a process called cellular respiration, which happens in the mitochondria of both plant and animals cells.

 

 

 

Let's watch a video explaining mitochondria in more detail.

 

 

 

Cytoskeleton

The eukaryotic cell has a network of filaments known as the cytoskeleton, which not only supports the plasma membrane and gives the cell and overall shape, but also aids in the correct positioning of organelles, provides tracks for the transport of vesicles and allows the cell to move. In eukaryotes, there are three types of protein fibers in the cytoskeleton: microfilaments, intermediate filaments, and microtubules.

Of the three types of protein fibers in the cytoskeleton, microfilaments are the narrowest. They have a diameter of about 7 nm (nanometers). These filaments serve as highways inside the cell for the transport of proteins and vesicles. Microfilaments can assemble and disassemble quickly, and this property allows them to play an important role in cell motility.

Intermediate filaments are a type of cytoskeletal element made of multiple strands of fibrous proteins would together and have an average diameter of 8 to 10 nm. Unlike microfilaments, which can grow and diassemble quickly, intermediate filaments are more permanent and play an essentially structural role in the cell. They are specialized to bear tension, and their jobs include maintaining the shape of the cell and anchoring the nucleus and other organelles in place.

Desprite the "micro" in the name, microtubules are the largest of the three types of cytoskeletal fibers, with a diameter of approximately 25 nm. They are made up of proteins arranged to form a hollow, straw-like tube. The microtubules help the cell resist compression forces as well as provide tracks for proteins which transport vesicles around the interior of the cell.

Let's watch a video explaining the cytoskeleton in more detail.

 

 

Flagella and Cilia

Microtubules are also key components of two more specialized eukaryotic cell structures: flagella and cilia. Even though prokaryotes also have flagella, which they use to move, eukaryotes also have them. They have pretty much the same role, but a very different structure.

Flagella are long, hair-like structures that extend from the cell surface and are used to mvoe an entire cell. If a cell has any flagella, it usually has one or just a few. Motile cilia are similar, but are shorter and usually apear in large numbers on the cell surface. When cells with motile cilia form tissues, the beating helps move materials across the surface of the tissue. Despite their difference in length and number, flagella and motile cilia share a common structural pattern. In most flagella and motile cilia, there are 9 pairs of microtubules arranged in a circle, along with an additional two microtubules in the center of the ring. This arrangement is called a 9 + 2 array, seen below.

 

 

 

 

Extracellular Matrix and Cell Wall

We've looked at what's inside the cell. Let's think about what's on the outside of the cell. Plants and fungi have a tough cell wall for protection and support, while animal cells can secrete materials into their surroundings to form a meshwork of macromolecules called the extracellular matrix. The extracellular matrix is directly connected to the cells it surrounds. This matrix can help the cells detect both chemical and mechanical cues from the extracellular matrix and trigger signaling pathways in response.

Plant cells have their own supportive extracellular structure: the cell wall. The cell wall is a rigid covering that surrounds the cell, protecting it and giving it support and shape. Fungi also have cell walls, as do some protists.

 

 

 

Eukaryotic Cell Reproduction

Eukaryotes can reproduce both sexually and asexually. Asexual reproduction is often referred to as cell division and it occurs through mitosis. On the other hand, sexual reproduction occurs through meiosis.

 

 

Mitosis

Mitosis is a process of cell division in which a mother nucleus is divided into two identical daughter nuclei. It is completed in 4 stages: prophase, metaphase, anaphase and telophase. The goal of mitosis is to make sure that each daughter cell gets a perfect, full set of chromosomes. So, when cells undergo mitosis, they don't just divide their DNA at random and toss it into piles for the two daughter cells. Instead, they split up their duplicated chromosomes in a carefully organized series of steps. These phases occur in strict sequential order, and cytokinesis (the process of dividing the cell contents to make two new cells) starts in anaphase or telophase.

 

 

 

Prophase

In early prophase, the cell starts to break down some structures and build others up, setting the state for division of the chromosomes.

 

The mitotic spindle begins to capture and organize the chromosomes:

 

 

 

Metaphase

In metaphse, the spindle has captured all the chromosomes and lined them up at the middle of the cell, ready to divide.

 

 

Before proceeding to anaphase, the cell will check to make sure that all the chromosomes are correctly attached to microtubules.

 

 

Anaphase

In anaphase, the sister chromatids separate from each other and are pulled towards opposite ends of the cell.

 

 

 

Telophase

In telophase, the cell is nearly done dividing, and it starts to re-establish its normal structures as cytokinesis (division of the cell contents) takes place.

 

 

When cytokinesis finishes, we end up with two new cells, each with a complete set of chromosomes identical to those of the mother cell.

 

 

Let's watch a video explaining mitosis.

 

 

Meiosis

In many ways, meiosis is a lot like mitosis. The cell goes through similar stages and uses similar strategies to organize and separate chromosomes. In meiosis, however, the cell has a more complex task which is accomplished using a two-step division process. Since cell division occurs twice during meiosis, one starting cell can produce four gametes (eggs or sperm). In each round of division, cells go through four stages: prophase, metaphase, anaphase and telophase.

First cell division:

 

 

Second cell division:

 

 

Let's watch a video explaining the difference between mitosis and meiosis.

 

 

 

 

Procaryotes and Eucaryotes: How They Are Similar

Despite their apparent differences, procaryotes and eucaryotes have a lot in common. They perform most of the same kinds of functions, and in the same ways. Both are enclosed by plasma membranes, filled with cytoplasm, and loaded with ribosomes. Both have DNA which carries the instructions for operating the cell. Despite all of these similarites, the differences are also clear. Eucaryotic cells are much larger and much more complex than procaryotic cells.

Similarities

Both types of cells use and/or contain:

 

Differences:

 

Now that we know a little more about eukaryotic cells, let's take a look at some of the species we'll be dealing with in the treatment process.

 

 

Protozoa

Protozoans are found in all moist habitats within the United States, but we know little about their specific geographic distribution. They are unicellular organisms that range in size from a few microns to several hundred microns. Polluted waters often have a rich and characteristic protozoan fauna. The relative abundance and diversity of protozoa are used as indicators of organic and toxic pollution.

Although protozoa are frequently overlooked, they play an important role in many communities where they occupy a range of trophic levels. As predators upon unicellular or filamentous alge, bacteria, and microfungi, protozoa play a role both as herbivores and as consumers in the decomposer link of the food chain.

Both in organic pollution of the natural environment and in the biological processing of human and domestic animal sewage, the ceaseless activity of the protozoa, particularly the colonial ciliates, in the extraction and digestion of bacteria and other suspended particles is the main element of the natural process by which the water supply is rendered once again fit for consumption by humans and other creatures. Any change in our environment which threatens the life of a balanced community of protozoans threatens also the continuity of a clean water supply for humans. This is particularly relevant in the light of our current over-use of kitchen and lavatory disinfectants and their effect upon the ciliates at sewage processing plants and in the waterways beyond.

Protozoa are members of the Kingdom Protista. There are about 20,000 known species of protozoa that live in water and soil. Some feed on bacteria while others are parasites and feed off their hosts. Most protozoa are asexual and reproduce in one of three ways: fission, budding, and multiple fission. Some protists are sexual and exhange genetic material from one cell to another through conjugation, which is the physical contact between cells.

 

A protist can survive in an adverse environment by encapsulating itself with a protective coating called a cyst. The cyst defends the protist in extreme temperatures against toxic chemicals and even when there is a lack of oxygen, moisture, and food.

 

 

Factors Affecting Growth and Distribution

Most free-living protozoa reproduce by cell division (exchange of genetic material is a separate process and is not involved in reproduction in protozoa). The relative importance for population growth of biotic versuc chemical-physical components of the environment is difficult to ascertain from the existing survey data. Protozoa are found living actively in nutrient-poor to organically rich waters and in fresh water varying between 0°C (32°F) and 50°C (122°F). Nonetheless, it appears that rates of population growth increase when food is not constrained and temperature is increased.

 

 

Nutrition

Animal-like protists are heterotrophs. This means that in order to get food and nutrition, these protists must eat/ingest food from their environment. They can do this in a few ways: Endocytosis, also called phagocytosis, is the most common method for heterotrophic protists. This is where the protists physically engulf or swallow their prey. Amoebas, for example, are animal-like protists that engulf their prey and break them down inside their cell in order to get their nutrition. These types of protists are also called phagotrophs. They can also intake liquid from the surrounding environment. Tiny pockets form along the membrane, fill with liquid, and pinch off. This is known as pinocytosis.

 

 

 

 

Other animal-like protists are filter feeders. They'll often use their flagellum to whip back and forth and create a flow or a current around them to filter through and absorb food from their environment. This type of heterotroph is also called an osmotroph, which means they absorb food to eat from the environment instead of engulfing it whole like a phagotroph.

Plant-like protists are autotrophs. This means that they create their own food without having to eat or engulf other organisms/organic material in the environment. Plant-like protists have chloroplasts in their cells in order to perform photosynthesis to convert sunlight into food. Common plant-like protista include microscopic algae as well as multicellular seaweeds like kelp.

Fungi-like protists are also called mold. The two major types of fungi-like protists can be divided into water molds and slime molds. These types of protists are heterotrophs, which means they absorb their food from the space, environment and organisms around them.

 

 

Protozoa in the Wastewater Treatment Process

Wastewater treatment is fundamentally a biological process. When influent enters the microbial ecosystem of a treatment plant, nutrient removal is accomplished through the consumption of organic matter by microorganisms. The bulk of all nutrient removal is performed by bacteria, however, protozoa and metazoa balance these bacterial populations and offer insight into the conditions of the wastewater. Operators who understand the varying roles of wastewater microbes and the conditions that favor their growth, can foster an ecosystem that promotes optimal treatment.

Protozoa are one of the most common components in the wastewater purification process. They are responsible for improving the quality of the effluent and maintaining the density of dispersed bacterial populations by predation. By consuming free bacteria and small, unsettled floc, protozoa enhance the clarity of the final effluent. Observing protozoa populations under a microscope can also alert operators of treatment conditions and sludge age.

Protozoa are considered to be the most important bacterivorous grazers. Even though some protozoa (crawling ciliates and other forms) can eat flocculated bacteria, most protozoa can only graze on suspended bacteria and particles; in this way they have a significant effect on the effluent quality. It is generally assumed that the primary role of protozoa in wastewater treatment is the clarification of the effluent. In the presence of ciliates, a reduction of the density of viable E. coli was observed. It has been observed that the presence of protozoa increases the per-cell nitrification rate, probably because of the ability of protozoa to influence bacterial growth. Protozoa release inorganic and organic products into their surroundings, including recycled nutrients, such as nitrogen, phosphorous, and organic carbon.

Protozoa in the activated sludge process commonly are placed in one of five groups: amoebae, flagellates, free-swimming ciliates, crawling ciliates and stalked ciliates. Amoebae and flagellates are considered "lower" life forms, while crawling ciliates and stalk ciliates are considered "higher" life forms. Free-swimming ciliates are considered "intermediate" life forms. Some treatment plant operators often perform routine microscopic examinations of the protozoa in the activated sludge to determine the health of the activated sludge process.

 

 

Amoeba

Amoeba are predominat under a young sludge age because they require high nutrient levels or low competition to grow. Under shock loads of biochemical oxygen demand (BOD), high concentrations of particulate matter, toxic conditions, or low dissolved oxygen (DO), amoeba can also dominate. The latter two conditions generally trigger the amoeba to develop a protective gelatinous shell that gives them an advantage over other microbes. Furthermore, their slow movement reduces oxygen demand required for growth and reproduction.

 

 

Flagellates

Flagellates are typically present under a young sludge age as well. Since flagellates compete poorly with bacteria for the same soluble nutrients, their growth is favored at the younger sludge age before bacteria have had a chance to populate. As such, a wastewater sample high in flagellates can indicate high soluble nutrient levels also known as a high food to microorganism (F/M) ratio.

 

 

Ciliates

Ciliates are favored under a healthy sludge age. While they do not consume organic matter, they do feed on bacteria, making them excellent indicators of healthy floc formation and useful claryifying agents. Without ciliates, bacteria and algae populations can grow out of control in the wastewater microbial ecosystem. Among the three types of ciliates (stalked, free-swimming, and crawling), each group has different conditions under which their populations are favored.

 

Typically, operators base the health of the process on the protozoan groups that are dominant as revealed by microscopic examination (Table below). For example, if lower life forms are dominant, the activated sludge is considered unhealthy and unacceptable. An unhealthy activated sludge produces an aeration tank effluent having a BOD greater than 30 mg/L. If the higher life forms are dominant, the activated sludge is considered healthy and acceptable. A healthy activated sludge produces an aeration tank effluent having a BOD less than 30 mg/L.

 

Protozoan Groups and Bioindicator Values
Amoebae Rarely predominant except for start-up conditions and conditions that mimic start-up such as over-wasting, recovery from toxicity, washout, and organic overloading
Flagellates, plant-like Dominant under high organic loading, dispersion of floc particles, such as through chlorination, and start-up conditions or conditions that mimic start-up. Also may dominate in the presence of excess soluble phosphorus.
Flagellates, animal-like Except for the presence of excess soluble phosphorus, these are dominant for operational conditions listed for plant-like flagellates and usually follow plant-like flagellates as the dominant group.
Free-swimming ciliates Transition group that dominates between healthy and unhealthy conditions and proliferates when large numbers of free-swimming bacteria are present.
Crawling ciliates Dominant in the presence of mature floc particles and low BOD in the bulk solution. Alternate with stalked ciliates as the dominant group.
Stalked ciliates Dominant in the presence of mature floc particles and low BOD in the bulk solution. Alternate with crawling ciliates as the dominant group.

 

 

 

Operators should exercise caution when correlating final effluent quality with the health of activated sludge as indicated by the dominant protozoan groups. For example, loss of secondary solids from the clarifier due to a bulking condition would increase the BOD above that achieved in the aeration tank.

Let's watch a video introducing you to cilia, flagella, and pseudopodia on protozoans.

 

 

Diseases Caused by Protozoa

Several protozoa can cause waterborne diseases.  The most common of these pathogenic protozoa are Cryptosporidium , Entamoeba , Giardia , and Toxoplasma .  These species are all able to form cysts , or protective coatings, which allow them to survive outside a host for extended periods of time.  Protozoan cysts also protect the organism from chlorine, so these species are not effectively controlled by chlorination.  Instead, protozoa are usually removed from water by filtration. 



Cryptosporidium

Cryptosporidium

Cryptosporidium is a sporozoa which is one of the most common pathogens responsible for waterborne diseases in the United States.  Scientists estimate that between 1% and 5% of Americans are infected with Cryptosporidium at any one time.  Cryptosporidiosis, the disease caused by Cryptosporidium infection, results in diarrhea, abdominal cramps, and fever.  Healthy adults usually do not require treatment for cryptosporidiosis since the body will heal itself naturally within a couple of weeks. 

Cryptosporidium , like most other waterborne pathogens, is spread to new hosts through the ingestion of contaminated food and drinking water.  In addition, several outbreaks have been found to result from fecal accidents in swimming pools.  Water treatment plants find it difficult to prevent the spread of Cryptosporidium since the cysts are very resistant to chlorination and can sometimes pass through filters.  Instead, the cysts can be killed by ozonation or by boiling the water for at least a minute. 


Entamoeba histolytica

Entamoeba histolytica

Entamoeba histolytica is an amoeboid protozoan which lives in anaerobic environments.  Like the other pathogenic protozoa, Entamoeba is capable of forming cysts which can remain dormant for extended periods of time in the water, in soil, or in food.  These cysts spread to new hosts when we ingest contaminated food or water. 

In humans, Entamoeba causes amoebic dysentery, which is usually treated with antibiotics.  Entamoeba outbreaks are rare in the United States but are quite common in developing countries when raw sewage contaminates drinking water supplies or when the soil is fertilized with untreated wastes.  In the water treatment plant, Entamoeba can be removed from water using a sand filter. 



Giardia lamblia

Giardia

Giardia lamblia is a flagellate protozoan which is capable of forming cysts.  The species is the most important cause of waterborne disease outbreaks in the United States. 

Infection with Giardia lamblia results in a disease called giardiasis, which is also known as traveler's diarrhea or Montezuma's Revenge.  Symptoms of giardiasis include diarrhea, abdominal cramps, fatigue, and weight loss.  Although most people who contract giardiasis heal naturally within a week or two, the illness sometimes lingers for up to a year, in which case antibiotics may be prescribed. 

Giardia lamblia is spread when people ingest contaminated food or water.  In the water treatment plant, chlorine is somewhat effective at inactivating the cysts at a dosage of 1.5 mg/L chlorine with a contact time of 10 minutes.  Filtration and boiling are more effective at killing Giardia .



Toxoplasma gondii

Toxoplasma gondii is a sporozoa which causes a disease known as toxoplasmosis.  In most cases, toxoplasmosis has mild flu-like symptoms which are often unnoticed or undiagnosed and do not require treatment.  However, a pregnant woman may pass on the infection to her unborn child, in which case the child will have much more serious symptoms. 

Toxoplasma gondii has a complex life cycle involving at least two different hosts.  The protozoan reproduces sexually within its primary host, the cat, and releases cysts in the cat's feces.  The cysts are then ingested by intermediate hosts, which include a large number of vertebrate species such as pigs, cows, and humans.  In the intermediate host, the cysts develop into an active form of the protozoan which reproduces asexually.  The offspring produced in the intermediate host then infect a cat, completing the cycle.

In most cases, humans contract toxoplasmosis by eating contaminated meat or by handling cats or changing cats' litter boxes.  However, a recent outbreak of toxoplasmosis linked to drinking water in Canada has alerted us to the possibility of Toxoplasma gondii being spread in water. 

 

 

Amoebae

Amoebae, like those shown below, are unicellular eukaryotic organisms classified as a protozoa. They appear as jelly-like blobs as they move about. Amoebas move by changing their shape, exhibiting a unique type of crawling motion that has come to be known as amoeboid movement. They can form temporary extensions of their cytoplasm known as pseudopodia or "false feet" which can be used for locomotion or capturing food. They acquire their food through phagocytosis and commonly reproduce through binary fission, a process in which the cell divides into two identical cells.

Amoebae itself is found in freshwater, typically on decaying vegetation from streams, also found in salt water, in wet soil, and in animals (including people); but large, naked Amoebae are not especially common in nature. However, because of the ease with which they may be obtained and kept in the lab, they are common objects of study, both as representative protozoa and to demonstrate cell structure and function.

A good method of collecting amoebae is to lower a jar upside down until it is just above the sediment surface. Then one should slowly let the air escape so the top layer will be sucked into the jar. Deeper sediment should not be allowed to get sucked in. It is possible to slowly move the jar when tilting it to collect from a larger area. If no amoebae are found, one can try introducing some rice grains into the jar and waiting for them to start to rot. The bacteria eating the rice will be eaten by the amoebae, thus increasing the population and making them easier to find.

Amoebae usually are present in high numbers during start up of a treatment plant recovery from a toxic discharge to the treatment plant or low dissolved oxygen levels. If amoebae are present as the dominant protozoan group, this could indicate an unstable wastewater environment and a sludge that is in poor health. Some general types of amoebae commonly found in wastewater are Arcella, Euglypha, and Centropyxis all of which are known as testate amoebae.

 

 

 

Anatomy

Amoebas are simple in form, consisting of cytoplasm surrounded by a cell membrane. The outer portion of the cytoplasm is clear and gel-like, while the inner portion is granular and contains organelles, such as nuclei, mitochondria, and vacuoles. Some vacuoles digest food, while others expel excess water and waste from the cell through the plasma membrane.

The most unique aspect of amoeba anatomy is the formation of temporary extensions of the cytoplasm known as pseudopodia. These "false feet" are used for locomotion, as well as to capture food such as bacteria, algae, and other microscopic organisms. Pseudopodia may be broad or thread-like in appearance with many forming at one time or one large extension may form when needed.

 

 

Amoebas don't have lungs or any other type of respiratory organ. Respiration occurs as dissolved oxygen in the water diffuses across the cell membrane. In turn, carbon dioxide is eliminated from the amoeba by diffusion across the membrane into the surrounding water. Water is able to cross the amoeba plasma membrane by osmosis. Any excess accumulation of water is expelled by contractile vacuoles withinthe amoeba.

 

Nutrient Acquisition and Digestion

Amoebas obtain food by capturing their prey with their pseudopodia. The food is internalized through a type of endocytosis known as phagocytosis. In this process the pseudopodia surround and engulf a bacterium or other food source. A food vacuole forms around the food particle as it is internalized by the amoeba. Organelles known as lysosomes fuse with the vacuole releasing digestive enzymes inside the vacuole. Nutrients are obtained as the enzymes digest the food inside the vacuole. Once the meal is complete, the food vacuole dissolves.

 

 

 

Reproduction

Amoebas reproduce by the asexual process of binary fission. In binary fission, a single cell divides forming two identical cells. This type of reproduction happens as a result of mitosis. In mitosis, replicated DNA and organelles are divided between two daughter cells which are genetically identical.

 

Some amoeba also reproduce by multiple fission. In multiple fission, the amoeba secretes a three-layered wall of cells that harden around its body. This layer, known as a cyst, protects the amoeba when conditions become harsh. Protected in the cyst, the nucleus divides several times. This nuclear division is followed by the division of the cytoplasm for the same number of times. The result of multiple fissions is the produciton of several daughter cells that are released once conditions become favorable again and the cyst ruptures. In some cases, amoebas also reproduce by producing spores.

 

 

 

 

Parasitic Amoebas

Some amoeba are parasitic and cause serious illness and even death in humans. Entamoeba histolytica cause amebiasis, a condition resulting in diarrhea and stomach pain. These microbes also cause amebic dysentery, a severe form of amebiasis. They travel through the digestive system and inhabit the large intestines. In rare cases, they can enter the bloodstream and infect the liver or brain.

Another type of amoeba, Naegleria fowleri, causes the brain disease amoebic meningoencephalitis. Also known as the brain-eating amoeba, these organisms typically inhabit warm lakes, ponds, soil and untreated pools. If they enter the body through the nose, they can travel to the frontal lob of the brain and cause a serious infection. The microbes feed on brain matter by releasing enzymes that dissolve brain tissue. A N. fowleri infection in humans is rare but most often fatal.

Acanthamoeba cause the disease Acanthamoeba keratitis. This disease results from an infection of the cornea of the eye. This disease can cause eye pain, vision problems, and may result in blindness if left untreated. Individuals who wear contact lenses most often experience this type of infection since they can become contaminated with the amoebae if they are not properly disinfected and stored.

 

 

The Most Common Types of Amoebae Found in Wastewater

The most common types of amoebae found in wastewater systems include Arcella, Euglypha, and Centopyxis.

Arcella

The Arcella is a species with a more or less circular shell with central, invaginated aperature, in many species surrounded by a collar and/or a circle of pores and is one of the largest and most common genera. Over 50 species, subspecies and varieties have been described, but many descriptions are not adequate enough, making it difficult to identify species. An important characteristic is the shape, which is like an umbrella. Young shells on Arcella are colorless, while older ones become brown due to iron and manganese storage in the cells.

Arcella vulgaris

 

Arcella species have an umbrella-shaped shell with a central invaginated aperture from where the pseudopods emerge. In a dorsal view, the shell ranges from circular or broad elliptical up to an irregular square shape. The aperature can be circular and in some species, or elliptical, and in many species surrounded by a tube and/or a ring of pores.

 


Morphology of Arcella.

 VA – ventral surface, C –  apertural collar, DA – dorsal surface, F – funnel-shaped invagination, 
– aperture, – basal border (modified after Deflandre, 1928).

 

 

Arcella nourish themselves on diatoms, unicellular green algae or animal protozoa such as flagellates and ciliates.

Three Arcella specimens joined together in one large pseudopodium.

 

 

 

Euglypha

This group of amoebae is a prominent group that produces shells from siliceous scales, plates and sometimes spines. These elements are created within the cell and then assembled on its surface in a more or less regular arrangement, giving it a textured appearance. There is a single opening for the long slender pseudopods, which capture food and/or moves the microbes across the surface.

 

Usually we consider amoebae protozoa as indicators of poor water quality since many tolerate low dissolved oxygen and can function well along with flagellates, in which F/M ratio is relatively high. One big exception to this rule is the testate amoebae (most common is Euglypha), which are much easier to see under a microscope because it has a shell surrounding the true amoeboid organism inside the shell. In wastewater, the most common testate amoeba is from the Euglypha, pictured above. This amoeba usually increases in number as soluble BOD falls below 40 mg/L and nitrification is occurring. This amoebae population tends to trend upwards with longer sludge ages.

While usually seen with good water quality, the shell can remain visible after the amoebae is dead or inactive. Therefore, we should not rely on Euglypha amoebae as an ideal indicator protozoa. For example, if you see this type of amoebae as abundant, but do not see activity of ciliated protozoa, there is a need to look further to see if any toxicity or other stress is occurring as the ciliates are much more sensitive to environmental changes than Euglypha.

 

 

Centropyxis

Centropyxis is a genus of amoeba that has a shell variable in contour and size, cap-shapes, yellow or brown, oval or circular and usually with four or more lateral spines. In lateral view the amoebae is spherical and tapering towards the aperature, resembling a beret, often covered with quartz grains and/or diatom frustules.

 

Lateral view

 

 

 

 

 

Wastewater Treatment Environment

Amoebae are found in many different types of wastewater, including activated sludge, trickling filters and lagoons. They grow well on particulate organic matter and are able to tolerate low dissolved oxygen environments. Testate amoebae are often found in lightly loaded plants or in plants where nitrification occurs. They are usually found in young sludge ages but they can be found at any age if all of a sudden a high BOD loading has occurred. Amoebae can be found during plant start-ups or often following upsets and can be used as an indicator for a process control tool.

If you see an increase in amoebae within your system, it depends upon what the rest of the biomass looks like before you can determine what the increase means. If the floc is small, weak, and dispersed, you may have a very young sludge age. Typically the presence of amoebae indicates a high loading of food versus the amount of biomass available to eat the organic material. You may have an older sludge age, but a recent high loading of BOD is forcing the sludge age to be younger. Usually you can expect high solids in the effluent and higher BOD levels if amoebae are present in significant numbers.

 

We've covered the amoebae that may be present in our wastewater, so let's look at another protozoa of importance: the flagellates.

 

 

Flagellates

 

Flagellates are single-celled protists (protozoan) with one or more flagella, which are whip-like organelles often used for propulsion. Eucaryotic flagella are not the same as flagella of bacteria. The flagella found in flagellates has an internal structure composed of small tubules of protein called microtubules. Some flagellates, such as the Euglena, can make food by photosynthesis like plants, while others, such as the Trypanosomes, are parasitic and cause disease. The word flagellate also describes a particular construction and their means of motion.

Flagellates belong to the class Mastigophora and range in size from 5-20 micrometers (µm) in diameter. They are commonly ovoid or pear-shaped with one to four flagella, hair-like projections used for locomotion, attached to one or both ends of the cell. The flagella can usually be observed at 1000X magnification. Some flagellates may form colonies in which the cell bodies are clumped together with their flagella projecting outward. The two major classes of flagellates are the phytoflagellates (resemble plants and obtain energy through photosynthesis and zooflagellates (resemble animals and obtain energy through feeding). Due to the particular characteristic of resembling plants, they are often classified as flagellated algae rather than protozoa.

The locomotion of flagellates is usually fast and they seem to flip and twist as the flagella are “whipped” around to propel them. Some flagellates have multiple flagella. This makes them appear “bouncy” and unorganized due to their locomotion mechanism while other higher life forms such as free-swimming ciliates present a more organized locomotion mechanism. This is sometimes helpful in identifying flagellates under the microscope. When counting flagellates under the microscope, a 400X magnification should be used in order to identify them better due to their small size.

Like their relatives the amoebae, flagellates are usually present when there are large amounts of soluble food available (high F/M or high BOD). They are found during start up when the sludge is young or after an upset, but will quickly predominate over the amoebae because they are more efficient feeders. They are often found in trickling filters, oxidation ponds, lagoons and activated sludge. Flagellates are one of the few protozoan form present in sludges that are strongly loaded. Their presence may indicate high soluble BOD levels. Flagellates usually are present in very large numbers during initial start-up of a wastewater treatment plant, during recovery from a toxic discharge to the treatment plant, or at low D.O. levels. If flagellates are present as the dominant protozoan group, this could indicate an unstable wastewater environment and a sludge biomass that is very young. Usually found in low MCRT or low HRT for activated sludge systems. Lagoon systems are different and flagellates are often found in lagoons since it is harder to develop an older sludge in a lagoon with high flows.

Flagellates prefer soluble nutrients and dead or decaying material. They compete with bacteria for food in the activated sludge process, but can only dominate when the nutrient level is high. Some of the larget flagellates eat bacteria in the sludge but can not keep up with the logarithmic growth rate of the bacteria in the activated sludge. Therefore flagellates can only dominate early in the treatment process when the soluble organic nutrients are high enough for both flagellates and bacteria to eat. In a continuosly fed batch process, the dominance of these microorganisms is short lived.

Flagellates feed on soluble organic matter and dispersed bacteria. and are more common in heavily loaded plants or during startups. They predominate when there is a high dispersed (singled-celled) bacteria population density. They are sometimes associated with turbid effluents produced during toxic upsets. They also bloom when septic sludge or inadequate aeration causes anaerobic conditions in the aeration basin. Their presence indicates high soluble BOD levels, low dissolved oxygen, and high organic load. Typically higher life forms disappear when a chemical upset goes through a wastewater system Flagellates are the first higher life form to come back after a chemical upset in the system. They can indicate when the system is getting healthier or if it is still overcoming a chemical upset.

 

 

Types of Flagellum

The flagellar structure consists of three different parts: rings embedded in the basal body, a hook near the surface of the organism to keep it in place, and the flagellar protein filaments. Every flagellum has these three things in common, regardless of organism. However, there are four distinct types of bacterial flagellum based on location:

Types of Flagellum

 

A. Monotrichous: A single flagellum at one end or the other of the organism.

 

B. Lophotrichous: Several flagellum on one end or the other of the organism.

 

C. Amphitrichous: A single flagellum on both ends of the organism.

 

D. Peritrichous: Several flagellum attached all over the organism.

 

 

 

Structure and Function

Eukaryotic flagella are not the same as bacterial flagella. They have an internal structure comprised of nine doublets of microtubules forming a cylinder around a central pair of microtubules.

 

 

The peripheral doublets are linked to each other by proteins. These proteins include dynein, a molecular motor which can cause flagella to bend and propel the cell relative to its environment, or propel water or mucus relative to the cell. Many flagellates have a thin, firm pellicle (outer covering) or a coating of a jellylike substance.

Flagellate occurs in two different stages: Trophozoite (feeding stage) and the cystic stage.

During the trophozoite, or feeding stage, the flagellate possesses a bilateral symmetrical body with organelles occuring in pairs and measures 10-18 µm in length. The body shape during this stage is "tear-dropped" with a convex dorsal surface and a concave ventral one. Flagella occur in four pairs (anterior pair, posterior pair, ventral pair and caudal pair). Two nuclei occur at the broader end of the body.

 

During the cystic stage, the fully formed cyst is oval in shape and measures 12 µm long by 7 µm broad. The cyst wall is thin and the organism does not fill the entire cyst. There are four nuclei which may remain clustered at one end or lie in pairs at opposite poles. The remains of the disintegrated flagella forming a central streak, visible in iodine, and the margins of the sucking disc may be seen inside the cytoplasm.

Flagellate is actually used by cells and unicelluar organisms (protozoans) for movement, sensation and signal transduction and are just an extension of the cell or organism. Depending on the organism, a flagellum consists of different structures. In bacteria, flagellates are made of the protein flagellin. In eukaryotes, flagellate consists of microtubules surrounded by a plasma membrane. Prokaryotes and eukaryotes use different sources of energy to drive the flagella. Moving eukaryotic flagellates require ATP, which is produced during photosynthesis. Prokaryotes use energy from the proton-motive force, which is the ion gradient that lies across the cell membranes.

 

 

Nutrient Acquisition and Digestion

Flagellates, just like other protozoans, possess membrane cellular organelles such as nuclei, food vacuoles and lysosomes. Food is captured in may different ways, however in most cases, after capture, it is contained in a spherical bubble (food vacuole) that is formed from the cell membrane and released into the cell interior. This process is similar to amoebaes.

While flagellates are in motion they accidentally "hit" substrates. With decreasing numbers of suspended bacteria, flagellates find it more difficult to find more (substrate).

 

 

 

Reproduction

Flagellates reproduce through binary fission since it consists of a cell splitting in half.  As you can see in the animation above, the microorganism first makes a second copy of its DNA in a process known as replication .  Next, the cell begins to constrict in the middle, leaving one set of DNA and organelles on each side of the constriction.  Eventually, the cell splits apart into two identical daughter cells.  Once these daughter cells enlarge to adult size, each one is ready to split into two more daughter cells.  This occurs during the trophozoite, or feeding, stage. When conditions become unfavorable, encystment takes place in which a tough resistant wall is formed by the parasite and the cell undergoes division to produce the two daughter cells within the cyst.

 

 

Environment

You see flagellates at the early stages of wastewater treatment or when there is log phase growth of bacteria. In lagoon systems, you will often find flagellates predominating near the inlet where dissolved oxygen is low & soluble BOD high. If seen near the effluent, this indicates some type of upset condition. Common triggers include - (1) high hydraulic loads, (2) increased organic loadings, (3) pond turnover, and (4) problems with dissolved oxygen concentrations.

In activated sludge or fixed film systems, the sudden appearance of flagellates can indicate an increase in organic loadings or follow a toxic event that killed off significant portion of the living bacteria found in the MLVSS or biofilm. Either event merits further investigation into what is causing the increase in flagellate populations.

It you see an increase in flagellates in your treatment system, it depends on what the rest of the biomass looks like before you can determine what it means. If the floc is small, weak, and dispersed, you may have a very young sludge age. Typically, the presence of flagellates, similar to amoebae, indicates a high loading of food vs. the amount of biomass available to eat the organics. Flagellates possess one advantage over their amoeboid relatives in that they can swim. This enables them to invade and adapt to a wider range of environments unsuitable for other amoebae. Usually, this means that the sludge is a bit older than if only amoebae are present and there are probably less single-celled bacteria swimming around.

 

Parasitic Flagellates

Some flagellates are very harmful to humans, such as Giardia lamblia, which is a flagellated protozoan that colonizes and reproduces in the small intestine, causing the disease giardiasis. The protozoan attaches to the lining of the host's intestines where it reproduces through binary fission. Symptoms incude diarrhea, fever, and abdominal pain. Some of the symptoms may also be caused by large numbers of the parasites blocking nutrient absorption by the host. Mammalian hosts of the parasite include cows, beavers, deer and sheep. People pick up the protozoan by ingesting cysts from fecally contaminated food or water. During the past two decades, Giardia infection has become recognized as one of the most common causes of waterborne disease in humans in the U.S.

Common Types of Flagellates

The typical flagellate found in wastewater treatment plants appear as small ovoid shapes that move in the liquid phase surrounded by much smaller free bacteria. Using a single, or multiple flagella, these organisms are one of the first indicator microbes seen in a treatment system. Flagellates consume the free-floating bacteria cells and adsorb some soluble organics for thier nutritional requirements. There are some photosynthetic flagellates (Euglena) found, but they are not common in wastewater treatment.

The most common types of flagellates are the animal-like flagellate such as Bodo, Hexamitus, and Peranema; and the plant-like, or pigmented, flagellate, such as Euglena. Since they contain chlorophyll, they are known as motile algae.

 

 

Euglena

Euglena are single celled organisms that belong to the genus protist. As such, they are not plants, animal, or fungi. In particular, they share some characteristics of both plants and animals. While they can manufacture their own food, like plants, they are also capable of movement and consuming food, like animals. When viewed under a microscope, Euglena appear as elongated unicellular organisms that are rapidly moving across teh field surface. One thing to notice is that it usually has a blunt, or rounded, end and a pointed end, giving it the tear-drop shape. The blunt end is often the head part from which the whip-like flagella is attached. Although one flagellum is often seen, they actually have two flagella, one of which is often hidden in a part of the Euglena referred to as the reservoir. The longer, visible flagellum, located at the anterior end of the organism twirls rapidly, making it possible for these organisms to move through the water.

A closer observation will reveal a reddish spot at the anterior part of the Euglena. This is an important organelle, known as the eyespot, that contains carotenoid granules that allows the organism to sense and move towards sunlight. The eyespot also helps filter wavelength of light that reaches the body, which is the light detecting structure that lies at the base of the flagellum. In response to this, Euglena moves towards the source for photosynthesis. This bodily movement of the organism is commonly known as positive phototaxis. In addition to the red spot, you'll also notice dark green spots all over the body of the organism. Some of these spots are chloroplasts which contain chlorophyll, which is responsible for photosynthesis. Since they are capable of making their own food, this makes them autotrophic. They are also heterotrophic, which means that they also consume food through phagocytosis. Euglena also has a contractile vacuole that helps collect and remove excess fluids from the cell. This prevents the cell from taking in too much water that can cause the cell to rupture. These are usually found in ponds, lakes or reservoirs, but usually not in wastewater treatment.

 

 

Peranema

Peranema are small flagellates and range in size from 20-70 µm in diameter. They are very active predators and scavengers and are common in waters rich in organic nutrients, especially in water in which decay is taking place (like bacterial growth/death). The single flagellum projects straight forward, and a rapid rotation of its extreme end pulls the Peranema smoothly through the water. The body of the Peranema can undergo extreme contraction and distortion as it moves. They have been seen inside the bodies of dead rotifers (which we'll discuss in the next lesson) and are said to absorb nutrients through their outer pellicle. In addition, they can ingest quantities of detritus, bacteria, algae and even large organisms by expansion of the cytostome (a cavity which lies at the base of the flagellum).

 

Now let's move on to Ciliates, which uses a different form of locomotion.

 

Ciliates

The Ciliates are more complex organisms than the amoebae and flagellates. The three types of ciliated protozoa are free-swimming ciliates, crawling ciliates, and stalked ciliates. All of these have short hair-like structures or cilia that beat in unison to produce a water current for locomotion and capturing bacteria. The water current moves suspended bacteria into a mouth opening. Ciliates are favored under a healthy sludge age. While they do not consume organic matter, they do feed on bacteria, making them excellent indicators of healthy floc formation and useful clarifying agents. Without ciliates, bacteria and algae populations can grow out of control in the wastewater microbial ecosystem. Among the three types of ciliates common to wastewater, each group has different conditions under which their populations are favored.

 

 

Types of Ciliates

Free-Swimming Ciliates

 

Free-swimming ciliates start to form as flagellates disappear. Free-swimmers are usually found when no large flocs have been formed so that it is easier to swim around. They die back as floc particles increase and dispersed bacteria decreases, at which time crawling ciliates flourish. They range in size from 20-400 µm and have two kinds of nuclei. They may experience a spike in population when levels of free bacteria are abundant for predation. Free-swimmers swim faster than flagellates, so they can out compete them for food. While bacteria and flagellates compete for dissolved organics, cilates compete with other cilates and rotifers for bacteria. They are usually an indicator of good quality sludge and are typically found in young to medium age sludge. Some of the most commonly found free-swimming cilates in wastewater are Paramecium, Euplotes, and Colpidium.

The rapid beating of the cilia permits the organism to move in a straight line. Free-swimming ciliates are found in large numbers when the bacterial population and dissolved oxygen concentration of the treatment process are relatively high. If free-swimming ciliates are present as the dominant protozoan group, this could indicate a wastewater environment that is not yet stabilized and a sludge that is intermediate in health.

 

 

Crawling Ciliates

Crawling ciliates, such as Aspidisca, possess ciliate only on the ventral or belly surface where the mouth opening is located. The beating of the cilia gives the appearance that it is crawling as it moves across the surface of floc particles. Some of the cilia are modified to form "spikes" that help to anchor the organism to the floc particle. Crawling ciliates are found in large numbers when the bacterial population and dissolved oxygen concentration of the treatment process are high and the wastewater environment is stable. In order for crawling ciliates to  be dominant, there must be large floc structures present that impede the free-swimmers and flagellates movement and provide a surface for the crawlers to "walk" on. The dominance of crawling ciliates begins after most soluble nutrients have been removed. Floc begins to form from dispersed bacteria. As they they feed on bacteria, crawling ciliates can improve floc structure. A more mature sludge age with reduced BOD allows stalked ciliates to compete with crawling ciliates.


 

Stalked Ciliates

Stalked ciliates, such as Carchesium and Voticella, have cilia around the mouth opening only and are attached to floc particles. Stalked ciliates have an enlarged anterior portion or "head" and a slender posterior portion or "stalk". Some , such as Vorticella, have a contractile filament within the stalk that permits springing action. The beating of the cilia and the springing action of the stalk produce a water vortex that draws dispersed bacteria into the mouth opening. Of all ciliated feeding mechanisms, the stalked ciliates mechanism is the most efficient in capturing stray bacteria. Stalked ciliates are also capable of swimming freely. This may occur during low dissolved oxygen levels within the treatment process. Stalked ciliates are found in large numbers when the bacterial population and dissolved oxygen concentration are high, the wastewater environment is stable and a mature floc has developed. Stalked ciliates indicate a stable wastewater environment and a healthy sludge.

Stalked ciliates can be seen in single organism form or can grow in colonies. Each "head" in a colony of stalked ciliates is considered one organism. Therefore, when counting higher life forms for maturity index calculations, every organism is counted in the colony. Colonies can range from three to over three hundred organisms each. Stalked ciliates usually attach themselves to a piece of floc or inert material, but can occasionally be seen moving through the water, with or without the stalk. Each species resembles a tulip or tube shape with cilia around the opening. The cilia trap bacteria by creating a current that moves the bacteria toward the opening. The stalked ciliate then contracts in a quick motion, which pushes the food into the body. Once their food levels have diminished significantly, more stalked ciliates begin to branch into colonies to acquire food more efficiently.

Stalked ciliates usually indicate a stable, healthy system with a moderate to high maturity index. Because stalked ciliates attach to pieces of floc, they usually imply that the biomass (bacteria) is forming well-structured floc that is essential to settling and good effluent quality. However, one stalked ciliate, Voricella microstoma, is often indicative of high turbidity and poor effluent quality. This is because they consume single bacteria cells in open water, which means dispersed bacteria are present and turbidities are elevated. But, this particular stalked ciliate has a very small mouth opening compared to other species, which enables them to be identified easily.

 

 

 

Reproduction

Ciliates reproduce by conjugation. Each cell has a large macronucleus and a smaller micronucleus. Conjugation is a form of sexual reproduction in which the individual cells fuse together and swap nuclear DNA in the form of small micronuclei. Conjugation is unlike the fusion of gametes in other protists and animals. It involves the division and fusion of micronuclei from opposite paramecia. After conjugation each paramecium continues on its way, genetically altered from its brief encounter because of different chromosomal combinations. The genetically altered paramecia continue to produce clones of themselves by asexual cell division, a process known as fission

 

 

 

Activated Sludge Process and Protozoa

As an activated sludge process ages and bacterial and protozoan communities develop, treatment efficiency improves and three significant events occur:

These changes permit higher life forms, such as ciliated protozoa, to proliferate in number in diversity. Decreasing numbers of dispersed bacteria also contribute to the change in dominant protozoan groups as the more efficient food-gathering mechanisms of crawling ciliates and stalked ciliates permit them to out-compete other protozoan groups for the bacteria.

Therefore, a general trend in progression of lower to higher life forms can be observed with decreasing pollution or BOD, increasing DO concentration, and decreasing numbers of dispersed bacteria -- that is, crawling ciliates and stalked ciliates become dominant under healthy activated sludge conditions. Conversely, with deteriorating conditions, a regression from higher to lower life forms occurs. Unhealthy conditions include organic overloading, sludge discharges of soluble organic wastes, undesired changes in pH, inhibition, toxicity, and loss of solids.

 

Ciliated Protozoa and Bioindicator Values

 

Group of ciliates

Species indicative of healthy activated sludge

Species indicative of unhealthy activated sludge

Free-swimming

Paramecium Aurelia, Litonotus Anguilla

Paramecium trichium, Trachelophyllum pusillum

Crawling

Euplotes affinis

Oxytricha ludibunda

Stalked

Epistylis rotan, Vorticella elongate, Zoothamnium mucedo

Epistlylis plicatilis, Opercularia coarctaata, Vorticella alba

Tentacled

Podophrya mapasi, Sphaerophyra pusilla

Podophrya fixa,Sphaerophyra magna

 

Because the activated sludge process is a biological system, the protozoa in the process are affected by changes in composition of the wastewater and the microbial community, flow, organic loading, and temperature. The most significant factors affecting protozoa are flow and organic loading or carbonaceous BOD (cBOD) as illustrated in the table below. Flow determines the time available in the process for the protozoa to reproduce, while organic loading influences DO availability. These two factors exert the most influence on the succession of protozoa groups and species of protozoa that are dominant.

 

Protozoan Groups and Bioindicator Values

 

Amoebae

Rarely predominant except for start-up conditions and conditions that mimic start-up such as over-wasting, recovery from toxicity, washout, and organic overloading.

Flagellates, plant-like

Dominant under high organic loading, dispersion of floc particles, such as through chlorination, and start-up conditions or conditions that mimic start-up.
Also may dominate in the presence of excess soluble phosphorus.

Flagellates,animal-like

Except for the presence of excess soluble phosphorus, these are dominant for operational conditions listed for plant-like flagellates and usually follow plant-like flagellates as the dominant group.

Free-swimming ciliates

Transition group that dominates between healthy and unhealthy conditions and proliferates when large numbers of free-swimming bacteria are present.

Crawling ciliates

Dominant in the presence of mature floc particles and low biochemical oxygen demand (BOD) in the bulk solution.
Alternate with stalked ciliates as the dominant group.

Stalked ciliates

Dominant in the presence of mature floc particles and low BOD in the bulk solution.
Alternate with crawling ciliates as the dominant group.

 

Changes in dominant specific protozoa are indicative of favorable or unfavorable conditions occurring in the activated sludge process. Routine microscopic examination of the mixed liquor can provide an operator with an early warning of unfavorable loading conditions and inappropriate process control changes.

 

 

Review

Protozoans are found in all moist habitats within the United States and the relative abundance and diversity of them are used as indicators of organic and toxic pollution. Protozoans that are important in the activated sludge process in removing bacteria are the amoebae, flagellates and ciliates.

Amoebae are protozoans which move by extending finger-like protrusions of their cells called pseudopodia. Amoebae usually are present in high numbers during start up of a treatment plant recovery from a toxic discharge to the treatment plant or low dissolved oxygen levels. If amoebae are present as the dominant protozoan group, this could indicate an unstable wastewater environment and a sludge that is in poor health.

Flagellates are single-celled protists with one or more flagella, which are whip-like organelles often used for propulsion. Like their relatives the amoebae, flagellates are usually present when there are large amounts of soluble food available (high F:M or high BOD). They are found during start up when the sludge is young or after an upset, but will quickly predominate over the amoebae because they are more efficient feeders. Flagellates can only dominate early in the treatment process when the soluble organic nutrients are high enough for both flagellates and bacteria to eat.

The Ciliates are more complex organisms than the amoebae and flagellates. The three types of ciliated protozoa are free-swimming ciliates, crawling ciliates, and stalked ciliates. All of these have short hair-like structures or cilia that beat in unison to produce a water current for locomotion and capturing bacteria. Ciliates feed on bacteria, not on dissolved organics. They are usually an indicator of good quality sludge and typically found in young to medium age sludge. They are important because they work with the bacteria in the activated sludge process by feeding on them and helping to clarify the effluent.

Free-swimming ciliates possess ciliate on all surfaces of the body and are usually found when no large flocs have been formed so that it is easier to swim around. Crawling ciliates possess ciliate only on the ventral or belly surface where the mouth opening is located. In order for crawling ciliates to  be dominant, there must be large floc structures present that impede the free-swimmers and flagellates movement and provide a surface for the crawlers to "walk" on. Stalked ciliates have cilia around the mouth opening only and are attached to floc particles. Stalked ciliates have an enlarged anterior portion or "head" and a slender posterior portion or "stalk". Stalked ciliates are found in large numbers when the bacterial population and dissolved oxygen concentration are high, the wastewater environment is stable and a mature floc has developed. Of all ciliated feeding mechanisms, the stalked ciliates mechanism is the most efficient in capturing stray bacteria.

As an activated sludge process ages and bacterial and protozoan communities develop, treatment efficiency improves and three significant events occur:

 

Let's watch a video that shows you all the organisms that are present during the treatment of wastewater.

You will learn about the Metazoas mentioned (Rotifers, Nematodes and Tardigrades) in the next lesson.

 

 

Assignments

Complete the assignment for this lesson and send to your instructor via email.

 

 

Lab

There is no lab associated with this lesson.

 

 

Quiz

Answer the questions in the Lesson 6 Quiz .  When you have gotten all the answers correct, print the page and either mail or fax it to the instructor. You may also take the quiz online and directly submit it into the database for a grade.