Environmental Pollution Control Microbiology - Chapter 6 pdf

Stalked ciliated protozoa and crawling ciliated protozoa require the least amount of food for survival as far as ciliated protozoa are concerned.. a Free-Swimming Ciliate b Stalked Cilia

Chapter 6

PROTOZOA AND OTHER ANIMALS

Microscopic animals differ from microscopic plants by their ability to metabolize solid particles directly Actually, the protozoa and higher animals hydrolyze the solid organics internally rather than externally The animals have complex metabolic systems that allow them to metabolize the nutrients and release the inert portions of the suspended organic solids back into the environment Microscopic animals range from single cells organisms to multicell animals that approach the macroscopic size The microscopic animals are a part of the organic matter that forms a link in the food chain for macroscopic organisms The larger organisms use smaller organisms as their source of nutrients Although the microscopic animals

do not metabolize waste materials, they play an important role in the organic waste stabilization process For this reason it is important for environmental microbiologists to learn to recognize the different groups of microscopic animals and the role they play in maintaining the environmental balance in both aqueous systems and soil systems

PROTOZOA

Protozoa are the simplest microscopic animals, being single cell organisms In nature, bacteria form the major food supply for protozoa The bacteria concentrate various nutrients into their protoplasm, making them the perfect food for the protozoa A portion of the organic matter from the bacteria is oxidized to yield energy for the synthesis of new protoplasm from the remaining organic matter The

synthesis relationships for protozoa are similar to the bacteria energy-synthesis relationships, 38% oxidation and 62% new cell mass The large protozoa can also eat small algae Most of the protozoa are aerobic, requiring dissolved oxygen as their electron acceptor There are a few anaerobic protozoa The problem with anaerobic protozoa is even more acute than with anaerobic bacteria Anaerobic organisms must process considerable quantities of organic matter for energy since most of the energy remains in the partially metabolized organic compounds Anaerobic protozoa will only be found in environments having very high organic concentrations and high concentrations of bacteria Until more research is carried out on anaerobic metabolism in protozoa, environmental microbiologists will deal with protozoa as if they are strict aerobic microorganisms

DESCRIPTION

Protozoa are identified entirely from their physical characteristics Microscopic examination of the protozoa at 100 X allows observation of the major characteristics used to identify the different organisms Protozoa are much larger than bacteria, ranging in size from about 10 um to several hundred microns Since

the protozoa have a discernable nucleus, they are classified as Eucarya There are five families of protozoa: (1) Sarcodina, (2) Mastigophora, (3) Sporozoa, (4)

Ciliata and (5) Suctoria Figure 6-1 shows sketches of the five families of protozoa, illustrating their major physical characteristics

(a) Sarcodina (b) Mastigophora (c) Sporozoa (d) Ciliata (e) Suctoria

Figure 6-1 SKETCHES OF THE FIVE FAMILIES OF PROTOZOA

The Sarcodina are the simplest protozoa They have flexible bodies and move by

pseudopodia created by streaming protoplasm within the cell while attached to a

surface The Sarcodina must live on solid surfaces in order to move under control.

If they lose contact with a solid surface, the Sarcodina have no control over their

movements and simply drift with the fluid currents The nucleus and food vacuoles

are easily observed in the Sarcodina They eat by engulfing their food As the cell

wall moves over the solid food, the food goes from outside the cell to inside the cell where it can be solubilized by enzymes The soluble nutrients are taken inside

the cell and used for energy and synthesis The Amoeba is the most common Sarcodina and is widely distributed in the environment Unfortunately, the Sarcodina are not efficient food gatherers compared to the other protozoa and are

not able to compete efficiently against the other protozoa They will be found growing on solid surfaces where bacteria and algae are attached Some of the

Sarcodina have the ability to create solid shells that can protect them from small predators A few of the Sarcodina are pathogenic The Entamoeba histolytica is

one of the most famous pathogens It was responsible for an epidemic at the 1933

Chicago World's Fair Entamoeba were carried by a cross-connection in a large

hotel in Chicago to a number of rooms from the sewage of a contaminated guest The cross-connection allowed the sanitary sewage to enter the water distribution system within the hotel by mistake The newly contaminated guests of the hotel carried the protozoa back home when they left Chicago, making it very difficult to trace the magnitude of the epidemic One of the positive aspects of this epidemic was to focus attention on the elimination of cross-connections between sanitary

sewage pipes and water distribution pipes The Sarcodina form cysts when the

environment becomes unfavorable The cysts are quite similar to bacterial spores and protect the nucleus with a hard coating When the cysts return to a favorable environment, the nucleus stimulates normal protozoa growth Growth of

Entamoeba inside animals results in the discharge of large numbers of cysts in

feces from the infected animal In countries where untreated sewage is applied directly to agricultural fields as a fertilizer, the cysts become attached to the crops

If the crops are eaten without adequate treatment, the cysts are ingested and grow

again, allowing the cycle to continue unabated The Endamoeba are parasitic

pathogens, drawing all their nutrients from their hosts The parasites sap the

strength of people and reduce their ability to work Endamoeba are seldom fatal,

except for people who have a damaged immune system Sewage treatment can remove the parasites from human wastewaters and break the growth cycle of this parasite Medical treatment of the infected individual can also destroy the pathogen Individual treatment is a more difficult and expensive way to control the spread of the pathogen in large populations than wastewater treatment

Mastigophora are the flagellated protozoa They have from one to four flagella that are used for motility and for gathering food The Mastigophora are divided into two groups, Phytomastigophora and Zoomastigophora The Phytomastigophora

are flagellated protozoa that are the transition phase between bacteria and algae Like bacteria, the phytoflagellates metabolize soluble nutrients Because of their large size the phytoflagellates cannot compete against the bacteria and can survive only in concentrated organic environments before the bacteria begin to grow and

predominate Protozoologists consider flagellated algae as a major part of the phytoflagellated protozoa; but environmental microbiologists have kept the photosynthetic phytoflagellates with the algae and the non-photosynthetic phytoflagellates with the protozoa Growth of bacteria permits the zooflagellates to grow since they eat bacteria for their food The inefficiency of the zooflagellates in obtaining nutrients keeps their populations low except in very high bacteria

populations Mastigophora are easily recognized under the microscope because of their size and their slow undulating motion Mastigophora range in size from 10

um to over 100 um It is possible to see the flagella under the optical microscope

The Sporozoa are parasitic protozoa that have complex life cycles The formation

of spores is the chief characteristic of the Sporozoa Plasmodium vivax is the most common Sporozoa It is the causative agent for malaria, one of the most common

diseases around the world Malaria is transmitted by mosquitos from person to person The mosquito plays an important part in the growth of this parasitic protozoa, as well as in its movement in the environment Limiting mosquito populations has provided control over this disease since it cannot be transmitted without the host mosquito

The Ciliata use short cilia for motility and gathering food They are grouped as

free-swimming ciliated protozoa, crawling ciliated protozoa, and stalked ciliated protozoa The free-swimming ciliates move very quickly and require lots of food for energy Dispersed bacteria are the primary source of food for the free-swimming ciliated protozoa They come in many sizes and shapes, ranging from 20

um to 300 um in length Paramecium is the typical free-swimming ciliated protozoa that most people recognize; but the smaller Tetrahymena is more common, since it requires less food for survival Blepharisma is an easy free-swimming ciliated protozoa to recognize because of its pink color Paramecium bursaria is also interesting with the algae, Chlorella, growing inside the protozoa.

The protozoa and algae find the relationship suitable for both organisms

Stylonychia and Euplotes are more complex free-swimming ciliates with cirri on

their underneath side The cirri allow these free-swimming ciliates to crawl over solid surfaces in search of food The crawling ciliates require less energy than the free-swimming ciliates and survive better as food becomes limiting The stalked ciliates are ciliated protozoa that have stalks to permit them to attach to surfaces The cilia located near the mouth are primarily for food gathering, but can be used for motility The stalked ciliates can be found as single cells or as colonies of cells Some of the stalked ciliates have stalks that contract and some have rigid stalks Stalked ciliated protozoa and crawling ciliated protozoa require the least amount of food for survival as far as ciliated protozoa are concerned Under adverse environmental conditions, the stalked ciliated protozoa form cilia around the bottom of the cell near where the stalk is attached The stalked ciliated protozoa

can detach itself from the stalk and becomes a large, free swimming ciliated protozoa It can become attached again when environmental conditions are suitable Figure 6-2 shows sketches of the three different groups of ciliated protozoa

(a) Free-Swimming Ciliate (b) Stalked Ciliate (c) Crawling Ciliate Figure 6-2 SKETCHES OF THREE MAJOR GROUPS OF CILATED PROTOZOA

Suctoria are interesting protozoa that look like stalked ciliated protozoa Instead of having open mouths for feeding, the Suctoria use hollow tubes to suck their nutrients inside the cell Suctoria are parasites, using free-swimming ciliates as their source of food The Suctoria are more complex protozoa, having two phases

in their life cycle The stalked growth is one phase and a free-swimming ciliate is the other phase Since the free-swimming ciliated protozoa used for nutrients are

quite large, it takes considerable time for the Suctoria to capture and to eat a free-swimming ciliate Suctoria will be observed only when there are large numbers of

free-swimming ciliated protozoa in the environment The growth of protozoa is similar to the growth of the other microorganisms Nutrients and environment determine which protozoa grow and the extent of that growth

METABOLISM AND GROWTH

Protozoa are primarily aerobic organisms, requiring dissolved oxygen as their electron acceptor Although protozoa can be grown in concentrated, complex nutrient media, they prefer to use bacteria as their source of nutrients The bacteria are concentrated nuggets of nutrients The protozoa metabolize the biodegradable portion of the bacteria for energy and synthesis and excrete the non-biodegradable fraction back into the environment The protozoa must continuously ingest nutrients or they will have to consume their own cell mass and die A number of studies have been carried out over the years evaluating the relationships between the growth of bacteria and protozoa

Butterfield, Purdy, and Theriault carried out one of the earliest studies on bacteria-protozoa metabolism in 1931 They were able to obtain a pure culture of

Colpidium, a small, free swimming ciliated protozoa, varying in size from 50 to 70

um in length They used a dilute glucose-peptone solution as a growth media and

quickly found that Colpidium could not survive in a 5 mg/L solution of each

nutrient, a total of 10 mg/L nutrients, unless the media became contaminated with bacteria Increasing the glucose-peptone concentration to 5,000 mg/L of each

nutrient, 10 g/L total nutrients, provided sufficient nutrients for the Colpidium to grow quite well without bacteria in a batch-fed bioreactor It took the Colpidium 23

days to reach its maximum population, 15,600/ml, when grown at 20°C in the

concentrated nutrients The Colpidium used 245 mg/L oxygen, about 0.016 ug/cell Once the Colpidium reached their maximum population, they began to slowly die

while using dissolved oxygen to remain alive by endogenous respiration They used

55 mg/1 DO over the next 4 days, 3.9 x 10"5 ug/hr/cell It appeared that oxygen transfer limited metabolism in both the growth and the endogenous phases Using a

dilute glucose-peptone solution containing 5 mg/1 of each nutrient and Aerobacter

aerogenes as the bacteria together with Colpidium, they found that the bacteria

metabolized the organics to new cells with the utilization of oxygen in the batch-fed

bioreactor The Colpidium growth lagged the bacteria growth, but quickly began to

reduce the bacteria population The protozoa reached its maximum population in 5 days incubation at 20°C Both the protozoa and bacteria populations slowly

decreased after 5 days The numbers of Colpidium reached 180/ml in 5 days and

dropped to 10/ml by Day 10 The bacteria population reached 6.9 x 106/ml after one day and was down to 0.7 x 106/ml by Day 10 Their study gave some

additional data The growth of Aerobacter aerogenes in the dilute glucose-peptone

solution used 3.0 mg/1 DO in 5 days incubation at 20°C Adding the protozoa,

Colpidium, to the A aerogenes gave an oxygen uptake of 4.8 mg/L in 5 days.

Using a mixture of several different bacteria in the same substrate gave 4.3 mg/L

oxygen uptake With Colpidium the mixture of bacteria used 5.2 mg/L DO Finally,

river water with mixed bacteria and mixed protozoa used 6.4 mg/L DO under the same conditions These data showed that the protozoa were dependent upon the bacteria to concentrate nutrients in dilute organic environments and that mixtures of microorganisms were more efficient at metabolism than pure cultures This is not surprising since the most efficient microorganisms grow and provide the greatest stabilization in the shortest time

In 1972 Tsuchiya, Drake, Jost, and Fredrickson published the results of their study

on the interaction of the amoeboid protozoa, Dictyostelium discoideum, and

Escherichia coll In a continuously fed bioreactor with 500 mg/1 glucose as the

substrate, E coli metabolized the glucose and produced about 1.5 x 109 bacteria/ml The amoeboid protozoa began to eat the E coli and increased in numbers The protozoa metabolism produced a major drop in the E coli population

and a rise in glucose concentration after several days of operation As the E coli

population decreased, the protozoa population also began to decrease The

increased glucose concentration stimulated the E coli to grow The glucose concentration soon dropped quite low and became growth limiting for the E coli.

The protozoa found a ready supply of food again and began to grow at the expense

of the E coli The oscillations in bacteria and protozoa populations eventually

became damped and the system operated with a balance between substrate fed and

the growth of both microorganisms At 25°C the (VK for D discoideum was 0.24/hr and 0.25/hr for E coli With continuous feeding of nutrients, one would expect that the \i for both organisms growing together would be the same The

half-saturation constant, Kj, was 4 x 108 bacteria/ml for the protozoa and 0.5 mg/1 glucose for the bacteria Quantitative evaluation of the data indicated that it took 1.4 x 103 bacteria to create an amoeba and 3.3 x 10"10 mg glucose to produce a bacterium

This same group continued their study of competition between protozoa and

bacteria by examining two bacteria, E coli and Azotobacter vinelandii.

Azotobacter vinelandii are nitrogen-fixing bacteria that can use glucose as its

substrate, the same as E coli The two bacteria are quite different in size E coli

has a mean cell volume of 0.3 umj; while the Azotobacter has a mean cell volume

of 3.0 umj Azotobacter has to metabolize much more glucose to produce a single cell than E coli Size differential is a major factor affecting competition between

microorganisms The microorganisms with the greatest surface area to mass ratio have a distinct advantage over the other microorganisms Growth of the two

bacteria in a simple, continuous feed system without the protozoa resulted in the E.

coli displacing the Azotobacter in a short period of time Theoretically, the E coli

should not completely displace the Azotobacter since both can compete for the soluble substrate The smaller, faster growing E coli should have been and was the

predominant bacteria In this study, a free-swimming ciliated protozoa,

Tetrahymena pyriformis, was used, since they were far more efficient at gathering

food than the amoeboid protozoa used in the previous study In the presence of the

ciliated protozoa, both bacteria groups survived E coli could not displace

Azotobacter when the protozoa ate the bacteria Together, the E coli had a

population around 1 x 109 cells/ml with Azotobacter around 3 x 107 cells/ml and

the Tetrahymena around 6 x 10J cells/ml The faster growing E coli appeared to

provide most of the nutrients for the protozoa, depending upon the accuracy of the bacteria numbers and their corresponding volumes Predator-prey relationships and competition between microorganisms for nutrients are very important in allowing the various groups of organisms to survive in the real world An interesting reaction was observed when they attempted to grow the microorganisms at fluid retention periods greater than 15 hours They noted that the bacteria aggregated, making it impossible to distinguish between the two organisms Aggregation of bacteria is

very important in environmental microbiology and has an important impact on bacteria survival This study clearly demonstrated that flocculation occurred when food became limiting in the environment, hi an excess of nutrients the two bacteria species remained dispersed with the protozoa

Growth of Tetrahymena pyriformis on pure cultures of bacteria was the subject of a study by Holm and Smith They used the bacteria, Citrobacter, which contained

8.6 x 10"11 mg carbon/cell The Tetrahymena contained 1.1 x 10"6 mg carbon/cell The free-swimming ciliated protozoa required 3 x 104 bacteria to produce a single new cell The overall metabolic efficiency was about 42% The same year, Sudo and Aiba reported on the isolation and growth of the stalked ciliated protozoa,

Vorticella microstoma They used Alcaligenes faecalis as the source of food for the Vorticella They found that the weight of Vorticella averaged 3.85 x 10"* mg/cell

and had a u^ of 2.2 days The cell yield of protozoa was about 47%, based on mass of bacteria metabolized

The metabolic data from these studies demonstrated that the aerobic growth of the protozoa followed the same general relationships of metabolism as the other microorganisms The difference was the bacteria supplied both the energy and the components for cell synthesis The energy content of bacteria is less than 100% based on VSS The net result is less synthesis of protozoa than would be expected

As large organisms, the protozoa must metabolize large numbers of bacteria to make a new cell Fenchel reported protozoa used 50% to 60% of their nutrients for cell synthesis The endogenous respiration rate proceeded at 2% to 5% of the normal growth rate in protozoa The dispersed bacteria in the environment form the best source of nutrients for the protozoa; but bacteria on the surface of soil particles

or on the surface of bacteria floe also can be used for nutrients

Although the majority of protozoa are aerobic organisms, there are anaerobic protozoa Like their bacteria counterparts, the anaerobic protozoa must eat tremendous quantities of nutrients in order to obtain sufficient energy for cell synthesis The low bacteria growth in anaerobic environments means anaerobic protozoa will only be found in high organic concentration environments Fenchel and Finlay reported that anaerobic protozoa had an overall yield of about 10% of their nutrients They indicated that there were anaerobic protozoa with methane bacteria growing inside the protozoa As the protozoa produced organic acids, the methane bacteria converted the organic acids to new cells and methane gas The methane metabolism removed potentially toxic organics from the protozoa and supplied the protozoa with additional nutrients There are both flagellated and free-swimming ciliated, anaerobic protozoa with the predominant numbers being ciliated protozoa The free-swimming ciliated protozoa predominate over the flagellated protozoa in anaerobic environments for the same reason that they

predominate in aerobic environments The free-swimming ciliated protozoa are simply more efficient in capturing bacteria for food than the flagellated protozoa Because of the limited environments for anaerobic protozoa, protozoa were considered as being strictly aerobic for many years More research is definitely needed on anaerobic protozoa to establish their relationships in the environment

POPULATION DYNAMICS

In the natural environment the different groups of protozoa compete for nutrients All of the major groups of protozoa will be found living together in numbers proportional to their ability to obtain nutrients The natural environment is more dynamic than static and does not allow a static equilibrium to exist for any extended period of time The addition of organic nutrients to the aquatic environment stimulates the growth of bacteria best equipped to metabolize the specific organic compounds The growth of the bacteria will be aerobic as long as there is sufficient dissolved oxygen in the water If the bacteria remove the dissolved oxygen, metabolism shifts from aerobic to anaerobic Since most protozoa will not grow under anaerobic conditions, there will be no significant growth of protozoa until the rate of bacteria metabolism slows and the system becomes aerobic again Figure 6-3 is a schematic diagram of the population dynamics of microbial growth in a batch fed system following the addition of organic nutrients to stimulate the bacteria Both the time scale and the numbers of

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TIME

Figure 6-3 SCHEMATIC DIAGRAM OF POPULATION DYNAMICS OF MICROBIAL GROWTH IN A BATCH FED SYSTEM

organisms are distorted to show relative growths of the different groups of organisms Small, flagellated protozoa will appear first since there are many bacteria to eat Larger flagellated protozoa appear next as sufficient food is available growth The amoeboid protozoa appear on solid surfaces where there are attached bacteria for them to use as food The amoeboid protozoa do not approach the numbers of the flagellated protozoa as the amoeboid protozoa are not as efficient food gatherers as the flagellated protozoa The small ciliated protozoa appear quickly as the DO rises The small, free-swimming ciliated protozoa move quickly through the solution, harvesting as many bacteria as possible The numbers

of free-swimming ciliated protozoa increase rapidly with larger species appearing

in smaller numbers The numbers of bacteria decrease as the ciliated protozoa

grow If there are enough small free-swimming ciliated protozoa, a few Suctoria

will appear As the bacteria decrease, the free-swimming protozoa give way to the crawling ciliated protozoa that find their food on solid surfaces The crawling ciliated protozoa are more efficient food gatherers than the amoeboid protozoa at finding the bacteria on the solid surfaces Stalked ciliated protozoa appear when the bacteria numbers drop lower and lower The free-swimming protozoa rapidly decrease as they expend too much energy trying to find enough bacteria to remain alive The stalked ciliated protozoa require many bacteria to grow; but they expend less energy in obtaining those bacteria As the bacteria population drops to very low levels, the stalked ciliates die off

REPRODUCTION AND SURVIVAL

Protozoa undergo reproduction by fission, splitting into two cells along the longitudinal axis Division starts with the nucleus splitting and creating the basis for two separate cells It takes several hours for the two cells to completely split Growth continues as long as environmental conditions are favorable When environmental conditions begin to turn bad for continued growth of the protozoa, they form cysts Each cyst is produced by coating the nucleus with a hard shell, allowing the nucleus to survive in adverse environments The rest of the cell tissues become nutrients for additional bacteria growth When the cyst finds a reasonable environment for growth, the nucleus begins to expand, creating a new protozoa

Environmental factors such as pH and temperature have the same relative effect on protozoa as on bacteria Protozoa grow best at pH levels between 6.5 and 8.5 Strongly acidic or strongly alkaline conditions are toxic to the protozoa As far as temperature is concerned, protozoa can be either mesophilic or thermophilic, the same as bacteria Most protozoa are mesophilic, having a maximum temperature for growth around 40° C Fenchel indicated that a few protozoa have been found in hot springs at 50° C There do not appear to be many thermophilic protozoa Part of