microscopic examination of activated sludge -...

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I. Abstract.This operator education course explains differences between basic types of microorganisms present in activated sludge, their metabolism and correct distribution to achieve optimum wastewater treatment. In addition, the Settling test and food to microorganism (f/m) ratio is explained and sample calculations are provided to aid the operator in optimizing treatment of the wastewater prior to discharge. Optimizing the treatment will as-sist the operator to be in compliance with its National Pollutant Discharge Elimination System (NPDES) Permit. Questions and answer section is also provided to determine if the operator un-derstood the material.

II. Keywords.Mixed Liquor Suspended Solids (MLSS) - The volume of activated sludge in the aerator.

Activated Sludge - A brownish floc-like substance made up largely of organic matter derived from the sewage. This substance is pop-ulated by millions of bacteria and other forms of biological life.

Solids Settling Test - A test to determine the settling capability of activated sludge after 30 minutes.

Food to Microorganism (F/M) ratio - A ratio of food per day to the amount of microorganisms calculated from the aeration tank.

Heterotrophic and Autotrophic bacteria - Bacteria which are re-sponsible for purifying the wastewater.

Protozoa - Microorganisms that metabolize different nutrients and clarifying the wastewater by consuming the bacteria.

Amoeboid - Amoeboid protozoa has extremely flexible mem-branes that allow food to be absorbed through them. They move throughout the MLSS by the movement of protoplasm within the cell. Generally, Amoeboids are found in high numbers in “young” MLSS and is associated with poor settling sludge.

Flagellates - Flagellate protozoa move throughout the MLSS by the corkscrew movement of the tail (Flagella) which extends from their round or elliptical cell configuration. Generally, Flagellates are found in high numbers where the Amoeboid population is low, and a high organic load (BOD). As the MLSS ages and exhibits good settling, the Flagellate population is reduced to one-eight (1/8) of the protozoans.

Ciliates - Ciliate protozoa move throughout the MLSS by rotat-ing hair-like membrane (cilia) which cover all or part of their cell membrane. Additionally, they use the cilia to pull food into their gullet. There are two types of Ciliates, free swimming and stalked Ciliates. Ciliates are associated with fair to good settling of the activated sludge.

Settleometer - A clear cylinder with calibration markings of 50 mL intervals, starting at the bottom, and ending at the mouth with 1000 mL.

III. IntroductionWastewater is a mixture of solids and water, with water represent-ing 99.9% of wastewater. That means that only .1% represents a pollution and health problem, unfortunately that is more than sufficient to cause human health problems. Wastewater must be sufficiently treated to remove possible threats to human health. The typical wastewater treatment plant three portions, Pre-liminary treatment, biological, secondary treatment and tertiary treatment.

Microscopic Examination of Activated Sludge

Educational ObjectivesUpon completion of this course, the operator should be able to use the microscope to view microorganisms present in activated sludge, understand their differences, metabolism and correct distribution to achieve optimum wastewater treatment. In addition, the operator will understand the importance of the microorganism groups as they relate to determining the food to microorganism (f/m) ratio and settling characteristic. Once the operator becomes proficient with these operational tests, the wastewater treatment plant effluent will be of higher quality.

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The preliminary treatment of the raw wastewater, termed, influent, includes physical or chemical removal of grit and screenings to be disposed of in a landfill. Grit and screening represents the inorganic portion of the wastewa-ter. The removal of the inorganic portion, allows efficient breakdown of the organic pollutants, such as, Carbona-ceous Biochemical Demand, Nitrogen compounds, Phos-phorus, fecal coliform to name a few. The breakdown and settling of these organics is accomplished in the biological/secondary treatment mainly be micro-organisms called, protozoans.

The biological/secondary treatment equipment gener-ally consists of three components; treatment unit, settling unit, sludge digester/holding unit. In the treatment unit the sludge and preliminarily treated influent, are mixed with dissolved oxygen for a specified amount of time. The sludge consisting of micro-organisms, termed Mixed Liquor Suspended Solids, (MLSS) metabolize and reproduce, over time, when combined with dissolved oxygen. The time needed, termed detention time, the MLSS require to adequately metabolize the organic matter and reproduce is dependent on the quantity and quality of MLSS popu-lation and strength of the influent. To determine quantity and quality of the MLSS, the operator must examine the sludge under a microscope. After sufficient treatment is achieved, the wastewater is transported via gravity or pumps to another unit for settling. The settling unit allows time with no agitation for the MLSS to settle to the bottom, allowing clear wastewater to leave the tank and progress to the chlorine contact tank. The MLSS may then be removed from the bottom of the tank, with one portion “returned” back to the treatment unit, and the other portion “wasted” to sludge digestion/holding. The returned MLSS, again, mixes with preliminarily treated influent and dissolved oxygen to allow the micro-organisms time for metabolizing the organic matter for reproduction. The wasted portion goes to digestion and is not provided any organic matter, only dissolved oxygen. In this way, the wasted sludge is volatized by the dissolved oxygen and is considered stable once the volatiles are reduced by 37%. The stable sludge may be used on farm fields as a fertilizer and can be stored in sludge holding. The tertiary treatment of the treated wastewater from the settling tank consists of chlorination and sand filtration.

The chlorination is designed to lower the bacterial counts so the effluent does not harm the stream biota. Af-ter chlorination the wastewater may be transported to sand filtration where unsettled solids are removed. The unsettled solids cause the effluent to be turbid. The turbidity is gen-erally dead MLSS that did not settle in the settling tank. The amount of turbidity can be reduced by adjusting the return rate and the wasted rate of the MLSS in accordance with the examination of the MLSS to determine the age, diversity and quantity of the micro-organisms.

Microscopic examination of the MLSS enables the op-erator to determine the level of treatment and indicate set-tling abilities in a matter of minutes. The microorganisms within activated sludge “treat” the wastewater by reducing the biochemical oxygen demand, organic and nitrogenous demands. The most important of these microorganisms are the heterotropic and autotrophic bacteria and the protozoa in treatment and clarification of the wastewater.

The protozoa will clarify the wastewater in a tank that provides “food” (the bacteria) and dissolved oxygen, the aeration tank. With dissolved oxygen and food they will reproduce to form a “floc particle”. Once the MLSS is moved to a “settling, or Clarifier” tank, the floc particles will be able to adhere to one another to form a “blanket” of sludge. This blanket becomes heavier as it thickens which allows it to “settle” to the bottom of the tank as the treated and clarified water exits the settling tank from the top. It is important to note that these protozoa must be returned to the aeration tank before they die and float to the surface of the settling tank.

The most important microorganisms for the operator to have in the MLSS for clarification and setting are the proto-zoa. As discussed previously, the protozoa eat the bacteria and help to provide a clear effluent. There are three groups of protozoa, each of which have significance in the treat-ment of wastewater.

The three groups include the:1. Amoeboids2. Flagellates3. Ciliates

Amoeboid

Amoeboid protozoa has extremely flexible membranes that allow food to be absorbed through them. They move throughout the MLSS by the movement of protoplasm within the cell. Generally, Amoeboids are found high in numbers in “young” MLSS. Generally, Amoeboids are found high in numbers in “young” MLSS and is associ-ated with poor settling sludge. Once the activated sludge is allowed to age and exhibits good settling, the Amoeboids population is about one sixteenth (1/16) of the total number of important protozoans.


Flagellates

Flagellate protozoa move throughout the MLSS by the corkscrew movement of the tail (Flagella) which extends from their round or elliptical cell configuration. Generally, Flagellates are found in high numbers where the Amoeboid population is low, and a high organic load (BOD) exists. If these protozoa predominate the MLSS the settling abil-ity of the MLSS is poor and the clarity is only fair. As the MLSS ages and exhibits good settling, the Flagellate popu-lation is reduced to one-eighth (1/8) of the total number of important protozoans.

CiliatesThese protozoa move throughout the MLSS by rotating

hair-like membrane (cilia) which cover all or part of their cell membrane. Additionally, they use the cilia to pull food into their gullet. There are two types of Ciliates, free swim-ming and stalked Ciliates. Ciliates are associated with fair to good settling of the activated sludge and comprise three quarters (3/4) of the total number of important protozoans.

Free-Swimming Ciliates

Free-swimming ciliates are numerous when there are large numbers of bacteria in the activated sludge. These protozoa consume the bacteria thereby clarifying the efflu-ent. The predominance of these ciliates indicates that the wastewater treatment process is approaching an optimum degree. In addition, their presence indicates that the set-tling of the activated sludge will be increased.

Stalked CiliatesStalked Ciliates present in high numbers in the MLSS

indicate that the wastewater treatment has surpassed the

reduction of carbonaceous biochemical oxygen demand and is satisfying the nitrogenous oxygen demand. These ciliates indicate that optimum treatment of the wastewater is occurring . In addition, ciliates attach themselves to each other along with suspended solids, forming large floc par-ticles that settle well. Large numbers of Stalked Ciliates are associated with “good settling sludge” and clear effluent water above the settled sludge blanket.

In an ideal world, the operator should examine the MLSS under the microscope on a daily basis, however for monitoring purposes two or three times per week should be sufficient. Additional examination should be performed if the treatment or settling abilities of the wastewater treat-ment plant appears to be compromised.

Each time the operator collects a sample of MLSS it should be collected from the same well-mixed portion of the aeration tank. In addition the operator should be fill out a worksheet so that the abundance of organisms can be correlated with treatment and settling.

Two other operational tools to compliment the micro-scopic examination are the “30 minute Settling Test” and calculation of the Food to Microorganism ratio.

The 30 minute settling test will confirm the microscopic examination results by providing further information on which group of microorganisms predominate the MLSS. The operator must perform this test directly before, after or concurrently with the microscopic examination to correlate the results. A good settling Sludge will settle to fill half of the Settleometer within 5 to 10 minutes. The blanket/water interface should be even and the water above the blanket should be very clear.

The food to microorganism ratio (f/m) can be computed from Pounds (Lb) of Biochemical Oxygen Demand (BOD) of the raw wastewater / Lbs of Volatile solids (MLSS x Volatile solid%) in the aeration tank. The f/m ratio should be kept around 0.2 - 0.5 to provide optimum treatment, however every wastewater treatment system is different and the operator should determine what ratio provides the best treatment and settling. This test relies on the B.O.D.,


therefore the f/m result is 5 days old and can only be cor-related with the 5 day old microscopic examination and 30 minute settling test worksheets. Once the operator calcu-lates the f/m and compares it to the microorganisms and settling, the operator can determine the correct amount of MLSS to maintain in the aeration tank to provide optimum treatment and clarity. Due to the fact the operator can not generally control the strength of raw wastewater, the opera-tor must adjust the amount of MLSS through returning the correct amount back to the aeration tank from the settling tank and removing or “wasting” any excess out of the sys-tem to a digester or holding tank.

IV. Procedure

Microscope Selection Note: A relatively low cost microscope is suitable, how-

ever a dissecting microscope does NOT have the magnifica-tion ability needed.

Features that are desirable:1. Built-in illumination or an external system which allows

variations of light intensity.2. A condenser system.3. A movable stage. Stage should be controlled by coaxial

handle rather than a manual push-pull.4. 10 X and 40 X objectives.5. 10 X eyepiece.

Needed additional equipment includes:1. Slides2. Coverslips3. Several small dropping pipettes (disposable are most con-

veinent)4. (Storage box5. Dust cover.

Use of the MicroscopeProcedures for preparing slides:

1. Clean cover slip and slide.2. Use pipette to grab a sample of MLSS. Put finger on top of pi-

pette until the immersed end of a wide tip pipette reaches the bottom of sludge sample. Release your finger to allow sludge into the pipette. Replace your finger on top of pipette and remove the pipette from the sampler beaker. A long tipped eyedropper may also be used.

3. Place one drop of MLSS from the pipette to the middle of the glass slide.

4. Pick up cover slip by two corners.5. Pull cover slip along glass slide towards drop of MLSS.6. As soon as cover slip touches drop of MLSS allow cover slip

to fall onto glass slide.7. Pick up glass slide. Place on microscope stage.8. Move stage up to within approximately 1/8 inch of objective

on 10 X.9. Use the coarse then fine adjustments on the microscope to

bring the MLSS into the field of focus.10. Turn to 45 X and repeat #9.11. Identify organisms in the MLSS.


Procedures for Microscopic ExaminationWhen performing a microscopic examination of MLSS,

the operator should fill out a worksheet that at a minimum records:1. Date and time of sample2. Sample location3. Name of operator4. Type of microorganism5. Approximate number within field view6. size of floc particle (use 10 x to determine)

The operator should examine at a minimum three slides per sample. Ideally the MLSS should be examined under the microscope on a daily basis, however for monitoring purposes twice to three times per week should be sufficient. Additional examination should be performed if the treat-ment or settling abilities of the wastewater treatment plant appears to be compromised.

Sample Worksheet for Microscopic Analysis

Procedure for 30 minute Settling testSettleometer

1. Collect 1 Liter of MLSS from the same sampling site used for the microscopic examination of MLSS above - a well mixed portion of the aeration tank.

2. Pour the sample into a Settleometer and slowly mix for 15 seconds.

3. After setting the timer for 30 minutes, allow to set and record the height of the settled sludge blanket at 5, 10, 20, and 30 min-ute intervals.

NOTE: Good settling sludge will settle to 500 mL, with clear liquid above the sludge blanket within 5 to 10 minutes.

Food / Microorganism (F/M) Ratio 1. Lbs BOD/Lbs MLSS x VS% in aeration2. Flow (MGD) x BOD (mg/L) x 8.34( MLSS x %Volatile) x aeration tank volume x 8.34 x 7.48 / 1000000

NOTE: Good F/M ratio is around 0.2 - 0.5.

EXAMPLE:1. Flow = 4.7 MGD2. Raw wastewater BOD = 149 mg/L3. 8.34Lb = weight of one gallon of water4. %Volatile = 77%5. MLSS = 1860 mg/L6. 5 Tanks = 210’ x 20’ x 14’7. 7.48 = gallons for 1 cubic foot8. 1,000,000 gpd = 1 MGD

Calculation: {4.7MGD x 149 mg/L x 8.34} = 5,840.502

0.77 x 1860 mg/L x [5(210 x 20 x 14) x 7.48 ] x [8.34 / 1,000,000] = 26,267.495

5,840.502 divided by 26,267.495 = .222

Conclusion:Evaluation of Microscopic Examination as related to the

30 min. Settling test and F/M ratio

If the operator identifies the Amoeboid as the most abundant, then the organic load in the aeration tank will be high and the settling abilities of the MLSS will be very poor.


The settling of this MLSS will be around 800 - 950 mL after 30 minutes. The F/M ratio will be close to or more than 1.

F/M = (5840.502 divided by 5267.495) = 1.1

Ideally the number of Amoeboid would be approxi-mately 1/16 of the total number of the three protozoa discussed. Due to the fact that these organisms do not adhere well to one another or other protozoa they remain suspended in the waster causing the clarity to be very poor. To help rectify this situation the operator should allow as much detention time in the aeration tank as possible to facilitate additional microorganism growth.

If the operator identifies the flagellates as the most abundant, the amoeboid population is low, and the organic load (BOD) is high and the settling ability is poor resulting in turbid water. The settling will be around 700 - 950 mL after 15 minutes. The F/M ratio will be 0.8 or more.

F/M = (5840.502 divided by 7267.495) = 0.8

Ideally the number of flagellates needed to treat the wastewater and exhibit good settling, the Flagellate popu-lation should be reduced to one-eighth (1/8) of the total number of important protozoans. Again, to help rectify this situation the operator should allow as much detention time in the aeration tank as possible to facilitate additional microorganism growth.

If the Ciliates,free swimming and stalked Ciliates are identified as most abundant, then the B.O.D. will be less than 10 mg/L and the nitrogenous demand will be satisfied reducing the ammonia to 3 mg/L or less.

The settling will be around 500 within 5 to 10 minutes and the F/M ratio will be around 0.5. Stalked Ciliates at-tach to one another and to other matter causing floc par-ticles to form and grow. In the clarifier the floc particles are able to adhere together forming a blanket that will settle to the bottom of the tank.

F/M =(5840.502 divided by 26,267.495) = .222

The most appropriate amount of Stalked Ciliates to treat the wastewater and exhibit good settling is three quar-ters (3/4) of the total number of important protozoans. The operator should monitor the situation to keep the balance of food to microorganism as it is now.

microscopic examination of activated sludge -...

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