Topic Microbes in Wastewater (Laboratory Report) Student`s

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Topic:Microbes in Wastewater (Laboratory Report)



Thisexperiment was performed to analyze the microbiological compositionand physicochemical properties of wastewater samples that had beentreated aerobically and anaerobically for 48 hours at 20°C. Thenumber of aerobic and anaerobic bacterial colonies, dissolved oxygen(DO), oxidation-reduction potential (ORP), ammonia, pH, andconductivity from wastewater sampled from oxygen-rich (stirred) andoxygen-deficient (static) locations were obtained. The results showedthat aeration improves the action of aerobic bacteria, while the lackof aeration improves the action of anaerobic bacteria. The action ofaerobic bacteria is accelerated by a neutral pH of 6.5-7.5. Aerationalso improves the removal of ammonia from wastewater. The resultsalso showed that the aerobic design of a wastewater treatment plantis the best in comparison to one that is anaerobic. The design oughtto incorporate stirrers to agitate and aerate the wastewater alongwith measures to bring the pH of the wastewater before treatment to6.5-7.5.

Microbesin Wastewater (Laboratory Report)

Theincrease in urban populations in many cities all over the world hasled to an increase in domestic water use, and this has, in turn, ledto an increase in the amounts of wastewater being generated. As perthe United Nations Environment Programme, the treatment of wastewaterhas two key goals: to eliminate organic and inorganic pollutantmatter from water and to eliminate pathogenic organisms from water(UNEP, 2016).

Thetreatment of wastewater benefits the public health as it eliminatesany disease-causing microorganisms in the wastewater. Prior totreatment, wastewater typically has infectious microorganisms(bacteria such as E.coli,viruses, and protists). Their elimination ensures any human beingswho consume the effluent are not at risk of diseases caused by themicroorganisms.

InCanada, the quality of wastewater effluent is measured using severalparameters. Its biochemical oxygen demand (CBOD, less than 25 mg/l),suspended solids (less than 25 mg/l), total residual chlorine (lessthan 0.02 mg/l) and unionized ammonia (not exceeding 1.25 mg/l) (Foodand Agriculture Organization, 2017).

Thetype of biological process used in wastewater treatment affects themicrobial processes and quality of effluent. These operations can begrouped into aerobic or anaerobic processes. Aerobic processes useoxygen in the wastewater to metabolize organic material. Anaerobicprocesses use microorganisms that can function without oxygen. Theseprocesses convert organic matter into products such as carbon dioxideand methane (UNEP, 2016).

Inthis analytical laboratory, we tested the microbiological compositionand physicochemical properties of wastewater samples that had beentreated aerobically and anaerobically for 48 hours at 20°C.

Materialsand Methods

Tendilutions of two soil samples (one had been stirred for 48 hours at20°C, and the other had not) the soil samples containingmicroorganisms was carried out using Peptone-Saline diluent. Threeconsecutive dilutions (10-2,10-3,and 10-4)of both samples were spread plated in duplicate on two R2A plates.One set of plates was inverted and incubated aerobically at 25°C for3 days. The other set was incubated anaerobically at the sametemperature and duration. The number of colonies on the plates with10-300 colonies were counted after three days.

Thenumber of presumptive colonies of E.coli surroundedby fluorescent halos under UV-light was determined after using thesame dilution and spread plating techniques as above. However, onlyone agar plate was used for each dilution. The plates were incubatedat 35°C for 3 days.

Thetemperature, pH, conductivity, dissolved oxygen (DO), andoxidation-reduction potential (ORP) readings for each wastewatersample were obtained using the YSI 650 Sonde probe. The ammonia levelin the samples was obtained using a handheld Thermo Orion 5-starsensor. Each reading of every parameter measured was noted down.


  1. Aerobic and Anaerobic Plate Count.

Table1. Aerobicand anaerobic heterotrophic plate counts (HPC) in stirred andstatically incubated (48 h, 20°C) synthetic wastewater. The numberof colonies obtained in each case was converted into cfu/ml byadjusting for the serial dilution.

Plate Counts




Aerobic HPC (cfu/ml)

6.9 × 109

1.2 × 108

Anaerobic HPC (cfu/ml)

5.2 × 107

0.9 × 109

Stirredwastewater samples accounted for a higher proportion of colonies inaerobic HPC, while static wastewater samples accounted for a higherproportion of colonies in anaerobic HPC.

  1. Number of presumptive E. coli colonies

Table2. PresumptiveE.colicounts observed under UV-light in stirred and statically incubated(72 h, 35°C) synthetic wastewater.


Colony count (A, cfu)

Volume (V, 0.1/1 ml)

Dilution(D, 1/dilution)

Result (N=cfu/ml)























*TNTC– too numerous to count (more than 300 colonies per plate)

Theresults obtained were converted into presumptive E.coliper ml of the wastewater. Stirred wastewater samples resulted in ahigher presumptive E.colicount.

  1. Results of pH, ORP, DO and ammonia

Table3. DO,ORP, PH and ammonia measurement in stirred and static wastewatersamples.





Conductivity (mSv)

Ammonia (ISE mg/L)













Stirredwastewater samples had higher proportions of ORP and pH, whereasstatic wastewater samples had higher proportions of DO, conductivityand ammonia.


Theresults obtained show a huge variation in the number of coloniesobserved in the samples incubated under oxygen-rich andoxygen-deficient conditions. Those incubated under oxygen-richconditions showed a higher colony count in aerobic counts (6.9× 109).The stirring of the wastewater samples introduced dissolved oxygen(DO) into them. The aerobic bacteria contained in the samples usethis oxygen for their respiration. They use the dissolved oxygen toconvert the organic matter in the wastewater into energy. Therefore,a high colony number of aerobic bacteria indicates a lower amount oforganic matter in the effluent. With respect to the anaerobicbacteria, there is a high colony count in the oxygen-deficient sample(0.9× 109).Anaerobic bacteria thrive in oxygen-deficient environments. Theybreak down organic matter into oxygen, carbon dioxide, and methane.The lack of agitation and stirring in the static samples mean thatthey have the lowest amounts of oxygen, and as a result offer optimumconditions for anaerobic bacteria to thrive, hence their huge numbers(Food and Agriculture Organization, 2017).

Theremoval of ammonium nitrogen from a wastewater sample occurs throughthe process of nitrification carried out by bacteria (such asNitrosomonasand Nitrobacter).These bacteria thrive in oxygen-rich conditions and are classified asbeing obligate aerobes (they need free molecular oxygen) (Food andAgriculture Organization, 2017). This is why the results show thatstirred (oxygen-rich) samples have almost half the amount of ammonia(20.6 ISE mg/L) compared to static (oxygen-deficient) samples (41.1ISE mg/L)

E.coli isused in tests to check for the removal of fecal pathogens fromtreated wastewater because of its ability to thrive in both aerobicand anaerobic conditions, as well as in high temperatures reaching44.5°C(Food and Agriculture Organization, 2017).These conditions are similar to those that exist inside humans’ andanimals’ bodies. Therefore, the presence of fecal coliform such asE.coliin treated wastewater shows that the treatment was not done optimallyas the water might be contaminated by pathogens that might lead towaterborne diseases such as typhoid and hepatitis A.

Anaerobic design offers the best wastewater treatment according to theresults obtained. To optimize it further, it will be important to addstirrers to the tanks that would agitate and stir the wastewater toensure equal distribution of dissolved oxygen in the wastewater. Aneutral pH of around 6.5-7.5 is considered optimum for aerobicbacteria in a wastewater treatment setting. Therefore, before aerobictreatment commences the pH of the wastewater has to be adjusted tofit with this pH value. The temperature is also crucial to theperformance of the aerobic bacteria. A study stated that aerobicbiological activity decreased when the temperature rose from 30°C to60°C. Hence the temperature of the water has to be maintained below30°C (Shohreh, Valipour, &amp Sithebe, 2013).


Foodand Agriculture Organization. (2017, 3 22). 1.Wastewater characteristics and effluent quality parameters.Retrieved from Food and Agriculture Organization:

Shohreh,A., Valipour, A., &amp Sithebe, T. (2013). Evaluation of DifferentWastewater Treatment Processes and Development of a Modified AttachedGrowth Bioreactor as a Decentralized Approach for Small Communities.TheScientific World Journal,456-459.

UNEP.(2016). Sanitation,Wastewater Management and Sustainability: From Waste Disposal toResource Recovery.Nairobi: United Nations Environment Programme.