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Updated: Oct 9

Technical Review Article | Open Access | Published 11th October 2024

Good bugs, bad bugs, and unwanted effects: Microorganisms and wastewater treatment


Tim Sandle, Ph.D., CBiol, FIScT | EJPPS | 293 (2024) | Click to download pdf   


Wastewater treatment plants are important to many types of pharmaceutical processing. An important functional basis of such plants is through microorganisms, where biological activity is essential for breaking down chemicals and ensuring the water passed into a municipal system meets required cleanliness standards. There is an additional element in relation to microorganisms; this is with minimising the release of antimicrobial compounds or genetically modified organisms, which can promote antimicrobial resistance in the community. 

 

This paper follows on from a general overview of wastewater plants (published in Vol.29.2; July 2024), focusing on the chemical and physical operational aspects, including an assessment of some of the reasons why wastewater plants go wrong. This companion paper examines another key reason for wastewater system failure - when the microorganisms in the process fail to function as intended. When a loss of the desired microbial community occurs, restoring this can be complex, and the paper discusses some ways to resolve this. 

 

The paper also considers some alternative technologies. 


How microorganisms treat water

The successful removal of wastes from the water depends on how efficiently the bacteria consume the organic material and on the ability of the bacteria to stick together, form floc, and settle out of the bulk fluid. The flocculation (or clumping) characteristics of the microorganisms in the inactivated sludge enable them to amass to form solid masses large enough to settle to the bottom of the settling basin. As the flocculation characteristics of the sludge improves, settling improves, resulting in improved wastewater treatment. 

 

Following the aeration basin, the combination of microorganisms and wastewater (mixed liquor) flows into a settling basin or clarifier where the sludge is allowed to settle. Some of the sludge volume is continuously recirculated from the clarifier (this is called Returned Activated Sludge), back to the aeration basin to ensure an appropriate quantity of microorganisms are maintained in the aeration tank. The microorganisms are then mixed with incoming wastewater where they are reactivated to consume organic nutrients. The process the begins again. 

 

Microorganisms play an important role in secondary wastewater treatment. Microbes degrade the biological content of the waste through aerobic biological processes. There are different ways to achieve this, including biofiltration, oxidation, and aeration. With oxidation there are different options, including lagoons, activated sludge, trickling filters, rotating biological contactors, and biofilters. The most common approach is activated sludge. 

 

At a later stage, microorganisms undertake the metabolization of organic matter as part of the wastewater in biological treatment. This includes aerobic processes (the decomposition of organic matter), anaerobic processes (fermentation) and through composting.  


Activated sludge 

Activated sludge refers to a biochemical process for treating sewage and industrial wastewater that uses oxygen and microorganisms to biologically oxidize organic pollutants, producing a waste sludge (sometimes termed floc) containing the oxidized material. In the activated sludge process, the dispersed-growth reactor is an aeration tank or basin containing a suspension of the wastewater and microorganisms, the mixed liquor. The contents of the aeration tank are mixed vigorously by aeration devices which also supply oxygen to the biological suspension. Aeration devices commonly used include submerged diffusers that release compressed air and mechanical surface aerators that introduce air by agitating the liquid surface. Hydraulic retention time in the aeration tanks usually ranges from 3 to 8 hours but can be higher with high BOD (biochemical oxygen demand) wastewaters. Following the aeration step, the microorganisms are separated from the liquid by sedimentation and the clarified liquid is secondary effluent. A portion of the biological sludge is recycled to the aeration basin to maintain a high mixed-liquor suspended solids (MLSS) level. The remainder is removed from the process and sent to sludge processing to maintain a relatively constant concentration of microorganisms in the system. Several variations of the basic activated sludge process, such as extended aeration and oxidation ditches, are in common use, but the principles are similar. 

 

This process requires ¹: 

 

  • An aeration tank where air (or oxygen) is injected and thoroughly mixed into the wastewater. 

  • A settling tank (called a clarifier or "settler") to allow the waste sludge to settle. Part of the waste sludge is recycled to the aeration tank and the remaining waste sludge is removed for further treatment and ultimate disposal. 

A trickling filter or biofilter consists of a basin or tower filled with support media such as stones, plastic shapes, or wooden slats. Wastewater is applied intermittently, or sometimes continuously, over the media. Microorganisms become attached to the media and form a biological layer or fixed film. Organic matter in the wastewater diffuses into the film, where it is metabolized. Oxygen is normally supplied to the film by the natural flow of air either up or down through the media, depending on the relative temperatures of the wastewater and ambient air. Forced air can also be supplied by blowers but this is rarely necessary. The thickness of the biofilm increases as new organisms grow. Periodically, portions of the film slough off the media. The sloughed material is separated from the liquid in a secondary clarifier and discharged to sludge processing. Clarified liquid from the secondary clarifier is the secondary effluent and a portion is often recycled to the biofilter to improve hydraulic distribution of the wastewater over the filter. 

 

For the trickling filter, this is formed of a bed of rocks, gravel, slag, peat moss, or plastic media over which wastewater flows downward and contacts a layer (or film) of microbial slime covering the bed media. Aerobic conditions are maintained by forced air flowing through the bed or by natural convection of air. 

 

The most important microbial control parameter is the biochemical oxygen demand (BOD). This refers to the amount of dissolved oxygen needed (or ‘demanded’) by the aerobic organisms present so they can effectively break down the organic material present in the wastewater, at the required temperature and across a specific time period. The activated sludge process, under proper conditions, is very efficient. This removes 85 to 95 percent of the solids and reduces the biochemical oxygen demand (BOD) about the same amount. Aerobic bacteria are used within an aerated environment. Such bacteria use the free oxygen within the water to degrade the pollutants in the wastewater and then convert this into energy to grow and reproduce. For these types of bacteria, oxygen must be added mechanically. This ensures the bacteria are able to function effectively and continue to grow and reproduce. 

 

Generally, when BOD levels are high, there is a decline in dissolved oxygen (DO) levels. This is because the demand for oxygen by the bacteria is high and they are taking that oxygen from the oxygen dissolved in the water. If there is no organic waste present in the water, there won’t be as many bacteria present to decompose it and thus the BOD will tend to be lower and the DO level will tend to be higher. High effluent BOD levels in the treated effluent can have a number of causes, such as: 

 

  • Incomplete wastewater treatment due to organic overloading. 

  • Low oxygen concentration. 

  • Low hydraulic detention time. 

  • Physical short circuiting. 

  • High algal or sulphur bacteria growth. 

  • Sludge accumulation and loss of old sludge to the effluent. 

 

Anaerobic bacteria are used in sewage treatment to reduce the volume of sludge and produce methane gas from it. These organisms are used more frequently than aerobic bacteria since the methane gas, if cleaned and handled properly, can be used as an alternative energy source. This can help to offset the high wastewater treatment energy consumption levels. Anaerobic organisms are able to gain sufficient oxygen from the food source. A key advantage is with  phosphorus removal from wastewater via the activities of anaerobic organisms. 

 

It is possible that the biota of a treatment plant may gradually become acclimatized to certain chemicals and therefore may degrade them more effectively given time ². The efficiency of the microbial aspect of the system is dependent upon several factors, including wastewater climate and characteristics. Toxic wastes that enter the treatment system can disrupt the biological activity. Waste materials heavy in detergents (as often occurs with pharmaceuticals) can cause excessive frothing and thereby create aesthetic or nuisance problems. In areas where industrial and sanitary wastes are combined, industrial wastewater must often be pre-treated to remove any toxic chemical components present. Hence, periodically assessing the microorganisms in terms of species and numbers represents an important activity for wastewater plant managers. This means the enumeration of metabolically active bacteria in environmental samples is frequently required to estimate system productivity, biomass turnover, or substrate utilization potentials (Coleman, 1980). One challenge is that many of the organisms are not culturable; this is often because it is not clear what facet of the environment is not being properly replicated (nutrients, pH, osmotic conditions, temperature, or many more), attempting to vary all of these conditions at once results in a multidimensional matrix of possibilities that cannot be exhaustively addressed with reasonable time and effort ³. However, those species that can be readily cultured can provide an indication of the overall health of the microbial community. 


Microbial control 

Everywhere, from the water arriving at the treatment plant to its outlet, microorganisms are used in the wastewater treatment process. There are variations, however, since the operating parameters defined in the treatment tanks influence the development of various microbial structures and species. This complex combination of micro-organisms, rich in species, achieves a high level of biodegradation over a wide range of substrates. Such is the importance of microorganisms that they are the main factor influencing the quality of treated wastewater. 

 

The microorganisms found in wastewater treatment processes come in contact with the biodegradable materials in the wastewater and consume them as food. In addition, most of the bacteria develop a biofilm which enables them to clump together to form bio-solids or sludge. In aquatic water systems, the majority of bacteria grow in biofilm communities and these sessile bacterial cells differ profoundly from their planktonic (floating) counterparts. These differences relate to the physiological attributes of the organisms, including such characteristics as altered growth rate and the fact that biofilm organisms transcribe genes that planktonic organisms do not. The biofilm is formed of communities of attached bacteria, encased in a “glycocalyx” matrix that is polysaccharide in nature, and this slime-like matrix material helps to mediate surface adhesion ⁴. 

 

These are then separated from the liquid phase. There are different organisms present, of which the most important for the treatment process are bacteria. The organisms required are: 

 

  • Bacteria, which are primarily responsible for removing organic nutrients from the wastewater. Usually, these organisms swarm and aggregate into a flake-like structure within the free culture called the ‘floc’. These flocs agglomerate around the suspended organic matter on which they feed. This is the case, for example, with activated sludge. In addition, in fixed cultures, biofilms develop on contact surfaces. For example, biofilters and biological disks. 

  • The common types of bacteria, albeit acknowledging there are regional variations, include Gram-negative bacteria of the proteobacteria type such as Betaproteobacteria (these are largely responsible for the elimination of organic elements and nutrients). The other types are Bacteroidetes, Acidobacteria and Chloroflexi. The most numerous types of bacteria are Tetrasphaera, Trichococcus, Candidatus, Microthrix, Rhodoferax, Rhodobacter, and Hyphomicrobium

  • Protozoa, which play a critical role in the treatment process by removing and digesting free swimming dispersed bacteria and other suspended particles. This improves the clarity of the wastewater effluent.  

  • Protozoa include: 

  • Amoebae - Little effect on treatment and die off as the amount of food decreases. 

  • Flagellates - Feed primarily on soluble organic nutrients. 

  • Ciliates -      Clarify water by removing suspended bacteria. 

  • Metazoa, which are multi-cellular organisms which are larger than most protozoa and have very little to do with the removal of organic material from the wastewater, apart from ingesting bacteria. A dominance of metazoa is usually found in longer age systems; namely, lagoon treatment systems.  These include rotifers, which clarify effluent; nematodes, which feed on bacteria, fungi, and small protozoa; and tardigrades. 

  • The presence of algae and fungi which are photosynthetic organisms and generally do not cause problems in activated sludge treatment systems, however their presence in the treatment system usually indicate problems associated with pH changes and older sludge. Common fungi include Ascomycetes

 

Filamentous bacteria can emerge when operational conditions significantly change. These bacteria grow in long filaments and may begin to gain an advantage. Changes in temperature, pH, oxygen, sludge age, or even the amounts of available nutrients such as nitrogen, phosphorus, oils and grease can affect these bacteria. The dominance of filamentous bacteria in the activated sludge treatment system can cause problems with sludge settling. At times excessive numbers of filamentous microorganisms interfere with floc settling and the sludge becomes bulky. This bulking sludge settles poorly and leaves behind a turbid effluent. Some filamentous microorganisms may cause foaming in the aeration basin and clarifiers. To take a real-case example (known to the author), one facility developed a wastewater plant biofilm composed primarily of only one bacterial species, and this resulted in the plant no longer functioning as required. The bacterium was called Shewanella putrefaciens. This a Gram-negative rod and hence not atypical for water. It is associated with aquatic environments and has been isolated globally from the sandstone used in water purification processing plants. The organism has a distinctive odour, described by some as resembling rotting fish (which accounts for the species name ‘putrefaciens’ ie. putrid) ⁵. 

 

Significantly this bacterium has the ability to reduce iron and manganese metabolically ⁶. The bacterium produces a by-product chemical called trimethylamine, from a reaction between ammonia and methanol to produce an amine, which is a flammable gas at room temperature.  It was reasoned that the organism was part of the sewage culture. The pharmaceutical company had changed one of the chemicals used in the plant, to ferrous chloride. It was speculated that an increase in levels of iron, from the chemical change, will have most probably either inhibited the growth of other organisms and / or created conditions that have allowed S. putrefaciens to grow exponentially and become the predominant species. The problem faced by the company was how to kill this organism without killing everything else. Starvation was found to be the best strategy, by reducing the iron supply and the then adding a new culture, where restored environmental conditions enabled the normal and desired population to flourish. An alternative solution would have bene to kill the organism using chlorine. With either strategy, it takes a few weeks for normal conditions to be reached again. 

 

The presence of the wrong types or combination of microorganisms can lead to the following problems ⁷: 

 

  • Low biogas efficiency of the anaerobic digester 

  • Poor flocculation and sedimentation 

  • An excess of filamentous bacteria 

  • Excess of phosphorus 

  • Low nitrogen removal efficiency (NH4, NO3) 

  • The production of unpleasant odours 

  • Excess consumption of chemical products 

  • In an anaerobic digester, foam production 

 

To address these problems, there are three mechanisms: 

 

  1. By changing the operating settings and waiting for the right species to colonize the environment again.  

  2. By completely removing the microorganisms in place when the first solution did not work.  

  3. Injecting specially selected, cultured and multiplied bacteria in order to recover the advantage over the undesirable bacteria present in the environment. 

 

Another concern is with the inhibition effect shown by certain drugs on nitrifying microorganisms, which decreases treatment plant efficiency. Moreover, this is of relevance as a signal of possible negative effects on aquatic organisms when a wastewater containing pharmaceutical leaves the facility and enters the general water system or environmental water sources. 


The problem of antimicrobial resistance 

The release of antimicrobials into the environment remains a major problem, although most of the release is due to people excreting by-products at home or carelessly flushing unwanted tablets down the toilet. The widespread presence of antibiotics not only impacts wildlife, but likely contributes to the problem of antimicrobial resistance. The risk with this practice is that over-exposure to antimicrobials leads to some bacteria acquiring resistance. Should sufficient numbers of the mutated bacteria enter the community, they could trigger an infection, and that infection may not be treatable with an antibiotic or other antimicrobial. The biggest risk group are those in hospital ⁸. 

 

Various studies have shown that wastewater contains a mix of antimicrobial agents, discarded by people at home, pharmaceutical companies and hospitals, along with a variety of pathogenic bacteria. Many of these bacteria are - unsurprisingly given where much of the water comes from - found in the intestines of people. Hence treatment plants contain many genes that can potentially trigger resistance to a range of antimicrobial agents ⁹. 

 

With the variation of different antibiotics, Ciprofloxacin, a frontline treatment for intestinal and urinary tract infections, is the most commonly recovered type. As an example of the impact, a study by Hirsch et al (1999) involved the analysis of various water samples for 18 antibiotic substances, from several groups, including macrolide antibiotics, sulphonamides, penicillins, and tetracyclines. Both sewage treatment plant effluents and surface-water samples were found to be frequently contaminated with sulfamethoxazole and roxithromycin (a degradation product of erythromycin) at concentrations up to 6 µg L−1. The highest concentrations detected for tetracyclines and penicillins were 50 and 20 ng L−1, respectively. In another case, New Delhi metallo-beta-lactamase 1 (NDM-1) is a gene that can cause antimicrobial resistance. Specifically, NDM-1 is an enzyme that makes bacteria resistant to a broad range of beta-lactam antibiotics. Beta-lactam antibiotics are a broad class of antibiotics and among the most commonly used worldwide. NDM-1 was first detected in a Klebsiella pneumoniae isolate from a Swedish patient of Indian origin in 2008 (the bacterium can cause destructive changes to human lungs via inflammation and haemorrhage). NDM-1 was later detected in bacteria in India, Pakistan, the United Kingdom, the United States, Canada, and Japan. It has also been found  in wastewater and sludge from two treatment plants in China ¹⁰.  

 

A further risk arises in relation to chemical alterations occurring with antimicrobials. One area of research looked at doxycycline, which is a broad-spectrum antibiotic, often sold under the brand name Vibramycin. It can be used as a defence against anthrax. The study revealed that chlorine used to treat wastewater can alter the chemical composition of the antibiotic, leading to an altered structure. The problem that inevitably arises is that some bacteria can become resistant to this altered structure and the risk is that this fuels a new form of antibiotic resistant organism ¹¹. 

 

While many pharmaceutical companies attempt to use their wastewater systems to prevent antimicrobials from entering the environment, not all companies have robust systems in place. As part of an investigation into the practices of pharmaceutical companies, the campaign group SumOfUs.org undertook a study. This indicated that the three Chinese companies, who supply active ingredients to pharmaceutical companies, including generic drug manufacturers, were found to have added high quantities of antimicrobials into water systems. Such findings infer a lack of regulation or an avoidance of regulation ¹². 

 

Alternative technologies 

 

While many aspects of wastewater treatment have remained unchanged over time, the sector is not without its innovations. From the management side, blockchain is being considered as a means to track and trace water treatment in order to maintain environmental and sustainability standards. This is through the application of a digital ledger, which cannot be altered. An example is with the WaterChain token, which is intended to help create a financial platform for the private water treatment systems that do not benefit from the millions and billions allocated to costly central water treatment systems ¹³. 

 

In terms of alternative technologies, one such development is with the use of hydrogel technology, a process that works in conjunction with sunlight. The technology brings together gel-polymer hybrid materials. The material has hydrophilic (attraction to water) qualities and semiconducting (solar-adsorbing) properties, and it is a form of “hydrogel”. The primary aim of the new technology is to remove salt from water; however, the process can also remove most other contaminants. 

 

Hydrogels are networks of polymer chains known for their high-water absorbency. Hydrogels are highly absorbent (they can contain over 90% water) natural or synthetic polymeric networks. As well as the current application, natural hydrogel materials are being investigated for tissue engineering and for use in agriculture, as they can release agrochemicals including pesticides ¹⁴. With the new development, researchers from the University of Texas have constructed a new hydrogel-based solar vapour generator. The device runs off ambient solar energy in order to power the evaporation of water to achieve desalination ¹⁵. Desalination refers to the removal of salts and minerals from a target substance; this can relate to soil or to water. Most commonly desalination is described in relation to saltwater (water that contains a significant concentration of dissolved salts), where the object is to desalinate the water in order to produce water suitable for human consumption or irrigation. 

 

A different type of water filter can be developed using graphene. Graphene in its basic form, is a one-atom thick sheet of carbon. The material is light, transparent, strong and very conductive. Graphene oxide is graphite oxide, which is an oxidized form of a compound of carbon, oxygen, and hydrogen in variable ratios, arranged in sheets. Once these sheets become monomolecular (that is, one molecule thick) graphene oxide is formed. Graphene oxide sheets can be used to prepare strong paper-like materials like membranes. 

 

Several laboratories are constructing graphene-based, laboratory-scale filters, intended to remove the natural organic matter that is left behind during conventional treatment processing of drinking water. Many of these projects are progressing towards scale-up technology.  

 

The main application of graphene technology is with natural organic matter contaminants. This is because these particles can affect the performance of filtration plants and over time the matter can reduce the capacity of the plant to function effectively, especially after periods of heavy rain. Conventional treatments involve the use of chemical coagulants, but these methods are not totally effective. Filtration has been shown to be a superior method. With the new filter, the researchers converted naturally occurring graphite into graphene oxide membranes. These membranes enable high water flow at atmospheric pressure. As an alternative, graphene-oxide membranes are attracting considerable attention as promising candidates for new filtration technologies, with the potential of filtering out small nanoparticles, organic molecules, and even large salts. With the filter, tiny capillaries of the graphene-oxide membranes function to block the salt from flowing along with the water as it passes through the filter ¹⁶. 

 

Alternative use of microorganisms is also being considered. As this paper has described, the use of microorganisms is key to the breakdown of materials through the wastewater process. However, the downside is that this process generates high levels of carbon dioxide and methane, both of which are atmospheric pollutants. This has led to research into a microbial fuel cell that uses only the power of microbes in the sewage lagoons to generate electricity. In a sense, a “microbial battery.” This fuel cell is a type of bio-electrochemical system that drives a current by using bacteria and mimicking bacterial interactions found in nature. 

 

A microbial fuel cell is made up of an anode and a cathode that are separated by a cation (positively charged ion) membrane. In the anode compartment, fuel is oxidized by microorganisms, generating carbon dioxide electrons and protons. Electrons are transferred to the cathode compartment through an external electric circuit, while protons are transferred to the cathode compartment through the membrane. Electrons and protons are consumed in the cathode compartment. Here they combine with oxygen to form water. 

 

With recent research, scientists have created favourable conditions for the types of microbes that can naturally generate electrons as part of their metabolic processes; in doing so they consume many of the gases that would ordinarily be released as pollutants. The microbes were able to successfully power aerators in a laboratory study for over a year. Based on this success, work is afoot on a full-scale pilot ¹⁷. 

 

Where water may be radioactive (as with the nuclear industry, although also with those pharmaceutical plants that develop certain radioactive medicines), an emerging technology is shock electrodialysis. This technology was originally intended to be an efficient way of separating salt from sea or brackish water by literally shocking the salt from the water, The system uses an electrically driven shockwave within a stream of flowing water that pushes salty water to one side of the flow and freshwater to the other. Latterly researchers are focusing on a more specific application, designed to improve the economics and environmental impact of radioactive water. Here a deionization shockwave in a tube of water has been designed to push electrically charged ions into a charged porous material that acts as the tube's lining. In other words, if the ions consist of the desired element for disposal, they can be selectively filtered out of the coolant water flow ¹⁸. One set of tests shows that 99.5 percent of cobalt and caesium radionuclides can be removed from water; while, at the same time, retaining about 43 percent of the cleaned water for reuse ¹⁹. 

 

Summary 

 

This article has continued the exploration of wastewater treatment, from a pharmaceutical perspective, focusing on the role of microorganisms in tackling pharmaceutical pollutants. This remains a matter of global concern ²⁰.  Microbial wastewater treatment utilises microorganisms as decontaminating agents in order to treat polluted wastewater, which is a particular worldwide concern in relation to pharmaceutical pollutants. Microorganism-based processes can treat the ever-increasing problem of polluted wastewater, provided that cultures and the process overall is effectively maintained. 

 

Failure to maintain a process consistently can lead to problems, either slowing down the treatment or leading to contaminated, and hence polluted, discharges. The article has looked at some of the situations by which systems can breakdown. The article has also considered the application of some alternative technologies. 



 

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Author Information

Corresponding Author: Tim Sandle, Head of Microbiology

                                          Bio Products Laboratory,  

UK Operations,                                           

England     

                                                                            



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