150 the sludge will have poor sedimentation properties, and the capacity of the treatment plant will be reduced as the index rises. SQI analysis To obtain relevant results it is essential to dilute the sludge for SV and SVI measurements. Even at relatively low concentrations, networks of filaments can form and the sludge will stick to the glass, thereby slowing sedimentation. The result will be misleading SV/SVI results. To avoid this, the sludge can be diluted in several stages. The following dilution procedure is carried out four times a year (in spring, summer, autumn and winter) as the sludge changes in nature over the course of the year. The sludge volume is determined after 30 minutes (SV30) in a graduated cylinder with a diameter of 6 cm and a capacity of 1,000 ml, for various sludge dilutions. The sludge is diluted with outlet (treated) water. • The graduated cylinders are placed indoors on a table that is in the shade and is not exposed to vibration • When all the samples are ready they are poured without delay into the graduated cylinders • After 30 minutes the sludge volumes and, if required, the floating sludge volumes, are read from each of the graduated cylinders • The results are plotted on a graph and the dilution factor is determined from the linear portion of the graph. As a rule of thumb it can be said that a sludge volume of 200 - 300 ml/l should be obtained after 30 minutes. Microscopic examination Because SVI and SQI only give a very rough idea of the properties of the active sludge it is important to carry out a microscopic examination. This will not always lead to optimisation of the operation of the treatment plant, however, but should be used to supplement the other results (SS, SVI, etc.) and hence contribute to a better picture of the sludge. The samples for microscopic examination are always taken from the same point in the process, and this must be a point where the active sludge is fully mixed, preferably through aeration. If there is any floating sludge or scum then samples of this should also be taken, since filamentous bacteria are often easiest to identify there. The test bottles should only be filled around 10% full, as this ensures that the microorganisms have a source of oxygen and will last longer. Active sludge that is to be evaluated for protozoa should preferably be examined immediately. Sludge samples for measuring filamentous bacteria and sludge floc composition can be kept for around 14 days in a refrigerator. The first characterization of the sludge floc is carried out at 100 - 200 times magnification. This permits evaluation of the sludge floc shape, size, structure, filament index, number of filaments where relevant, protozoa and organic and inorganic particles. The strength of the sludge floc is determined by applying light pressure to the coverslip. This is always done last, since the samples are unusable afterwards. Measurement of the filamentous bacteria is carried out at 400 times magnification. If Gram-staining or Neisser-staining is to be carried out this is done at 1,000 times magnification using an oil emulsion and a dry specimen. Some of the organisms and filamentous bacteria that can be found during microscopic examination are described below. Testate amoebae Testate amoebae Arcella vulgaris and Euglypha were previously very uncommon, but are now seen increasingly often, both in municipal plants and in industrial plants. Their natural habitat is among grass in damp meadowland, and they can therefore be found in wastewater from slaughterhouses and breweries. They cling to the sludge floc and begin to digest it by injecting floc-forming bacteria through openings in their tests (shells). The first signs that testate amoebae are at work can be found in the sedimentation basin, where small sludge flocs appear that are difficult to retain. This results in the loss of bacteria that are important for reducing nitrogen and organic matter. Testate amoebae, which are relatively heavy, give a low SQI, and most analysts believe that an SQI value of 60-70 ml/g does not cause any problems. Testate amoebae can be transferred from one treatment plant to another, since they work well in low-nitrogen treatment plants. Testate amoebae flourish in May and dwindle in September. They have, however, also been observed in large numbers at other times of the year. Protozoa In recent years there have been discussions about what role protozoa play in the treatment wastewater, since it is mainly bacteria that remove nitrogen, phosphorus and organic matter. Often they are simply used to ensure that the sludge looks healthy, but protozoa can also have a cleaning effect on runoff. A sharp increase in immobile ciliates has often been observed, and these are involved in taking up free cells from the water phase. A treatment plant can, however, function perfectly well without protozoa. The type of protozoa present gives some indication of the age of the sludge and the load on it. In lightly loaded plants, protozoa can primarily be used as an indicator of toxicity. Toxicity leads first to the inactivation of Vorticella, followed by free-swimming ciliates. This is generally followed by the flourishing of flagellates that live on dissolved matter, and a rise in their concentration in the water phase. Rotifers - wheel-like organisms - are typical where the load is decreasing and the sludge age is rising. They have a life cycle of 6-45 days and reproduce asexually, with the male having a considerably shorter life than the female. Nematodes, which look like small worms, can also be found in active sludge with a high sludge age, and also reproduce asexually. In turnover terms, nematodes have relatively little significance however. Filamentous bacteria in active sludge at municipal wastewater treatment plants In the majority of cases, poor sedimentation properties are due to the growth of filamentous bacteria. Filamentous bacteria are distinguished by the fact that they live together, in contrast to other bacteria, which often live apart. Because of their different living requirements the composition of the wastewater is critical in determining which types are dominant. The concentrations of organic matter, oxygen and nutrients also play a role in this respect. Due to their larger surface area and the way that they project from the sludge flocs, filamentous bacteria can survive periods when some of the previously mentioned substances are absent. The types of filamentous bacteria that are most often present in municipal wastewater treatment plants are: Microthrix parvicella, N. limicola, Type 0041/0675, and in recent years also Nocardia. In wastewater treatment plants with a high load of industrial wastewater the following types may occur: Type 021N, H. hydrosis, type 0914, S. natans, Thiotrix, type 0092, N. limicola and type 0803. Microthrix parvicella: Sharply curving and frequently arched filament that intertwines in and around the sludge floc and usually has a length of 200-400 µm and a diameter of 0.5 µm. The septa (cell walls) are difficult to detect under the microscope. Some clustering may occur. Microthrix parvicella belongs to the group of bacteria that can form foam or floating sludge on the surface of both the process basin and the clearing basin. This is because the surfaces of their cells are water-repellent (hydrophobic) and therefore try to avoid water. They do not exhibit this behaviour all year round however, but it is mostly seen at temperatures around 12ºC, in other words in spring and autumn. Studies carried out at Ålborg University by Kjær Andreasen and Per Haldkær, among others, have shown that Microthrix lives on palmitic acid and oleic acid, two long-chain fatty acids, which are also known as lipids. Studies have also shown that around 30% of the organic matter in wastewater is in lipid form, which means that Microthrix has sufficient nutrients. It also turns out that Microthrix can take up the long-chain fatty acids under all process conditions (aerobic, anoxic and anaerobic) and even create problems in biogas reactors. Microthrix can store large amounts of fatty acids under oxygen-free and nitrate-free (anaerobic) conditions, i.e. under conditions in which it cannot grow. There are only a few bacteria that have this ability to store a "fat layer" for later use in situations when oxygen or nitrates are available. This gives Microthrix an advantage over many other bacteria that can only make use of fat when oxygen is available. Type 021 N: Long, slightly curved, non-mobile filament. The septa are visible. The cell shape can vary somewhat. Can be seen in the water phase between sludge flocs. Type 021 N is not capable of denitrification and it should therefore be possible to control it using a selector. However, variants of 021 N have appeared that can only by identified using gene probes. These new variants cannot be controlled using a selector. Type 021 N can be controlled by dosing with PAX-14 in the recirculated sludge. Found most frequently in industrial wastewater from dairies, sweet manufacturers and chicken farms, etc. Thiotrix: Slightly curved, immobile filament that mostly projects from the sludge floc. Occurs in conditions where there is readily degradable source of carbon, especially low-molecular organic acids (butyric acid, acetic acid). Capable of oxidising sulphide to sulphate and absorb it into cells. Controlled by removing or binding sulphide and reducing the concentration of low-molecular organic acids. N. limicola: Occurs in three variants. Often seen in sludge of medium to high age and in conditions where there is a readily degradable source of carbon. Strongly dominant in a few locations (in Germany and Denmark), where it can cause floating sludge. Can possibly be controlled by reducing the sludge age. An alpha variant of N. limicola has appeared in recent years and attempts have been made to control this by polymer dosing. This resulted in N. limicola returning to the sludge floc, without killing it. Nocardia: Highly branched short filaments that are immobile. Until a few years ago it was uncommon in Denmark, and only appeared in warmer countries such as the southern US, Israel, Greece, Turkey, etc. Recently however, it has been seen in several locations in Denmark, in both industrial and municipal wastewater treatment plants. It becomes hydrophobic and hence results in floating sludge. Can be controlled with hydrogen peroxide. Control methods There is no universal method that can be applied to all filamentous bacteria. Often, attempts are made to control them with toxins such as chlorine or hydrogen peroxide. Some can be controlled by using a selector, and Microthrix parvicella and type 021 N can be controlled using PAX-14. If chlorine or hydrogen peroxide are used they must be added in small, controlled amounts to prevent the removal of "good" bacteria as well. In order to control Microthrix parvicella, PAX-14 may either be added to the recirculated sludge or at the outlet from the process basin. The quickest effect is achieved by dosing the recirculated sludge, where PAX is not intended to remove phosphorus, but it may attach to the surface of Microthrix and prevent it from taking up the long-chain fatty acids (this is not entirely proven yet, but we believe it to be the case). However, experience has shown that where aluminium products are added over an extended period (the whole year) Microthrix cannot be controlled in this way. This would therefore indicate that Microthrix becomes resistant to aluminium. To achieve the quickest and best effect from dosing with PAX-14 it must be added at the right point in time, i.e. precisely before Microthrix becomes hydrophobic and starts to cause foaming and floating sludge. There is still a long way to go before we fully understand the lives and behaviours of these microorganisms, and possibly this is a process that will never be complete. If a solution does appear, and succeeds in eliminating one type of filamentous bacteria, another one will soon appear and start causing problems, so we return to square one. Fortunately, extensive research is being carried out continuously in this field, in Denmark and in the rest of the world. References: Per Halkjær Nielsen, Jeppe Lund Nielsen and Kjær Andreasen. Trådformede bakterier - et problem i renseanlæg. From Naturens verden issue. 7 1999 Kjær Andreasen and Lars Sigvardsen. Letslam December 1989 David Jenkins, Michael G. Richard and Glen T. Daigger. Manual on the Causes and Control of Activated Sludge Bulking and Forming D.H.Eikelboom and H.J.J. van Buijsen. Handbuch für die mikroskopische Schlamm-untersuchung Lene Mikkelsen. Forbedring af slamegenskaber ved kalkdosering på Them Renseanlæg"> The composition and treatment of active sludge - WaterWorld

The composition and treatment of active sludge

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Feb. 19, 2004 -- The 1987 Action Plan for the Aquatic Environment led to a stiffening of wastewater treatment requirements for nitrogen, phosphorus and organic matter. As a result, many municipalities and industries also chose to use biological processes, often however in combination with a preliminary treatment such as chemical precipitation.

It is critical for the efficiency of an active sludge plant that the active sludge remains in the sedimentation basin. The transfer of active sludge, also known as sludge escape (increased levels of SS, etc.), means that approved emission levels are exceeded, and leads to additional charges for the removal of nitrogen, phosphorus and organic matter.

Sludge escape can be caused by hydraulic overload, but for many sewage plants, and in particular municipal plants, it is often associated with sludge that has poor sedimentation properties (sludge volume index, SVI >150 ml/g). Sludge escape is frequently due to the formation of filamentous bacteria, but can also be caused by colloid chemistry conditions, "Zoogloea bulking", viscous bulking, detergents, toxins and fat/oil. Microscopic examination of the active sludge can reveal whether it is due to filamentous bacteria (and possibly which types), Zoogloea (floc former) or toxins.

What is active sludge?

Active sludge consists of microorganisms and a larger or smaller proportion of inorganic particles, organic fibres, filamentous bacteria, extracellular polymer substances (EPS, biopolymers, exopolymers) and ions.

The sedimentation properties of the sludge depend to a high degree on the ability of the bacteria to "clump together" (form flocs). Bacteria that have this ability are known as floc-forming bacteria and are very important in wastewater treatment. Effective floc formation is the basis for satisfactory separation during the sedimentation phase.

Floc formation takes place as a result of the active bacteria precipitating out EPS, which then bind the various components together. In a good sludge floc, the filamentous bacteria and other components act as the "backbone" that holds the sludge floc together. In some cases, however, the presence of filamentous bacteria - whether they float freely between the sludge flocs or project between them - may prevent further flocculation from taking place, and the sedimentation properties from being drastically impaired.

The structure of the sludge floc and the concentration of filamentous bacteria in the active sludge can vary considerably from plant to plant, and reflect differences in the plant types, operation and wastewater composition. This often leads to variations in the sedimentation properties of the active sludge.

About SVI and SQI

Over the years attempts have been made to find a clear and simple method to describe a particularly important part of the treatment process, namely the separation of sludge, i.e. sedimentation.

Initially the sludge volume was measured with the aid of a conical measuring glass, known as an Imhoff cone. The sludge volume expresses the volume that a sludge suspension occupies after 30 minutes of sedimentation. The sludge volume is an indirect measure of the amount of suspended substances in the sludge, and is used to describe the sedimentation properties of the sludge in the sedimentation basin.

Back in 1934 it was, however, realized that the use of an Imhoff cone was not entirely without problems, since the sludge is excessively compressed in the tip of the cone, as well as having a tendency to stick to the sides. A switch was then made to the use of a 1,000 ml cylinder glass.

The sludge volume was used to calculate a sludge volume index (SVI), which describes, among other things, the organic content of the sludge, by determining the volume in ml that 1g of SS occupies after 30 minutes of sedimentation. Because the SVI can be affected by the sludge concentration it was recommended in 1964 that the sludge should be diluted with treated wastewater to give a sludge volume of between 200-300 ml/l.

On the basis of several years of experience with the active sludge process, sludge is defined as having normal sludge properties if SVI < 150 ml/g. If SVI > 150 the sludge will have poor sedimentation properties, and the capacity of the treatment plant will be reduced as the index rises.

SQI analysis

To obtain relevant results it is essential to dilute the sludge for SV and SVI measurements. Even at relatively low concentrations, networks of filaments can form and the sludge will stick to the glass, thereby slowing sedimentation. The result will be misleading SV/SVI results. To avoid this, the sludge can be diluted in several stages. The following dilution procedure is carried out four times a year (in spring, summer, autumn and winter) as the sludge changes in nature over the course of the year.

The sludge volume is determined after 30 minutes (SV30) in a graduated cylinder with a diameter of 6 cm and a capacity of 1,000 ml, for various sludge dilutions. The sludge is diluted with outlet (treated) water.

• The graduated cylinders are placed indoors on a table that is in the shade and is not exposed to vibration
• When all the samples are ready they are poured without delay into the graduated cylinders
• After 30 minutes the sludge volumes and, if required, the floating sludge volumes, are read from each of the graduated cylinders
• The results are plotted on a graph and the dilution factor is determined from the linear portion of the graph. As a rule of thumb it can be said that a sludge volume of 200 - 300 ml/l should be obtained after 30 minutes.

Microscopic examination

Because SVI and SQI only give a very rough idea of the properties of the active sludge it is important to carry out a microscopic examination. This will not always lead to optimisation of the operation of the treatment plant, however, but should be used to supplement the other results (SS, SVI, etc.) and hence contribute to a better picture of the sludge.

The samples for microscopic examination are always taken from the same point in the process, and this must be a point where the active sludge is fully mixed, preferably through aeration. If there is any floating sludge or scum then samples of this should also be taken, since filamentous bacteria are often easiest to identify there.

The test bottles should only be filled around 10% full, as this ensures that the microorganisms have a source of oxygen and will last longer. Active sludge that is to be evaluated for protozoa should preferably be examined immediately. Sludge samples for measuring filamentous bacteria and sludge floc composition can be kept for around 14 days in a refrigerator.

The first characterization of the sludge floc is carried out at 100 - 200 times magnification. This permits evaluation of the sludge floc shape, size, structure, filament index, number of filaments where relevant, protozoa and organic and inorganic particles. The strength of the sludge floc is determined by applying light pressure to the coverslip. This is always done last, since the samples are unusable afterwards.

Measurement of the filamentous bacteria is carried out at 400 times magnification. If Gram-staining or Neisser-staining is to be carried out this is done at 1,000 times magnification using an oil emulsion and a dry specimen.

Some of the organisms and filamentous bacteria that can be found during microscopic examination are described below.

Testate amoebae

Testate amoebae Arcella vulgaris and Euglypha were previously very uncommon, but are now seen increasingly often, both in municipal plants and in industrial plants. Their natural habitat is among grass in damp meadowland, and they can therefore be found in wastewater from slaughterhouses and breweries.

They cling to the sludge floc and begin to digest it by injecting floc-forming bacteria through openings in their tests (shells). The first signs that testate amoebae are at work can be found in the sedimentation basin, where small sludge flocs appear that are difficult to retain. This results in the loss of bacteria that are important for reducing nitrogen and organic matter.

Testate amoebae, which are relatively heavy, give a low SQI, and most analysts believe that an SQI value of 60-70 ml/g does not cause any problems. Testate amoebae can be transferred from one treatment plant to another, since they work well in low-nitrogen treatment plants. Testate amoebae flourish in May and dwindle in September. They have, however, also been observed in large numbers at other times of the year.

Protozoa

In recent years there have been discussions about what role protozoa play in the treatment wastewater, since it is mainly bacteria that remove nitrogen, phosphorus and organic matter.

Often they are simply used to ensure that the sludge looks healthy, but protozoa can also have a cleaning effect on runoff. A sharp increase in immobile ciliates has often been observed, and these are involved in taking up free cells from the water phase. A treatment plant can, however, function perfectly well without protozoa.

The type of protozoa present gives some indication of the age of the sludge and the load on it. In lightly loaded plants, protozoa can primarily be used as an indicator of toxicity. Toxicity leads first to the inactivation of Vorticella, followed by free-swimming ciliates. This is generally followed by the flourishing of flagellates that live on dissolved matter, and a rise in their concentration in the water phase.

Rotifers - wheel-like organisms - are typical where the load is decreasing and the sludge age is rising. They have a life cycle of 6-45 days and reproduce asexually, with the male having a considerably shorter life than the female.

Nematodes, which look like small worms, can also be found in active sludge with a high sludge age, and also reproduce asexually. In turnover terms, nematodes have relatively little significance however.

Filamentous bacteria in active sludge at municipal wastewater treatment plants

In the majority of cases, poor sedimentation properties are due to the growth of filamentous bacteria. Filamentous bacteria are distinguished by the fact that they live together, in contrast to other bacteria, which often live apart. Because of their different living requirements the composition of the wastewater is critical in determining which types are dominant.

The concentrations of organic matter, oxygen and nutrients also play a role in this respect. Due to their larger surface area and the way that they project from the sludge flocs, filamentous bacteria can survive periods when some of the previously mentioned substances are absent.

The types of filamentous bacteria that are most often present in municipal wastewater treatment plants are: Microthrix parvicella, N. limicola, Type 0041/0675, and in recent years also Nocardia.

In wastewater treatment plants with a high load of industrial wastewater the following types may occur: Type 021N, H. hydrosis, type 0914, S. natans, Thiotrix, type 0092, N. limicola and type 0803.

Microthrix parvicella: Sharply curving and frequently arched filament that intertwines in and around the sludge floc and usually has a length of 200-400 µm and a diameter of 0.5 µm. The septa (cell walls) are difficult to detect under the microscope. Some clustering may occur.

Microthrix parvicella belongs to the group of bacteria that can form foam or floating sludge on the surface of both the process basin and the clearing basin. This is because the surfaces of their cells are water-repellent (hydrophobic) and therefore try to avoid water. They do not exhibit this behaviour all year round however, but it is mostly seen at temperatures around 12ºC, in other words in spring and autumn.

Studies carried out at Ålborg University by Kjær Andreasen and Per Haldkær, among others, have shown that Microthrix lives on palmitic acid and oleic acid, two long-chain fatty acids, which are also known as lipids.

Studies have also shown that around 30% of the organic matter in wastewater is in lipid form, which means that Microthrix has sufficient nutrients. It also turns out that Microthrix can take up the long-chain fatty acids under all process conditions (aerobic, anoxic and anaerobic) and even create problems in biogas reactors.

Microthrix can store large amounts of fatty acids under oxygen-free and nitrate-free (anaerobic) conditions, i.e. under conditions in which it cannot grow. There are only a few bacteria that have this ability to store a "fat layer" for later use in situations when oxygen or nitrates are available. This gives Microthrix an advantage over many other bacteria that can only make use of fat when oxygen is available.

Type 021 N: Long, slightly curved, non-mobile filament. The septa are visible. The cell shape can vary somewhat. Can be seen in the water phase between sludge flocs. Type 021 N is not capable of denitrification and it should therefore be possible to control it using a selector.

However, variants of 021 N have appeared that can only by identified using gene probes. These new variants cannot be controlled using a selector. Type 021 N can be controlled by dosing with PAX-14 in the recirculated sludge.

Found most frequently in industrial wastewater from dairies, sweet manufacturers and chicken farms, etc.

Thiotrix: Slightly curved, immobile filament that mostly projects from the sludge floc. Occurs in conditions where there is readily degradable source of carbon, especially low-molecular organic acids (butyric acid, acetic acid). Capable of oxidising sulphide to sulphate and absorb it into cells. Controlled by removing or binding sulphide and reducing the concentration of low-molecular organic acids.

N. limicola: Occurs in three variants. Often seen in sludge of medium to high age and in conditions where there is a readily degradable source of carbon. Strongly dominant in a few locations (in Germany and Denmark), where it can cause floating sludge. Can possibly be controlled by reducing the sludge age. An alpha variant of N. limicola has appeared in recent years and attempts have been made to control this by polymer dosing. This resulted in N. limicola returning to the sludge floc, without killing it.

Nocardia: Highly branched short filaments that are immobile. Until a few years ago it was uncommon in Denmark, and only appeared in warmer countries such as the southern US, Israel, Greece, Turkey, etc. Recently however, it has been seen in several locations in Denmark, in both industrial and municipal wastewater treatment plants. It becomes hydrophobic and hence results in floating sludge. Can be controlled with hydrogen peroxide.

Control methods

There is no universal method that can be applied to all filamentous bacteria. Often, attempts are made to control them with toxins such as chlorine or hydrogen peroxide. Some can be controlled by using a selector, and Microthrix parvicella and type 021 N can be controlled using PAX-14. If chlorine or hydrogen peroxide are used they must be added in small, controlled amounts to prevent the removal of "good" bacteria as well.

In order to control Microthrix parvicella, PAX-14 may either be added to the recirculated sludge or at the outlet from the process basin. The quickest effect is achieved by dosing the recirculated sludge, where PAX is not intended to remove phosphorus, but it may attach to the surface of Microthrix and prevent it from taking up the long-chain fatty acids (this is not entirely proven yet, but we believe it to be the case).

However, experience has shown that where aluminium products are added over an extended period (the whole year) Microthrix cannot be controlled in this way. This would therefore indicate that Microthrix becomes resistant to aluminium.

To achieve the quickest and best effect from dosing with PAX-14 it must be added at the right point in time, i.e. precisely before Microthrix becomes hydrophobic and starts to cause foaming and floating sludge.

There is still a long way to go before we fully understand the lives and behaviours of these microorganisms, and possibly this is a process that will never be complete. If a solution does appear, and succeeds in eliminating one type of filamentous bacteria, another one will soon appear and start causing problems, so we return to square one. Fortunately, extensive research is being carried out continuously in this field, in Denmark and in the rest of the world.

References:

Per Halkjær Nielsen, Jeppe Lund Nielsen and Kjær Andreasen. Trådformede bakterier - et problem i renseanlæg. From Naturens verden issue. 7 1999

Kjær Andreasen and Lars Sigvardsen. Letslam December 1989

David Jenkins, Michael G. Richard and Glen T. Daigger. Manual on the Causes and Control of Activated Sludge Bulking and Forming

D.H.Eikelboom and H.J.J. van Buijsen. Handbuch für die mikroskopische Schlamm-untersuchung

Lene Mikkelsen. Forbedring af slamegenskaber ved kalkdosering på Them Renseanlæg

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