Activated Sludge Problems and the Filaments Responsible. Sphaerotilus natans type Haliscomenobacter hydrossis. Now that we know how certain microbes help clean wastewater in treament systems, let's take a look at some of the biggest issues that may occur if those microbes get out of hand: sludge bulking and foaming. In practical applications, the SVI for each plant where problems of solids loss over the clarifier weirs is site specific due to hydraulics and solids loadings rates.
In simple terms, if the sludge doesn't settle well in the clarifier, it may be a bulking sludge. So, what are the causes of sludge bulking? Some of the causes include:. Don't assume that if you have poor sludge settling that it is caused by filamentous bacteria. Non-filamentous bulking slime bulking can be due to proliferation of zooglea bacteria, or the buildup of polysaccharides under nutrient limited conditions. In these conditions, chlorination of the return activated sludge RAS line is not recommended, as this can produce serious foaming problems.
Note that zooglea and nutrient deficiency can both cause problems due to excessive polysaccharides, but they are caused by separate things. Zooglea bacteria, such as Zoogloea ramigera, has cells that are rod-shaped, stains gram negative, and can resemble fingers or branches of a tree.
Under normal conditions it is a major contributor to floc formation in activated sludge. A problem begins when the sludge has an excess, or overabundance, of this zooglea bacteria, causing slime bulking. This type of bulking causes the activated sludge to have a slimy, or jelly-like, consistency and texture.
There can be several reasons for this to occur, including:. Nutrient deficiency is caused by an improper balance of BOD food to macronutrients nitrogen and phosphorus.
This deficiency may be observed if there is a severe degree of industrial contribution or too much phosphate is precipitated through primary treatment with addition of iron salts.
Fixing a slime bulking problem depends on the cause s. Remedies can range from adjusting the pH and adding missing macronutrients to wasting slowly, or adding in good mixed liquor from another plant. Each situation is different. Filamentous bulking is caused by the proliferation of bacteria filaments that outcompete floc forming bacteria for food and occurs due to bad settling and thickening of the solids. The reason behind the unstoppable filamentous growth is usually the hydrophobic surface of the bacteria, which leads to flotation of the sludge.
Since a certain amount of filamentous bacteria is useful in the activated sludge process , lack of it may cause floc with good settling abilities but leaving behind a turbid effluent. This is because filaments are the backbone of a floc structure and a base for larger floc. Some filaments catch small particles during the settling process, therefore, lowering the effluent turbidity.
Too many filaments will, on the other hand, cause bulking. There has to be a happy medium! Ideal balance of filamentous and floc-forming bacteria. Shifts in the sludge environment, such as changes in pH, DO, nutrients, etc, willoften cause a change in bacterial behavior.
To solve any problem in the treatment system you have to involve microscopic examination of the activated sludge. This will reveal whether the problem is caused by filaments or not. If it is caused by filaments, identification of the strain will provide the approach to take for the remedy. The table below shows the filament types of concern and what they can indicate about a system. The activated sludge process is widely used in treating wastewater because of the low investment, high treatment efficiency and strong adaptability.
Complete biodegradation in the aeration basin and good separation of mud and water in the secondary sedimentation tank are critical in the activated sludge process. The sedimentation performance of activated sludge is often a problem. There are two kinds of sludge bulking: 1 filamentous sludge bulking induced by the mass proliferation of filamentous bacteria and 2 viscous sludge bulking caused by high viscous substances produced by bacterial micelles.
Factors such as water temperature, dissolved oxygen DO , sludge retention time SRT , pH, influent quality, nutrient ratio and sludge loading are responsible for filamentous sludge bulking. The microbial community responsible for sludge bulking varies depending on the water quality and operational conditions.
For example, for bacterial communities, Microthrix proliferated at low sludge loading and low temperature, wheras Eikelboom type N induced bulking at high sludge loading and high temperature. For fungal communities, excessive propagation of Trichosporon caused bulking at low DO.
Identifying bulking filaments is not a simple task, like foaming filaments, which are discussed next. The majority of filaments causing problems in the system, cause bulking. Using stains to identify bulking filaments is not as helpful as it is with foaming filaments unless you are lucky and you happen to have one of the few gram positive bulking filaments: Nostocoida limicola or type The majority of bulking filaments are gram negative and Neisser negative as well.
Let's take a look at those few gram positive bulking filaments first. The one bulking filament that stains strongly gram positive is Nostocoida limicola and it's easy to identify. When stained, it resembles a purple beaded necklace. It's easy to identify without a stain using a wet mount because of its unique oval cells. Distinguishing between the three really doesn't matter because they all thrive under the same conditions.
This particular filament is seen more often in industrial treatment systems, though they are still common in municipal utilities as well. Nostocoida limicola is also Neisser positive as well as filament type They are very easy to differentiate from each other. Type resembles short, slightly curved sticks and can be difficult to see without the Neisser stain.
Type is often mistaken for a foamer because it can become trapped in the foam caused by Microthrix. Identifying filaments would be a lot easier if we only had these gram positive filaments to worry about. Unfortunately, most of the bulking filaments are gram negative, and are much harder to identify.
As if things couldn't be more complicated! Let's add another type of filament to the mix. There are three gram-variable filaments, which are not considered to be gram positive or negative, but exhibit characteristics of both! Filaments type , type and type all stain gram-variable. In most cases we will not be able to rely on the gram or Neisser stain reaction to help identify which filament is causing problems in the system. We have to dive a little deeper into the type of microbe giving us issues before we know how to treat it.
Some of the other unique characteristics that can help simplify the identification process include:. Sometimes cells are stacked inside a sheath, like a chain, and sometimes there is one continuous cell inside the sheath.
Cells may escape leaving an empty space in the sheath. As you examine the sample, look up and down the length of filaments looking for "missing cells" or empty spaces between cells. Of all the filaments that are commonly found in activated sludge, seven of them have a sheath, including:. Not all filaments that are sheathed have attached growth, but those that do resemble a "bottle brush". Of the most common sheathed filaments, only four of them are often seen with attached growth.
The sheathed filaments that have a "type and number" scientific name are the ones that are commonly seen with attached growth. This makes it a little easier to remember. It's important to note that these types of filaments can also grow in the treatment system without the attached growth. If they do have the attached growth, this usually means that conditions in the wastewater are allowing them to grow quite rapidly, making it more difficult to identify them.
You can narrow your options down to seven by identifying the sheath, but it will take additional observations to determine which of these is dominating in the treatment system. Type , type and type are often seen with heavy attached growth.
The attached growth is only sparsely attached to type , which is gram-variable and grows in bundles. So even if you don't have a microscope, if you have type with attached growth, you're probably going to see if pretty easily. Types and type are also gram-variable and have square-shaped cells, with type being the larger of the two. Looking at the size and shape of the individual cells will also help to identify the filaments.
Type has cells that look like round-ended rods or "link sausages". The oval shaped cells are stacked in a tight fitting sheath. Type is a very thin filament. Even with attached growth, it's easy to identify. Sphaerotilus natans, Thiothrix and H. They are very different from each other and easy to identify.
It is also the only filament that exhibits false-branching. Another characteristic that distinguishes Thiothrix from S. Unlike the others, H. It is so thin that it is very difficult to detect the sheath. This increase is limited because the increased return flow to the system hydraulically pushes more sludge from the clarifier, making effluent TSS losses worse.
Polymer and Coagulant Addition There are different types of chemicals which can be added to enhance activated sludge settling. They are usually added to the MLSS as it leaves the aeration basin or to the secondary clarifier center well.
Chlorination Chlorine and hydrogen peroxide successful temporary control method for filamentous growth. Chlorine is cheap and available on-site at most plants so it is widely used. Sufficient chlorine damages the filaments extending from the floc surface while leaving organisms within the floc largely untouched. If the bulking problem is not caused by filamentous bacteria, adding chlorine can worsen the bulking — for slime bulking or poor floc development It is crucial to properly dose the chlorine and add it frequently to activated sludge.
In normal amounts, they aid floc formation and help to catch small particles during settling yielding a less turbid effluent. They are also excellent BOD degraders. The filament characteristics will enable a trained person to identify the type of filament and the root causes that are associated with that particular filament.
Intracellular offspring attach to absorptive epithelial cells via their holdfast and induce condensed actin rearrangements underneath the point of attachment while displacing some of the neighboring microvilli structures Chase and Erlandsen, Before luminal attachment, the nucleoid region of the intracellular offspring appears condensed, suggesting reduced amounts of transcription.
However, when the intracellular offspring attaches to the host, the nucleoid region decondenses and allows for genomic transcription Chase and Erlandsen, Once the filament reaches its maximum length, a second round of symmetric division begins from the distal end to divide each original segment in half into secondary undifferentiated cells.
These secondary cells range in length from 1 to 1. After elongation, the filament will often separate from the holdfast segment and enter into the ileum. These secondary segments then undergo differentiation form a mother cell and a daughter cell Klaasen et al. Differentiation of these filaments appears to be more pronounced in the presence of slightly aerobic conditions, when oxygen concentrations range from 1 to 2. Once differentiated, the mother cell engulfs and houses the daughter cell, where the daughter cell undergoes division into two intracellular offspring Klaasen et al.
The intracellular offspring contained within the mother cell are then subject to two fates, dispersal from the filament or sporulation Davis and Savage, ; Klaasen et al. These released offspring are then allowed to colonize additional host tissue and undergo filamentation and differentiation, thus completing the SFB lifecycle Martin et al. When an unfavorable or hostile environment is presented, the two intracellular offspring produce a single spore coat that covers both of the cells.
Once coated with the layer of peptidoglycan, the spore matures into a complete endospore inside of the mother cell, and is released from the filament Martin et al. Once shed, the spores can be transmitted to another host via horizontal transmission Davis and Savage, Though it was previously proposed that the entire SFB lifecycle occurred while attached to the host tissue Chase and Erlandsen, , filaments containing intracellular offspring have not been observed in published TEM and SEM images of filaments attached to enterocytes Caselli et al.
Alternatively, filaments containing differentiated intracellular offspring only appeared as detached and free-floating, indicating that maturation and differentiation of segments occur independently from host tissues. These filaments are separated from the holdfast segment, which then penetrates the epithelium until it undergoes endocytosis, phagocytosis, or transcytosis Caselli et al. The ingestion of the holdfast segment presents a great number of bacterial antigens to antigen presenting cells and lymphocytes contained within the ileal epithelium Caselli et al.
In rare cases, a small number of segments may remain attached to the holdfast, but these segments often exhibit irregular morphologies and present enlarged intrasegmental junctions Caselli et al. These SFB genomes are highly similar but do contain several species-specific genes of unknown function that may be involved in the species-specificity of SFB colonization Prakash et al.
All sequenced SFB have displayed a small genome size of 1. SFB possess a highly reduced genome similar to their genetic relatives within the genus Clostridium Ericsson et al. The biosynthetic pathways of most amino acids, vitamins and cofactors such as B 1 , B 2 , and B 12 , pyridoxine, nicotinamide, pantothenate, and biotin are incomplete or absent altogether in SFB genomes Kuwahara et al. SFB are also unable to synthesize nucleotides independently; instead they utilize alternative pathways that rely on the uptake of nucleotide bases Prakash et al.
To obtain nucleotides, amino acids, and peptides from the environment, SFB genomes contains genes encoding two extracellular nucleases as well as a list of proteases and peptidases, 20 of which are membrane associated and 4 to 6 that are thought to be secreted Kuwahara et al.
In addition, SFB genomes contain numerous open reading frames ORFs thought to encode a large number of transporters and permeases for small molecules and ions such as amino acids, oligopeptide, dipeptides, manganese, zinc, iron, and phosphate compared to other organisms with small genomes Sczesnak et al.
A particularly strong requirement for iron uptake was noted by Sczesnak et al. SFB also have several ORFs for phosphotransferase systems predicted for uptake of sugars such as mannose, cellobiose, mannitol, and fructose Prakash et al. Finally, the SFB genomes contain genes for the non-oxidative pentose phosphate pathway and a complete glycolysis pathway to convert glucose to pyruvate, but are deficient for genes encoding almost all components of the Krebs cycle, which is required for aerobic respiration Prakash et al.
However, SFB can tolerate small concentrations of oxygen and counteract oxidative stress, as SFB genomes contain genes predicted for two catalases, a peroxidase rubrerythrin , and an arginase, which might limit nitric oxide production through catabolism of arginine Kuwahara et al.
These protective mechanisms are likely essential, given the replicative niche of SFB at the surface of the small intestinal epithelium where the oxygen tension is estimated to be around 1. There are several factors that have been discovered about SFB that explain their auxotrophic nature.
The genome of SFB isolated from a rat host Rat-YIT contains 28 putative genes predicted to encode for proteases and 53 for peptidases along with many other genes through to be involved with sporulation and germination Prakash et al.
Peroxidase and catalase genes were also found, which explains the potential for SFB to exist in microaerophilic environments Prakash et al. Metabolic features of Candidatus Arthromitus through inference of genome contents from annotated genomes through use of Rapid Annotation using Subsystem Technology Reiland, An extensive body of work has evidenced the presence of SFB in a large number of animal species, such as horses, cattle, pigs, turkeys, chickens and even humans Ericsson et al. In nearly all of the mammalian species studied for the presence of SFB, the bacteria selectively colonize the ileum of the host, with the exception of fish species that lack a well-defined ileum Ericsson et al.
Attempts to colonize an animal species with SFB from another species have been unsuccessful. When germ-free mice and rats were inoculated with ileal homogenates containing SFB from both species, animals became colonized with SFB from their own species, indicating that SFB are host-specific and host selective Tannock et al.
Host-specificity of SFB may be due to differences in the sequences of flagella genes fliC3 and flicC4 , which show greater variability than fliC1 and fliC2 Chen et al. For most animal species, holdfast cells have the capability to attach to goblet cells, M-cells, absorptive enterocytes, and cellular junctions of the ileal epithelium Meyerholtz et al. Species to species variance exists in the preferred cell of attachment.
In mice and horse, attachment occurs primarily to follicle-associated epithelial cells and mainly to absorptive ileal villi for rabbits, cattle and canines Pamp et al. SFB are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells, but do not invade the host Sanford, The exact mechanism of host specificity remains unclear, but it is believed that initial binding of the holdfast segment to the host epithelium serves as a ligand-receptor interaction, triggering a response from the host Kuwahara et al.
SFB binding elicits actin polymerization and condensation at the point of attachment Chase and Erlandsen, , suggesting a specific host response. Host specificity of SFB suggests that these bacteria have coevolved with their hosts to promote the commensal relationship that exists between the two species.
Though initially considered to be common commensal members of the host microbiota, recent research suggests that SFB serve an important role in modulating host microbiome and immunity. SFB are unique amongst intestinal commensals and symbionts because they penetrate the intestinal mucus layer and intimately associate with host cells without invading the host Chase and Erlandsen, ; Lee et al.
The holdfast binding of SFB to the ileal mucosa does not elicit a strong inflammatory response Klaasen et al. Several interactions between the host and SFB have been investigated, indicating almost entirely positive associations between the SFB and the host, with rare exceptions. Recent advances have identified different methods to culture SFB in vitro , accelerating research to study the interaction of SFB with different members of the intestinal microbiota Ericsson et al.
A large amount of research has demonstrated the indispensable role that SFB play in the maturation of the host gut immune barrier, inducing both innate and adaptive immune responses Ivanov et al. Immune modulation of SFB may extend beyond the intestinal tract in the blood, or to other mucosal barriers McAleer et al.
Mice colonized with SFB were more resistant to sepsis secondary to experimental cecal ligation and puncture injury Cabrera-Perez et al. Oral vancomycin treatment to mice diminished the number of intestinal Gram-positive bacteria including SFB , which negatively impacted anti-fungal Th17 immunity in the respiratory tract McAleer et al. These data suggest that the composition of intestinal microbiota, especially SFB, is vital for impacting immunity to bacterial and fungal pathogens beyond the intestinal tract.
Ivanov et al. IL is a cytokine that enhances production of antimicrobial peptides from intestinal epithelial cell and prevent bacterial pathogens from inducing attaching and effacing lesions Schupf et al. Virulent Shiga-toxin producing E. ILA and ILF additionally aid in regulating the activation and differentiation of host neutrophils, and stimulate the production of host-defense peptides Schnupf et al.
Thus, neutrophil recruitment may lessen ILA and chemokine production, and serve as a negative feedback loop to limit SFB colonization. The regulation of these immune-stimulatory compounds and cell types is essential in combatting intestinal colonization and infection from microorganisms.
Th17 cells provide colonization resistance to other pathogenic bacteria present at mucosal barriers, such as Escherichia coli in the intestinal tract Zheng et al.
In newborn or germ-free mice, the presence of Th17 cells in the lamina propria is rare, appearing only after colonization by microbes Gaboriau-Routhiau et al. The role of SFB in Th17 cell production was initially demonstrated when mice were inoculated with mouse, rat, and human microbiota containing bacterial spores similar to that of the genus Clostridium.
Only the experimental mice inoculated with a mouse-derived bacteria were shown to produce Th17 cells in response to colonization. Mice colonized with rat- and human-derived bacteria produced much less of a Th17 response when compared to the mouse-derived microbiome treatment, indicating host-specific bacteria such as SFB as the causative agent of the immune response Gaboriau-Routhiau et al.
This association was also confirmed when 16S rRNA sequencing was performed on the gut microbiome of mice presenting ileal Th17 cells, revealing the presence of SFB Ivanov et al. In experiments testing the reactivity of mouse lamina propria against a SFB expression library, two proteins of unknown function elicited a Th17 cell response Yang et al.
It was predicted that these unknown proteins may serve as cell surface proteins, potentially elucidating the role that SFB attachment may serve in stimulating host immunity Yang et al. Proteins from SFB, secreted or bacterial-associated, are believed to interact with host cells and modulate immunity include ADP-ribosyltransferases and a myosin-cross reactive antigen Pamp et al. The exact antigen presenting cell responsible for immune modulation by SBF is controversial, but it appears that SFB antigens presented to both intestinal macrophages Panea et al.
Because of the intimal relationship of SFB with intestinal epithelium, it is possible that metabolites from SFB may also impact the differentiation of Th17 cells. Intestinal macrophages, and not intestinal dendritic cells, appear to be vital for generating SFB-specific Th17 responses in the murine ileum Panea et al. The addition of the Th1-indcing bacterial pathogen Listeria monocytogenes failed to impact induction of Th17 cells in SFB colonized mice Yang et al.
SAA also stimulates intestinal antigen presenting cells to secrete IL, which assists in Th17 activation and survival Schnupf et al.
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