Filamentous bacteria Eickelboom morphotype 0914 is one of the most common filament types observed in all activated sludge treatment processes. Morphological features to recognize this filament are a cell diameter of approximately 1 µm, and squarish cell shape. 

These filament types can occur within the flocs, extending from the flocs or dispersed in solution. In comparison to many other filamentous bacteria morphotypes, type 0914 tends to be relatively short in length, rarely extending beyond 200 µm.

Recognized growth conditions for filament type 0914 are elevated concentrations of low molecular weight organic acids and sulfide. Organic acids may be naturally occurring in many industrial wastes and septage or can be formed in areas of septicity/fermentation (equalization basins, lift stations, etc.) Organic acid (volatile acid) concentrations of greater than 100 mg/L are a recognized cause of type 0914 bulking. Sulfide is formed when sulfate is reduced under anoxic conditions.

Identification challenges

From a practical standpoint, in terms of filamentous bacteria identification, type 0914 may present several challenges. Among these challenges are the morphological features of the filament based on the type of substrate utilized, growth rates, and other conditions, in addition to the genetic diversity within the filament type. As an example it is fairly common for type 0914 cells to be difficult to distinguish between square and rectangular in shape. Also, type 0914 filaments may be present with or without attached growth and can be found over a wide range of sludge ages and food-to-microorganism (F/M) conditions.

Type 0914 filaments sometimes (but not always) include intracellular PHB granules or intracellular sulfur granules (sulfur granules, if present, are square to rectangular in shape). To further challenge the identification of filamentous morphotype 0914, filament type 0675 and 0803 possess basically identical morpholgoical traits. Dr. Michael Richard, an author of the Third Edition Manual on the Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems (2004) — who has over 40 years or experience as a pioneer in wastewater microbiology — has not recognized 0803 as a filament type (previously classified as a low F/M filament) for many years. Also, in various text references by Per Neilson and colleagues, type 0914 and type 0803 have been combined as the same morphotype. In the training handbook that we developed — which is currently in its first edition — type 0914 is referenced as type 0914/0803 with recognized causes of elevated organic acids and/or sulfide.

Where further challenge lies in the identification of filament type 0914/0803 is that filament type 0675 may either closely resemble type 0914 (especially if attached growth is present), or in some instances is believed to actually be the same organism as type 0675. Where this can cause confusion is that filament type 0675 is associated with higher sludge retention time values or lower F/M ratio, which are separate recognized causes from type 0914. 

In real practice, a thorough microscopy evaluation by a trained professional is needed to make these distinctions based on the big-picture diagnosis of the sample, which is obtained from the other filament types and other indicator organism phylotypes present, along with their rank, abundance, and growth causes, as well as a general familiarity of which filaments and microbe types typically occur together. All said, identification is more of an art than a science, and “birdwatching” techniques for identification tend to be the most successful for filaments such as type 0914. 

Morphology

From a genetic diversity standpoint, filament type 0914 may fall into either the Proteobacteria phylum or the Chloroflexi phylum. In general, Proteobacteria filaments tend to grow quickly and outcompete traditional floc forming bacteria at high concentrations of soluble carbonaceous material/organic acids while Chloroflexi tend to favor higher SRT, with many having the ability to store substrate under anaerobic conditions for later usage further down the aeration basin. 

Additionally, other Chloroflexi phylum filaments may be polysaccharide degraders (responsible for endogneous activity). From the Proteobacteria phylum, the Acidovorax genus has been recognized by Sevior labs as being able to possess the type 0914 morphotype, and we have confirmed this in our studies thus far. Depending on the database (naming and classification of microbes is an ongoing and ever changing process), Caldilinea (not recognized by MiDAS field guide) has been confirmed by Sevior labs as a Chloroflexi phylum member able to possess type 0914 morphology (Caldinea has also correlated well within our studies). 

In the MiDAS database, Candidatus Sarcinithrix (a genus with four currently unnamed separate species) is among Chloroflexi genera that have been correlated with type 0914 morphology. Sevior labs recognizes Villigracilis, Defluviifilum and Trichobacter genus as able to possess type 0914 morphology, however, in our studies, we have presently not seen these correlations, likely because database libraries and naming can vary depending what is being used for 16S rRNA sequencing). We have seen correlations with many taxonomical identification numbers that may represent currently unnamed genus or are the furthest down the from a taxometry standpoint that microbes can be recognized with current technology. These include candidates Aster Bio taxonomical identification numbers 292628, 292625, 475963, 475964, 2303486, 2303491, 1382929, 1382930, 200795 and 26216. Interestingly some of these may also be the lowest common denominator for other Flexibacter filamentous morphotypes, indicating that type 0914 is either very closely related or there is significant further research needed on the DNA end at this time to differentiate these at the lower and more specific taxometry levels.

From a microscopy standpoint, it is important to remember that we are looking at the big picture of what is occurring and utilizing this information in addition to process control variables as a tool to make decisions to optimize treatment, keep things as is, or  troubleshoot when these needs may arise. As an example, if we are not certain whether to call a filament type 0914 or type 0675, looking around at what else is present may be needed. If other filament types associated with organic acids/sulfide are present, then chances are that it is type 0914. If other filaments associated with high SRT and/or low F/M are present, then chances are that it is type 0675. This is highly important because these are two differently recognized growth causes and the first step of any decision making is highly dependent on a correct diagnosis.

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About the author: Ryan Hennessy is the principal scientist at Ryan Hennessy Wastewater Microbiology. He was trained and mentored by Dr. Michael Richard for over 10 years in wastewater microbiology, and serves as a microbiology services consultant. Hennessy is a licensed wastewater treatment and municipal waterworks operator in the state of Wisconsin and fills in as needed for operations at several facilities. He can be reached at ryan@rhwastewatermicrobiology.com.