Updated Handbook for Wastewater Microscopy Applications and Filamentous Morphotype ID Methods Acknowledging recent Genetic Findings Ryan Hennessy, Midwest Contract Operations Inc. 2020 ©2020 Midwest Contract Operations, All Rights Reserved. Table of Contents Content ......................................................................................................................................................... 3 Prelude ...................................................................................................................................................... 3 Terminology .............................................................................................................................................. 4 Explanations for Updated Training Methods ............................................................................................ 5 Explanations for New Filamentous Morphotype Listings ......................................................................... 6 Updated Morphotype Table ..................................................................................................................... 9 Currently Known Genetic Diversity within common Filamentous Morphotypes* ................................. 10 Morphology Traits (Visual Key at 1000x Oil Immersion) ........................................................................ 13 Other Morphology Traits ........................................................................................................................ 19 Filamentous Morphotype Identification Table (Key) .............................................................................. 23 Filamentous Morphotype Identification Bench Sheet ............................................................................ 25 Recommended Bench Sheet for lab use* ............................................................................................... 26 Picture Guide of Filamentous Morphotypes ............................................................................................... 27 Ranking the Abundance of Filamentous Bacteria ....................................................................................... 38 Gram Stain and Neisser Staining Procedures ............................................................................................. 39 PAOs (Polyphosphate Accumulating Organisms) ....................................................................................... 40 GAOs (Glycogen Accumulating Organisms) ................................................................................................ 41 Nitrifying Bacteria ....................................................................................................................................... 42 Additional Phenotypes, Traits, and Associated Causes* ............................................................................ 43 Known Genetic Diversity within Additional Phenotypes* .......................................................................... 44 Picture Guide of Additional Phenotypes and Traits 1000x ......................................................................... 45 Judging Polysaccharide (Reverse India ink Stain) ....................................................................................... 48 Appendices .................................................................................................................................................. 49 Midwest Contract Operations ................................................................................................................ 49 Author ..................................................................................................................................................... 49 Additional Acknowledgements ............................................................................................................... 49 References .................................................................................................................................................. 50 2 Content Prelude As a student of Dr. Michael Richard, I am extremely grateful and fortunate for his continued assistance and mentorship over the previous 10 years and counting in addition to his high amount of input on this document. As Dr. Richard has retired, I have personally evaluated, and continue to evaluate, a high volume of wastewater biomass samples from municipalities and industries throughout North America. Hands-on training requests often arise from various facilities to assist clients to optimize microscopy in- house, in order to make process control adjustments. Drawing upon my teaching experiences, along with minor adjustments of previous methods, updated information has become available and is included below. Updates include, but are not limited to: New filamentous bacteria morphotype worksheet (1000x oil immersion). Updated list of filamentous morphotypes and associated cause (s.) Elimination of the dichotomous key/ replaced by an identification table that can be used together with the updated filamentous bacteria morphotype identification worksheet. De-emphasis on staining reactions in many instances. De-emphasis on previous identification traits such as filament length, filament location (in floc out of floc etc.), and filament shape (smoothly curved vs. straight etc.) Increased “short cuts” and notes to help reach filament identification without as many steps. Personal observations and notes from training and evaluating a high amount of samples. Inclusion of some ongoing genetic research in this area. 3 Terminology • F/M: Food to Microorganism Ratio • SRT: Sludge Retention Time (often interchangeable with “sludge age”) • SVI: Sludge Volume Index • SBR: Sequencing Batch Reactor • MLSS: Mixed Liquor Suspended Solids • DO: Dissolved Oxygen • OUR: Oxygen Uptake Rate • Low molecular weight organic acids: Low molecular weight organic acids may be naturally occurring in many industrial wastes and septage or may be formed in areas of fermentation (including lift stations, collection systems, primary clarifiers, sludge handling return side-streams etc.) 100 mg/L of total volatile acids are a recognized cause of filamentous bulking (Jenkins et al., 2003). • Morphology: The relationship between organisms and their characteristics. • PHB: A storage mechanism for many bacteria. Often these bacteria include visible granules. • Phylum: A principal taxonomic category that ranks above class and below kingdom. • Genus: A principal taxonomic category that ranks above species and below family, and is denoted by a capitalized Latin name. • Species: A group of living organisms consisting of similar individuals capable of exchanging genes or interbreeding. The species is the principal natural taxonomic unit, ranking below a genus and denoted by a Latin binomial, e.g. Homo sapiens. • Taxonomic hierarchy: 1) Kingdom. 2) Phylum. 3) Class. 4) Order. 5) Genus. 6) Species. 4 Explanations for Updated Training Methods Elimination of the dichotomous key/ De-emphasis on staining for most morphotype identifications It is very easy to get on the “wrong path” as the dichotomous key was highly dependent on staining reactions. o Staining reactions can often be attributed to chemistry of the wastewater; often in industrial processes the staining reactions can be highly variable. o Staining reactions are highly dependent on technique of the lab tech and improper decolorizing can easily lead to inaccuracies. o Due to the high genetic diversity within the recognized morphotypes it is likely many genus with the same morphotype have different staining characteristics. Why Gram and Neisser stain still remain essential Identification of Microthrix morphotype must be gram positive (or can be confused with morphotype 0581). Often staining helps to confirm Actinomycetes and Microthrix abundance as sometimes abundance can be underestimated (or missed) at only phase contrast magnification. Actinomycetes morphotypes in wastewater stain gram positive when healthy. To positively ID morphotype type 0092 Neisser positive staining reaction is needed. Polyphosphate Accumulating Organisms (PAOs) stain deeply Neisser positive and their abundance can be estimated upon staining. De-emphasis on Filament Length and Location of Filaments While this is relevant for many morphotypes there are many exceptions to these guidelines and the visibility of cell septa, sheath, cell shape and diameter take priority for identification. Depending on growth conditions most filaments can grow dispersed in solution if they are growing fast enough. De-Emphasis on Filament Shape Filamentous morphotypes can be variable as to their shape (straight vs. smoothly curved, etc.) o Based on our experience with classroom training this often causes confusion. The visibility of cell septa, sheath, cell shape, and diameter take priority for identification. De-Emphasis on Attached Growth Presence/Absence Almost all filamentous morphotypes can incur attached growth if they are present in the system long enough for bacteria to grow attached to them. It remains true that high SRT/low F/M filaments more often contain attached growth, however these can easily be confused with other filaments with incidental attached growth (for example attached growth is very common with some Thiothrix morphotypes and the previous manual has negative attached growth traits for Thiothrix creating high potential for mis-identification. 5 De-Emphasis on PHB granules All filamentous