Updated Handbook for Wastewater Microscopy Applications and Filamentous Morphotype ID Methods Acknowledging Recent Genetic Findi

Updated Handbook for Wastewater Microscopy Applications and Filamentous Morphotype ID Methods Acknowledging recent Genetic Findings Ryan Hennessy, Midwest Contract Operations Inc. 2020

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

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.

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.

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.

De-Emphasis on PHB granules

 All filamentous bacteria have the ability to store PHB (Richard, 2020). It remains true that PHB granules are more commonly observed in certain filaments, however using PHB granules for identification can cause confusion. o PHB granules are most often observed in Sphaerotilus, type 1701, type 021N, Thiothrix, Nostocoida morphotypes, type 0914, and Beggiatoa.

Explanations for New Filamentous Morphotype Listings Based on recent genetic findings we now recognize that the bacterial diversity of filamentous bacteria in municipal and industrial facilities is much more extensive than previously recognized in earlier literature. The goal of microscopy in wastewater from a practical standpoint is not based on precise identification to the species level, but rather to determine a general understanding of conditions occurring within the plant, floc structure and filamentous bacteria impact on the flocs, determining general health of the system, monitoring and diagnosing dispersed growth, and determining at which points to make operational changes. Use of microscopic evaluation in combination with process control data can help make informed decisions on changes in sludge age, mixed liquor concentrations, return activated sludge rates, step feed configurations, internal recycling rates, adjustments to dissolved oxygen concentrations, modifications to cycles in SBR (sequencing batch reactor) processes, the use of chlorine and other disinfectants to selectively kill filamentous bacteria, and more. A well-trained lab tech can complete an accurate microscopy assessment in less than two hours, providing valuable and essential information that cannot currently be provided in other testing methods. Simply put, the microscope and its value when used properly for wastewater process control cannot be replaced.

Genetic testing (specifically 16S rRNA or shotgun sequencing) has value along with microscopy for certain applications, such as quantification of various genus with recognized causes (this literature covers genetic findings within various filamentous morphotypes), quantification of nitrifying bacteria, quantification of PAOs (polyphosphate accumulating organisms), and obtaining a more detailed understanding of the diversity and changes that occur within the system. Based on these understandings from a practical standpoint, simplification of morphotypes for microscopy purposes and identification techniques can help to simplify things, as correlating the “big picture” and causes is our ultimate goal. The following changes are based on practicality purposes with respect to traditional names to not change the context of previous reporting significantly.

The Changes

 Sphaerotilus natans has been shortened to Sphaerotilus as there are three known species within the Sphaerotilus genus (Sphaerotilus natans is only one of these species). Also Leptothrix morphology cannot be easily distinguished from Sphaerotilus so is included in this morphotype.

 Haliscomenobacter hydrossis has been shortened to Haliscomenobacter as there are three known species within the Haliscomenobacter genus. While the causes for Haliscomenobacter

morphotype remain the same, this morphotype has been observed in the Bacteroides and Chloroflexi phylum (Chloroflexi likely have several different genus and species with this morphotype). The cause for Haliscomenobacter is most likely to be low dissolved oxygen, but in certain instances may include low phosphorous availability.

 Thiothrix I and II shortened to Thiothrix as there are seven known species within the Thiothrix genus. The previous descriptions were based on diameter. However, from a practical standpoint, it is more important to associate these descriptions together as to not “dilute” the abundance associated with these causes (organic acids and/or sulfide). Leucothrix is also associated with having Thiothrix, as well as type 021N morphotypes/ causes. Further, Thiothrix and type 021N are no longer commonly classified with low nitrogen availability. This remains possible, however the listing of low nitrogen availability as a presumed cause of Thiothrix has led to high amounts of confusion during diagnosis. o Anthrone testing is recommended to determine nutrient availability for bacteria.

 Nostocoida limicola I and Nostocoida limicola II are combined as a single morphotype with a larger range of diameter recognized simply as Nostocoida or Nostocoida (I and II). The Nostocoida morphotype is recognized under the Actinobacteria, Firmicutes, and Planctomycetales phylum in a wide range of different genus types and over 30 different species. Based on the genetic diversity of this morphotype from a practical standpoint, it is more important to judge the abundance of these organisms together to help correlate the “big picture.” Note that professionals with extensive experience in wastewater microscopy can recognize certain previous Nostocoida II that often occur at higher sludge retention times, however this cannot be easily described or taught (birdwatching). As a result, the best evidence available suggests it is preferred to combine Nostocoida limicola I and II. Nostocoida Limicola III remains separate as this morphotype behaves differently than the other Nostocoida morphotypes. It often manifests at elevated organic acids and at high F/M (food to microorganism ratio) at the beginning of biological treatment (typically the first 15-30 minutes in the aeration basin) and/or at low phosphorous availability. Several different genus/species fall under the Nostocoida limicola III morphotype, within the Proteobacteria Alpha subclass, Firmicutes, and the Plantomycetales phylum that cannot be easily distinguished from morphological traits.

 Nocardioforms/Nocardia are broadened to the Actinomycetes morphotype. Actinomycetes can grow on organic acids and/or on fats, oils, and grease. Gordonia is a genus within this morphotype that has 12 known species. Nocardioides are a separate genus with 14 known species. Also sharing this morphotype are Mycobacterium (ten species- some filamentous), the Actinomyces genus (two species/ variable filamentous), and other genus such as Dietzia and Fondicola that can also possess this morphotype if growing in filamentous form. Generally, in municipal processes and food processing wastewater treatment plants, oil and grease are suspected as the cause for Actinomycetes. In oil refineries, textile applications, and paper mills,

Actinomycetes typically grow on organic acids. These changes were made to prevent instances of oil and grease being diagnosed incorrectly in certain applications as the cause of filaments.

 Type 0803 is no longer recognized as a low F/M filament. This morphotype has been combined with type 0914 and is now referenced as simply type 0914 or type 0914/0803 as FISH probes have found these genetically similar. There is high diversity of genus and species possessing this morphotype including the Sarcinithrix genus (four species with morphology trait) within the Chloroflexi phylum. The recognized cause for type 0914/0803 is organic acids and/or sulfide.

 Type 0675 and type 0041 have been combined as type 0675/0041, as these morphotypes are listed under the Chloroflexi and Saccharibacteria phylum. Due to difficulty growing these filaments in pure culture, little genetic information has been discovered at the time of this writing. However, it is suspected that the species within these morphotypes are broad. As with the Nostocoida morphotypes, the associated causes (low F/M filament and/or high sludge retention times) are more practical and combing these morphotypes helps prevent “dilution” or minor changes of naming based on diameter differences.

 Microthrix parvicella has been shortened to Microthrix as Microthrix is a genus with four recognized species (including Microthrix parvicella and Microthrix calida). The overall causes remain the same (oleic acid-fats, oils, and grease). However, it is incorrect to assume all Microthrix morphotypes are Microthrix parvicella species.

 As morphotypes reference “groupings” based on physical characteristics rather than specific genus or species these are non-italicized in print. For example, Microthrix parvicella is included in the Microthrix morphotype and the Microthrix genus.

Updated Morphotype Table

Common Filamentous Morphotypes and Their Associated Growth Conditions*

Cause Filamentous Morphotype Low Dissolved Oxygen Sphaerotilus Haliscomenobacter Type 1701

Elevated Organic Acids Thiothrix Nostocoida (I and II) Nostocoida limicola III Type 021N Type 0914/0803 Type 0211 Type 0961 Type 0581 Type 0092 Type 0411 Actinomycetes Beggiatoa

Sulfide Type 0914/0803 Thiothrix Type 021N Beggiatoa

High Sludge Retention Time Type 0675/0041 Type 1851

Fats, Oils, Grease Type 1863 Actinomycetes Microthrix

Low pH Fungi

Low Phosphorus Haliscomenobacter, Sphaerotilus, Nostocoida limicola III

*Courtesy of Dr. Michael Richard, 2020

Currently Known Genetic Diversity within common Filamentous Morphotypes*

Note: Table continued onto following pages.1

Morphotype Phylum Taxometry Additional Notes Sphaerotilus Proteobacteria-Beta Three known species within Leptothrix discophora Subclass genus. Sphaerotilus natans, may also possess Sphaerotilus montanus, and Sphaerotilus an unnamed. morphotype. Type 1701 Proteobacteria- Beta Suspected to belong to N/A Subclass Curvibacter genus (2 unnamed species) and likely Sphaerotilus genus. Haliscomenobacter Bacteroidetes Haliscomenobacter genus. 3 N/A recognized species are Haliscomenobacter hydrossis, and two unnamed. Haliscomenobacter Chloroflexi Various genus and species High diversity of genus (ongoing research). and species suspected. Thiothrix Proteobacteria- There are six known species N/A Gamma Subclass within the Thiothrix genus. Thiothrix lacustris, Thiothrix unzii, Thiothrix fructosivorans, Thiothrix eikelboomii, and two unnamed. Thiothrix Proteobacteria- Leucothrix mucor Nielsen, P., & Daims, H. Gamma Subclass (2009). Nostocoida Actinobacteria Tetrasphaera genus has 19 Not all Tetrasphaera limicola (I and II) known species. Many of are filamentous. All which may possess species are capable of Nostocoida morphotype. biological phosphorus removal. Nostocoida Firmicutes Streptococcus genus has nine Lactococcus genus has limicola (I and II) species with Nostocoida variable filamentous morphology. capabilities/ not all Trichoccocus genus has three Lactococcus are known species that can filamentous. possess Nostocoida morphology. Lactococcus has two species within genus of which can possess Nostocoida morphology. Nostocoida Planctomycetales Ongoing research. N/A limicola (I and II)

1 Table references Nierychlo et al. (2019). For more information see: https://www.midasfieldguide.org/guide/search

Morphotype Phylum Taxometry Additional Notes Nostocoida Proteobacteria- Alysiophaera genus has 4 Other possible genus limicola III Alpha Subclass species which are unnamed in and species: MIDAS field guide. Alysiomicrobium bavaricum? Monilibacter batavus? Combothrix italica? Nielsen, P., & Daims, H. (2009). Nostocoida Planctomycetales Isosphaeraceae family Ongoing research. limicola III (Isosphaera) and Nostocoida MIDAS field guide genus. Little known info. recognizes one species in the Nostocoida genus. Type 021N Proteobacteria- Meganema genus has one N/A Alpha Subclass known species. Meganea perideoroes. Type 021N Proteobacteria- Four species may possess Leucothrix species may Gamma Subclass type 021N morphotype. T. possess type 021N eikelboomii, T. disciformis, T. morphotype. Nielsen, flexilis, Leucothrix mucor P., & Daims, H. (2009). Type 0914/0803 Chloroflexi Sarcinithrix genus has four Also possible species unnamed species with type within Anaerolinea 0914/0803 morphotype. genus? Caldilineacea (unclassified)? Type 0211 Unknown N/A N/A Type 0961 Unknown N/A N/A Type 0411 Unknown Unknown Species within Runella genus of Bacteroidetes phylum? Actinomycetes Actinobacteria All filaments possess N/A Actinomycetes morphotype. Actinomycetes genus has 2 species with variable filamentous traits. Gordonia genus has 12 species. Mycobacterium genus has ten species (variable filamentous). Nocardioides genus has 14 species (some are single cell). Beggiatoa Proteobacteria- Beggiatoa genus. Individual N/A Gamma Class species within genus are ongoing research. Type 1863 Proteobacteria- Acinetobacter genus has 12 Not all species within Gamma Class known species (variable Acinetobacter genus filamentous) possess type 1863 morphology.

Morphotype Phylum Taxometry Additional Notes Type 1863 Bacteroidetes? Chryseobacterium genus? This Ongoing research. genus has six known species which may potentially possess type 1863 morphotype. Microthrix Actinobacteria Four species recognized N/A within genus including Microthrix parvicella, Microthrix calida, and two unnamed species. Type 0092 Chloroflexi Promineofilum genus has 10 Branchthrix is an species all of which possess alternate name for type 0092 morphotype. morphotype in some literature. Type 1851 Chloroflexi Seven species within the N/A Kouleothrix genus (all unnamed) possess type 1851 morphotype Type 0675/0041 Chloroflexi Unknown/ little available Ongoing research. literature. Type 0675/0041 Saccharibacteria Unknown/ little available Ongoing research. literature. Type 0581 Unknown N/A N/A *Table references Nierychlo et al. (2019).

Morphology Traits (Visual Key at 1000x Oil Immersion) Diameter (µm measurement): Diameter is a critical component to filamentous morphotype analysis. Measuring the diameter can quickly reduce the list of possibilities. A reticle is needed in the eyepiece of the microscope for diameter measurement. When using the diagnostic table, it is recommended to use 1.0 µm as a “measuring stick,” separating filaments that are <1 µm as skinny and those that are >1µm as wider. See the figure below where the red line represents the diameter.

Septa (Cross Walls Visible):

Cross walls not visible:

Sulfur Granules:

Cell Shape/Size: size= diameter measure by (individual) cell length

Cell Size (diameter by length)

Cell Size (cell diameter by cell length)

Sheath

Cell Shapes

Sausage:

Rectangle:

Discs, Ovals:

Elongated Rods:

Squares:

Irregular/ Barrels:

Oval Rods:

Other Morphology Traits True Branching:

False Branching:

Transparent:

Bundles:

“Pins in a Pin Cushion”:

PHB Granules:

Attached Growth:

Filamentous Morphotype Identification Table (Key)

Note: Table continued onto next page

Identification Table for Common Filamentous Morphotypes observed in Activated Sludge

Filament Notes Filament Septa/ Cell Can store Cell Shape/Size Sheath y/n Morphotype Diameter Walls sulfide (Size= diameter (typical observed y/n (potential by cell length) values) for sulfur granules) Sphaerotilus Commonly has 1.2-2.26 µm Yes No Sausage shaped. Yes false branching. 1.6 x 2.5 Type 1701 Thinner filament 0.8- 1.0 µm Yes- Usually. No Sausage shaped. Yes with sausage 1.0 x 1.5 shaped cells (sometimes cells are hard to see). Occasionally has attached growth. Haliscomenobacter 0.5 um diameter 0.5 µm No No Usually can’t see yes (skinny) individual cells. Often “pins in a pin cushion”. Sheath often difficult to see. Type 021N Large filament 1.6-2.5 µm Yes Yes Barrels, No that typically rectangles, extends from the discoid. flocs or bridges 1.6 X 2.5 flocs together. Irregular cell shape. Thiothrix Rectangular cell 0.8- 2.8 µm Yes Yes Rectangles. Yes shape. Typically 0.8-1.4 x 1.0-3.0 extends from flocs. Type 0914/0803 Occasionally has 1.0-1.2 µm Yes Yes Square. Yes square sulfur 1.0 x 1.0 granules. Beggiatoa Typically motile 2.0- 4.0 µm Sometimes. Yes Rectangles. No and usually 2.0-4.0 x 6.0 -8.0 contains either sulfur granules or PHB granules. Nostocoida “Hockey pucks”. 0.8-2.0 µm Yes No Discs, ovals. No limicola I and II 0.8 x 1.0-1.5 Nostocoida Individual discoid 1.7-2.5 µm Yes No Discs, Ovals No limicola III cells. 2.0 x 1.5 Type 0411 “Elongated rods/ 0.8-1.2 µm Yes No Sausage, Rods. No long sausage 0.8-1.2 x 2.0-5.0 links”.

Filament Notes Filament Septa/ Cell Can store Cell Shape/Size Sheath y/n Morphotype Diameter Walls sulfide (Size= diameter (typical observed y/n (potential by cell length) values) for sulfur granules) Type 0961 Transparent. 1.0-1.4 µm Yes No Rectangles. No (not a Often has “cuff”. 1.0-1.4 x 2.0-4.0 true sheath). Type 0092 Neisser positive. 0.6-1.0 µm Yes- No Rectangles. No (not a Need Neisser Sometimes 0.8-1.0 x 1.0 true stain to ID. Often but not sheath). difficult to see at always. phase contrast. Type 0675/0041 “Grainy” 1.0-2.2 µm Yes No Squares. Yes appearance. 1.0-2.0 x 2.0-3.0 Often has attached growth. Type 1851 Often forms 0.8 µm- 1 µm Often difficult No Rectangles. Yes “bundles/twisted to view septa. ropes”. Often has attached growth. Microthrix Gram positive. 0.5-0.8µm Very rarely No ------No Often has Neisser see septa Typically can’t positive granules. see individual Typically “coiled”. cells Actinomycetes “True branching”. 0.8-1.2µm Yes- most No Variable. No Almost all species species. 1.0 x 1.0- 2.0 in wastewater stain gram positive when healthy. Type 1863 Usually dispersed. 0.8-1.0 µm Yes No Oval rods. No “Chain of cells” 0.8-1.0 x 1.0-1.5 appearance. Type 0211 Grows dispersed. 0.3-0.4 µm Yes No Oval rods. No “Thin and 0.4 x 0.6 crooked”. Type 0581 Looks like 0.5-0.8 µm No No ______No Microthrix but gram positive. Fungi Very Large/True 5.0-10.0 µm Sometimes. No Variable. No (but branching. does often have thick cellulose cell wall).

Filamentous Morphotype Identification Bench Sheet  The bench sheet below is compatible with the identification table/key.

(For use at 1000x oil immersion phase contrast and 1000x brightfield for Gram and Neisser stain).

Recommended Bench Sheet for lab use*

*Courtesy of Dr. Michael Richard, 2020

Picture Guide of Filamentous Morphotypes

Microscope setup at MCO lab, 2020.

See pictures on following pages:

Sphaerotilus

Type 1701

Haliscomenobacter

Thiothrix

Nostocoida limicola (I and II)

Nostocoida limicola III

Type 021N

Type 0914/ 0803

Type 0211

Type 0961

Type 0411

Actinomycetes

Beggiatoa

Type 1863

Microthrix

Type 0092

Type 1851

Type 0675/0041

Type 0581

Ranking the Abundance of Filamentous Bacteria The following system is our recommended way of quantifying filamentous bacteria abundance. *

Procedure: Count the total number of filaments visible within the floc and extending the floc for a minimum of 20 flocs. For abundance take the average value per floc observed.

Numerical Value Rank Description

0 None No filaments observed.

1 Few Filaments observed in only occasional flocs. Filaments observed in approximately half the flocs. In 2 Some flocs that have filaments, filamentous abundance is low. 3 Common Average floc viewed contains 1-5 filaments per floc.

4 Very Common Average floc viewed contains 5-20 filaments per floc.

5 Abundant Average flow viewed contains >20 filaments per floc. Excessive Filamentous bacteria predominate and their abundance 6 (“Up the Wazoo”) is higher than flocculated bacteria abundance. *Ranking system courtesy of Dr. Michael Richard, 2020

 A filamentous morphotype or indicator organism must be of common or greater abundance for its respective cause (s) to be significant to the overall findings.  Generally changes to the SVI (sludge volume index) begin to be noticed once filamentous bacteria ranking approaches very common abundance.  The floc structure is vital to the impact filamentous bacteria will have on the SVI. o Stronger/firmer flocs have more weight and are able to accommodate higher filamentous bacteria abundance with less corresponding impact on settling rates.  Common abundance of filamentous bacteria are believed to often help to provide a supporting “backbone” structure for the floc.  In many instances in which the sludge settles too quickly (low SVI) there is little forgiveness for any dispersed growth and turbidity may result in the supernatant (liquor above the settled sludge) which has the potential to increase effluent total suspended solids concentrations.  State Point Analysis is a useful tool to help determine at what SVI values, MLSS concentrations, and flow rates clarifier failure/solids washout may occur. o For state point analysis assistance please email [email protected]

Gram Stain and Neisser Staining Procedures

Courtesy of Dr. Michael Richard, 2020. Slide Prep (for both stains)

1) At the edge of each slide label each slide as desired (example: G for Gram stain and N for Neisser stain). Note: Frosted edges on slides can be easier for labeling purposes. 2) Using a small transfer pipette place one small drop of mixed liquor sample on the edge of each slide. 3) Using the side of the pipette evenly spread the sample to cover approximately 70% of the slide (do not spread sample over where you wrote the label for each). 4) Allow sample to air-dry. This typically takes 10-15 minutes.

______Gram Stain

1) Stain the dried smear one minute with gram solution No. 1; rinse with water.

2) Stain the slide one minute with gram solution No. 2; pour off stain.

3) Flood the slide with No. 3 (alcohol) until no more blue stain is removed (typically about 10 seconds).

a. This is a critical step as over or under-decolorizing can change results.

4) Stain the slide for one minute with No. 4; wash with water; blot dry.

Blue/Purple= Positive Red= Negative

______

Neisser Stain

1) Stain the dried smear 30 seconds with No.1 reagent; wash with water.

2) Stain the slide one minute with No. 2; wash; blot dry.

Purple= Positive Brown= Negative

______

Notes: Both slides are examined at 1000x brightfield oil immersion (typically “0” on phase ring). Stain reagents can be purchased at https://nclabs.com/

PAOs (Polyphosphate Accumulating Organisms)

PAOs belong to a group of bacterial that facilitate the removal of phosphorus from wastewater by accumulating it within their cells as polyphosphate in the process of enhanced biological phosphorous removal. Genetic Diversity within PAOs*

Genus Phylum Species Notes Accumulibacter Proteobacteria 14 Typically one of the primary genus responsible for enhanced biological phosphorus removal. Also, some species can switch between PAOs and GAOs. Tetrasphaera Actinobacteria 19 Some species also may be involved in denitrification. Some (but not all) may possess Nostocoida limicola morphotype but do not appear to only rarely cause bulking issues. Obscuribacter Cyanobacteria 2 Unknown/ ongoing research. Corynebacterium Actinobacteria 1 Unknown/ ongoing research. Halomonas Proteobacteria 3 Typically small straight or curved rod shaped cells. Some species can grow on filamentous form. Dechloromonas Proteobacteria 14 Rods/ coccobacilli. Sometimes form microcolonies. Species within genus vary upon PAO capabilities. *Table references Nierychlo et al. (2019)

GAOs (Glycogen Accumulating Organisms)

GAOs are believed to compete with PAOs in the process of enhanced biological phosphorus removal. Genetic Diversity within GAOs*

Genus Phylum Species Notes Accumulibacter Proteobacteria 14 Many species are variable GAOs (can also be PAOs). Competibacter Proteobacteria 26 N/A Contendobacter Proteobacteria 6 N/A Defluviicoccus Proteobacteria 12 Some can possess Nostocoida limicola morphotype in filamentous form. Micropruina Actinobacteria 5 Cells appear as clusters or pairs with cocci shape. Propionivibrio Proteobacteria 7 N/A *Table references Nierychlo et al. (2019)

Nitrifying Bacteria

The process of nitrification involves oxidizing ammonia to nitrite and then to nitrate. Genetic Diversity and Functions of Nitrifying Bacteria*

Genus Phylum Species Function Brocadia Planctomycetes 3 AOB and NOB Nitrosomonas Proteobacteria 20 AOB Nitrosospira Proteobacteria 1 AOB Nitrobacter Proteobacteria 2 NOB Nitrospira Nitrospirae 6 NOB Nitrotoga Proteobacteria 3 NOV AOB= Ammonia Oxidizing Bacteria NOB= Nitrite Oxidizing Bacteria *Table references Nierychlo et al. (2019)

 Nitrosomonas and Nitrobacter are nitrifying bacteria that can be viewed at phase contrast 1000x oil immersion magnification. (Richard, 2020).

Additional Phenotypes, Traits, and Associated Causes*

Associated Cause/ Growth Condition Phenotype/Observational Trait

Organic Acids Spirillum

Spirochaetes

Flexibacter

Zoogloea bacteria

Tetrads

C1 Compounds (Methanol) Hyphomicrobium

Elevated Zoogloea bacteria abundance Elevated polysaccharide upon reverse India ink stain

Low Nutrient Availability Elevated polysaccharide upon reverse India ink stain

*Table courtesy of Dr. Michael Richard (2020).

Known Genetic Diversity within Additional Phenotypes*

Phenotype Genus Phylum Notes

Flexibacter Flavobacterium? Bacteroidetes Flexibacter has been reclassified several times. There are 27 species in the Flavobacterium genus, some of which we presume to have this phenotype.

Spirochaetes >20 recognized Spirochaetes Ongoing research occurring in this area. genus apply Genus include Leptonema, Leptospira, Salinispira, Spirochaeta 2, Treponema, Treponema 2, Turneriella, and many unnamed genus

Spirillum Unknown Proteobacteria Spirillum has been reclassified many Beta Subclass? times. It is generally accepted that this phenotype belongs to the family Spirillaceae.

Tetrads Various Various? Tetrads are a growth formation of 4 coccoid shaped bacteria with likely high applicable genetic diversity.

Hyphomicrobium Hyphomicrobium Proteobacteria 31 known species exist within the Hyphomicrobium genus possessing this phenotype.

Zoogloea Zoogloea Proteobacteria There are 6 species recognized within the Zoogloea genus including Zoogloea ramigera, Zoogloea oleivorans, Zoogloea caeni, Zoogloea oryzae, and 2 unnamed species.

*Table content references Nierychlo et al. (2019).

Picture Guide of Additional Phenotypes and Traits 1000x Flexibacter

Spirochaetes

Spirillum

Tetrads

Hyphomicrobium

Zoogloea

Judging Polysaccharide (Reverse India ink Stain)

Procedure: 1) Mix one drop of India ink and one drop of activated sludge sample on a slide. 2) Place cover slip over slide and blot lightly with paper towel to remove excess ink. 3) Examine at 100x, 200x, or 400x using phase contrast. 4) If polysaccharide is normal the ink penetrates the flocs almost completely. If polysaccharide is elevated large and clear areas will be visible. Normal Polysaccharide

Elevated Polysaccharide

Appendices

Midwest Contract Operations Midwest Contract Operations, Inc. (MCO) is a professional Contract Operations Firm specializing in management, operation, and maintenance of water systems, wastewater systems, and public works departments.

MCO was formed in 1987 to provide the necessary “hands on” technical support required today to meet the economic and technological challenges facing municipalities, utilities, and industries.

Contact info:

 https://mco-us.com/services/  [email protected]  (920) 751-4299

Author Ryan Hennessy is a Microscopy and Operations Specialist for Midwest Contract Operations (MCO). In addition to contract operations, MCO provides hands on training, troubleshooting, and microbial sample analysis. Our clients for microscopy services include a wide range of municipalities, engineering and consulting firms, chemical vendors, and industrial wastewater treatment plants throughout (but not limited to) the United States and Canada.

Contact info:

 [email protected]  (920) 573-2820  https://mco-us.com/wastewater-microbiology/

Additional Acknowledgements  All pictures courtesy of Ryan Hennessy, MCO 2020  Thanks to my sister Rose Hennessy for her assistance in editing this document.  Extra special thank you to Dr. Richard for all the continued years of mentorship and training and best wishes in retirement. We are honored to continue the legacy. o Dr. Richard has a fleet of refurbished phase contrast microscopes for sale . For microscope sales please contact Dr. Richard at [email protected]

References

Nielsen, P.H., & Daims, H. (2009). FISH handbook for biological wastewater treatment. IWA publishing.

Nielsen, P. H., Kragelund, C., Seviour, R. J., & Nielsen, J. L. (2009). Identity and ecophysiology of filamentous bacteria in activated sludge. FEMS Microbiology Reviews, 33(6), 969–998. doi: 10.1111/j.1574-6976.2009.00186.x

Nierychlo, M., Andersen, K.S., Xu, Y., Green, N., Albertsen, M., Dueholm, M.S., Nielsen, P.H. (2019). Species-level microbiome composition of activated sludge - introducing MiDAS 3 ecosystem- specific reference database and taxonomy. BioRxiv.

Jenkins, D., Richard, M.G., & Daigger, G.T. (2003). Manual on the causes and control of activated sludge bulking, foaming, and other solids separation problems, 3rd Edition. CRC Press.

Seviour, R., & Nielsen, P. H. (2010). Microbial ecology of activated sludge. London: IWA Publishing.