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Example Filament ID

Activated Sludge Microbiology Evaluation

Paper Mill
Midwest USA
July 14, 2009

General Summary

The samples were submitted by the environmental engeneer of the paper mill for evaluation of microscopic health of the system for background information on the sequencing batch reactors. The samples were collected on July 14th and shipped over night to the EBS laboratory. Analyses were performed on July 15, 2009. This report is based on our examination of the sample and discussions with the engeneer. This is the first sample from this system that we have seen in a while. Therefore, our comments will be somewhat generic in nature. According to the engeneer, the system is running well and the analyses are for background information to be used to help troubleshoot any future problems that the plant may have. Settling and loading have been good for a while and the biomass appears healthy.

Floc Structure, Settling and Water Clarity

Floc was variable in size, ranging from medium to large dense floc in both samples. The samples settled well leaving behind a fairly clear supernatant. The MLSS for the samples was 4300 mg/L for the North SBR and 4530 mg/L for the South SBR. At these MLSS values the SVI calculated to 42 and 43 mLs/g, respectively, which indicates a quick settling rate. Filament abundance was a 2.5 (some) on the 0 – 6 scale with all floc containing filaments within the floc and extending out into the bulk solution as well as some filaments that were free in solution. The filaments had little to no effect on the floc structure. There was no interfloc bridging by the filaments and they did not cause open floc structure. Therefore the sludge settling remains reasonable. The samples had a fairly clear supernatant after 30 minutes of settling. The supernatant TSS was 128 mg/L for the North SBR and 126 mg/L for the South SBR due primarily to the pin floc left behind by the quick-settling sludge. The summary of the microscopic examination information for both samples is shown in Table 1. Table 2 contains the analytical testing data. The photos below provide examples of the various characteristics observed.

In addition to the floc and filaments noted, several other significant observations were made. The system contained a small amount of tetrads and Zoogloea. Tetrads are clusters of four cells commonly found in wastewater systems that are not completely understood by microbiologists and wastewater treatment specialists yet. Zoogloea are a specific type of bacteria that produce a collection of extracellular polymer strands composed of polysaccharides. This makes them “sticky” so that they form floc. They can be good for settling in normal amounts, but can hinder settling if they become excessive. These samples had normal amounts of Zoogloea, which did not inhibit the settling of the sample. The system also had a fair amount of bacteria that contained sulfur granules. This indicates some septicity upstream that produces sulfides that the bacteria can incorporate and utilize in respiration. This could be coming from the primary clarifier, the EQ tank, or in the mill itself. Photos of the sulfur containing bacteria are included below.

North SBR, Phase Contrast, 100X and 400X, respectively. Note dense floc structure.
South SBR
South SBR, Phase Contrast, 100X and 400X, respectively. Note dense floc structure.
South SBR
South SBR, Phase Contrast, 1000X, showing tetrads.
North and South SBR
North and South SBR, Phase Contrast, 1000X, showing sulfur containing bacteria.

Table 1: Summery of microscopic observations

North SBR South SBR
Floc Structure Compact and dense with no filament effect on floc structure Compact and dense with no filament effect on floc structure
Dispersed Bacteria (0-3) 1 1
Pin Floc (0-3) 0 0
Filament Rating (1-6) 2.5 2.5
India Ink Stain for Polysaccharides (1-3) 2 2
Zoogloea Abundance (0-3) 1.5 1.5

Table 2: Analytical testing data

North SBR South SBR
pH 7.39 7.32
NH3 as N (mg/L) 0.36 0.27
oPO4 as P (mg/L) 1.56 0.60
MLSS/TSS (mg/L) 5540 5800
MLVSS (mg/L) 4300 4530
% MLVSS (%) 77.6 78.1
30 minute settling (mLs) 233 250
SVI (mLs/g) 42 43
Supernatant TSS (mg/L) 128 126
DOUR (mg/L/hr) 18.8 19.2
SOUR (mg/L/hr/g) 81 77
sCOD (mg/L) 335 328

Filament Type and Abundance

Filament abundance was some to common. The predominant filament types were Beggiatoa, Thiothrix Type II, and H. hydrossis. These three species indicate low DO, septicity, and the presence of H2S in a system. Thiothrix II can also indicate organic acids, nitrogen deficiency, and complete mix according to the Manual on the Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems, 3rd edition, by David Jenkins, Michael Richard, and Glen Daigger. This relationship is also noted in Wastewater Biology: The Microlife, 2nd edition, published by The Water Environment Federation. Table 3 below is a summary of the causes associated with each of the filamentous species identified in your sample.

Table 3: Filamentous Bacteria Causes

Filament ID Causes
Filament Rank Identification Low DO Complete Mix Nutrient Deficiency Low Molecular Weight Organic Acids H2S
1 Beggiatoa X
2 Thiothrix II X X X X
3 H. hydrossis X X
Thiothrix II and Beggiatoa
North SBR, Phase Contrast, 1000X, showing Thiothrix II and Beggiatoa (with sulfur granules) filaments.
H. hydrossis and Beggiatoa
South SBR, Phase Contrast, 1000X showing H. hydrossis and Beggiatoa filaments along with tetrads.
Beggiatoa and Thiothrix II
North SBR, Gram Stain, 1000X, showing Beggiatoa and Thiothrix II filaments.
Beggiatoa and H. hydrossis
South SBR, Gram Stain, 1000X, showing Beggiatoa and H. hydrossis filaments.
North and South SBR, Neisser Stain, 1000X, showing Beggiatoa filaments.
H. hydrossis and Thiothrix II
South SBR, Neisser Stain, 1000X, showing H. hydrossis and Thiothrix II filaments.

India Ink Stain and Polysaccharide Abundance

The India Ink stain is an excellent tool for estimating the amount of extracellular polysaccharides in the biomass. This is useful in diagnosing various problems, including nutrient deficiency. The India Ink stain for this sample was normal with fair floc penetration by the ink. The white areas seen in the photos below are areas where the Zoogloea colonies are present in higher numbers. The Zoogloea were not numerous enough to hinder settling.

North SBR, India Ink Stain, 100X, showing areas of elevated polysaccharides due to Zoogloea.
India Ink Stain
South SBR, India Ink Stain, 100X, showing areas of elevated polysaccharides due to Zoogloea.

Higher Life Forms

The higher life forms observed in these samples were numerous and diverse in types. We saw a few flagellates, a free-swimming ciliate, and lots of stalked ciliates, rotifers. We also saw a few oligochaete worms, also called bristle worms. The type and number of higher life forms present in this sample indicate a very mature sludge that has low loading, no recent upsets, and good BOD/COD reduction efficiencies. A summary of the higher life forms observed is in Table 4 below along with a few pictures taken from your samples.

Table 4: Higher Life Forms

North SBR South SBR
Flagellates 0 3
Free Swimming Ciliates 1 0
Stalked Ciliates 32 27
Rotifers 9 6
Maturity Index 3.2 3.0
flagellate and a stalked ciliate
Phase Contrast, 100X and 400X, showing a flagellate and a stalked ciliate.
Phase Contrast, 400X and 100X, showing two types of rotifers.
Oligochaete (bristle) worm
Phase Contrast, 1000X and 100X, showing rotifer and an Oligochaete (bristle) worm.

Conclusions and Recommendations:

The pH was neutral on both samples. Phosphorus residuals were sufficient but ammonia residuals were low. MLSS was a bit higher that normal for a system of this type. However, the samples settled very well and left fairly clear supernatants that had relatively low COD. The microscopic evaluation revealed a very heathy, mature sludge with dense, heavy floc and a good diversity of higher life forms. Filamentous bacteria were at a low rating and did not hinder the settling of the sample at all. Zoogloea were present, but not in sufficient numbers to cause bulking problems in the sludge. Overall, the system looks very good at this point.

Continue to monitor the nutrients and biomass to ensure that the bacteria remain healthy and have all that is needed to degrade the BOD present in the system and to enable them to handle an upset condition. Also, continuing to send regular samples like these will enable us to better troubleshoot your system when upsets occur. This will reduce the amount of time needed to get the system back in shape so that you continue your environmental regulatory compliance.

Keep an eye out for septicity upstream of the SBRs. This will be important in controlling filamentous bacteria when settling problems occur. The types of filaments and the sulfur containing bacteria observed can indicate upstream septicity.

While microscopic evaluation and microbiology optimization are key components to successfully addressing activated sludge performance issues, there are operational and monitoring strategies that are equally important. Please continue to learn from the system and make adjustments as needed to maximize the efficiency of the system. EBS has extensive experience in auditing and troubleshooting industrial activated sludge systems, as well as developing and delivering operator training programs. Please let us know if we can assist you in any of these endeavors.