Activated Sludge Troubleshooting Chart

The Microscope
SSVI<90 SSVI>120 No SSVI

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Examining Activated Sludge

1) On receipt of sample, spread a drop evenly over each of two microscope slides and allow them to air dry, whilst undertaking steps 2 and 3. 2) Spread a drop of sample over a third slide, cover with a cover slip and gently press it down. 3) Examine under high power and phase contrast, and note: the size and shape of the flocs presence of protozoa and an estimate of the predominant types Then at a low power note: presence of filamentous bacteria and an estimate of their abundance (on a scale of 0 to 4) 4) After examining the wet mount, the air dried slides are used for filament identification. One of them is stained with the Gram stain, the other using the Neisser stain.

The Test

SSVI
Symptoms

A clean, well adjusted microscope is essential for identification of sludge microfauna. When using the microscope remember to: ensure the condenser is setup correctly keep the lenses clean (always use lens paper - ethanol is good for cleaning very dirty lenses) never scratch the lens on the slide, watch from the side when making large adjustments watch from the side when changing from 10X to 40X objectives.

Protozoa Identification
Protozoa can comprise up to 5% of the mass of a healthy activated sludge, with as many as 20,000 organisms/ml. There are over 200 species encountered and so identification to species level is rarely carried out! They are divided up into four easily identifiable groups for the purpose of diagnosing problems with an activated sludge plant. Flagellated protozoa Small (5-20m) and very fast moving protozoa with one or more whip-like flagella. Because they move so fast they are difficult to see clearly. Free swimming ciliated protozoa Much larger (20-100m) and slower swimming than the flagellates. With short, hair-like cilia which aid their mobility. Many of these protozoa can be observed crawling over the surface of sludge flocs. Stalked ciliates Very easily recognised bell-shaped protozoa, attached by a stalk to the sludge floc. Each stalk can have one, two or numerous bells attached to each stalk. Rotifers These are very large (100-500m), slow-moving and easily recognised. They are not actually protozoa, but metazoa.

Filament Identification
Correct identification of the prevalent filament types requires a wet mount which is examined under phase contrast, an air-dried Gram stain and an air dried Neisser stain. A limited number of characteristics are needed to identify filaments, and most of them can be seen with the wet mount at a magnification of 1,000X. These characteristics are: Branching (present or absent, if present true or false) Filament shape (straight, smooth-curve, bent, chains, coiled, mycelial) Attached growth (present or absent) Sheath (present or absent) Cross walls (present or absent) Shape of cells (square, rectangular, barrel, sausage shaped) Size (length and width of cell in m) Sulphur deposits In addition to the above, the staining characteristics of the filaments are determined from the slides which received the Gram stain and the Neisser stain, which are both examined under direct illumination (bright field). These characteristics are: Gram +ve (organisms stain a blue colour) Gram -ve (organisms stain a red colour; most filaments are gram -ve) Neisser -ve (whole filament stains light brown) Neisser +ve (whole filament stains blue) Neisser +ve granules (filament stains light brown, with dark blue granules clearly visible)

Good settling sludge, many protozoa in mixed liquor, especially stalked ciliates, and free-swimming protozoa with some rotifers.

Poor settling sludge, many filamentous bacteria, dispersed floc with few stalked protozoa but abundant flagellates and free-swimming protozoa. Unable to control sludge blanket in clarifier.

No discernible settlement, highly dispersed floc, few or no protozoa - mainly flagellates.

Vorticella

Carchesium

Paramecium

Opercularia

(sulphur granules)

Thiothrix

Nocardia
(unstained)

(+ve purple/blue; -ve yellow/brown)

Neisser Stain

Filament Shape

Straight

Smooth Curve

Causes
Ideal operating conditions. Filamentous sludge bulking or foaming. May be due to: low dissolved oxygen (DO), nutrient deficiency, inappropriate F/M, septicity. Toxic material in influent, organic shock loading, operating F/M too high, chronic nutrient deficiency.
Coiled Mycelial

Aspidisca

Euplotes

Chilodonella

(unstained)

O21N

M. parvicella
(gram stained)

Gram Stain
(+ve and -ve)

Crosswalls Present

Square

Rectangular

Cures
Continue this operating regime. Optimise operation of secondary sedimentation tank (use the WRc nomograph) check DO, nutrients and sulphide levels. Undertake toxicity evaluation, monitor influent loading, check nutrient levels.

Barrel

Presence of Sheath

Flagellated Protozoa

Suctoria

Rotifer

Nematode

S.natans
(unstained)

H. hydrosis
(unstained)

M. parvicella
(unstained) Present Absent

Attached Growth

Loading Rates
Is your activated sludge plant operating well? If a treatment plant receives adequate organic material for the biomass present in the reactor (i.e. it has the correct F/M ratio), with enough oxygen, and no toxic substances present, then a healthy sludge will develop. If the F/M ratio is too high, or too low, then the result will be sub-optimal effluent treatment. This is illustrated below.
Organic Loading Rate (F/M) Too Low Ideal Predominant Protozoa Stalked ciliates and rotifers, with some nematode worms present. A large number of protozoa and a wide range of species. Dominated by free-swimming and stalked ciliates. Large number of flagellates and small, free-swimming ciliates.
Absent Present

Yara Industrial offers a range of products and dosing controllers for biological effluent treatment. The SciSol and Vitamax ranges offer a source of balanced nutrients and trace metals; Nutriox will prevent the odour and treatability problems caused by hydrogen sulphide. Our customers are supported by a network of field-based technical service staff.

Regular monitoring of protozoa

Changes in protozoal populations can indicate a wide range of operating problems such as: toxicity, shock loading, inadequate nutrients and oxygen deficit. However to interpret these it is important to have baseline conditions regarding your own plant. The figure opposite is typical of an activated sludge plant - construct one for your own plant as follows: 1) Dilute the sample 1:1 with tap water and prepare a wet mount. A Lund cell allows sufficient depth of liquid for protozoal mobility, and can be calibrated to obtain a quantitative assessment of numbers. 2) Count five fields of view in a vertical plane. 3) Count protozoa in the order: flagellates, free-swimming protozoa, rotifers, stalked ciliates. 4) Record each group as numbers/litre mixed liquor and also note the plant sludge age, loading rate and effluent BOD. Plot them graphically and display the results. 5) Carry out the test at least three times each week (it takes only 20 minutes from receipt of sample). 100 90 80 70 60 50 40 30 20 10 0
Free-swimming ciliates - Flagellated protozoa -| | | | | 0 1 2 Stalked ciliates

Naming filaments
Filaments are identified and named using a dichotomous key, and those produced by Eikelboom or Jenkins are commonly employed. However in the UK a limited range of filament types are routinely encountered, and with experience these are quickly recognised. They are: Nocardia, Type 021N, Type 0041, Microthrix parvicella, Sphaerotilus natans and Haliscomenobacter hydrosis. Their key characteristics are summarised opposite: Filament type Gram stain Nocardia M. parvicella S. natans H. hydrosis 021N 0041 + + + Neisser stain
Filament Granule

Shape & size (m) Branched 1.0 x 1.0 - 2.0 Irregular coils 0.6 x 100 -400 Rounded rods 1.4 x 2.0 Straight and rigid 0.5 x 20 - 100 Barrel to ovoid 1.0 - 2.0 x 1.5 - 2.0 Straight, smooth curve or bents 1.4 x 1.5 - 2.0

Cross walls + + +

Sheath + + +

+ + -, + -, +

Rotifers

Too High

4 6 8 10 16 18 20 24 Sludge age (d) Toxicity increasing Loading Rate increasing

This chart was prepared by Dr Nigel Horan and the photomicrographs supplied by Dr Louise Hornsby (School of Civil Engineering, University of Leeds).

Yara Industrial, Immingham, N. E. Lincolnshire, DN40 2NS Tel: 01469 554711 Email: yarauk.info@yara.com

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