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9th European Waste Water Management Conference 12-13 October 2015, Manchester, UK

MICROSCOPY AND FILAMENT IDENTIFICATION AS A TOOL FOR TROUBLESHOOTING BIOLOGICAL TREATMENT PROCESSES

Morley, E.Aqua Enviro, UK

Corresponding Author Email. [email protected]

Abstract

Aqua Enviro have microscopically examined thousands of activated sludge samples in the last 15 years developing an overview of conditions across a wide range of plants. While Microthrix parvicella is still widespread in the municipal sector, increased sludge handling is leading to Type 021N becoming more common on some plants as return liquors become stronger and septicity is introduced to the head of the works. Industrial plants have a myriad of filaments depending on their process conditions and influent types. Legislative pressures are meaning that influents are typically becoming more concentrated and effluent limits tighter so there is more pressure on the effluent plants to perform to a high level. Understanding the health of the biomass is vital in maintaining the effluent quality and optimising expenditure.

Keywords

Activated Sludge, Biological Treatment, Bulking, Microscopy, Microthrix parvicella, Type 021N, Wastewater Treatment

Introduction Despite the long history of activated sludge there are still operational problems which occur on occasion. This is true for other biological treatment processes whether they are a new innovative design or a more traditional trickling filter. All the biological systems depend on maintaining a healthy and often varied consortium of bacteria and protozoa within the process which is not always easy to do.

A wide range of pressures apply within the municipal and industrial sectors. Many of these are the same across both types of wastewater treatment plants. The Environment Agency can be acting to ensure that new directives and regulations are met by municipal and industrial operators while the municipal operators can themselves be the regulator for trade effluent discharges from industry. There are financial pressures to reduce operating costs, which can include man power, energy and chemical dosing, and to minimise capital expenditures which might be delaying asset renewal or upgrades or having wait until the next financial year or even AMP cycle. Municipal plants are at the mercy of new developments, the impacts of climate change; such as dealing with rainfall and flooding on a more frequent basis, and on-site sludge handling issues. Sites could have effluents that are vastly different from original plant designs with industrial inputs being replaced by domestic sewage over the asset life. The same applies to industrial plants in which the water usage and the manufacturing process have changed considerably over since the plant was first constructed.

These are all likely to have an impact on the microbiome that is present in the treatment plant, as operating parameters such as sludge age and F/M ratios, as well as the chemical constituents of the effluent, change. The upshot of this could be poor settlement and foaming in an activated sludge plant and under or over growth of a biofilm in an attached growth process. The ultimate outcome could be consent limits being threatened due to solids carryover or incomplete treatment.

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Microscopic examination of the mixed liquor or biofilm can be used to identify the biotic causes of many operational issues. Once the microbiological reason has been established, actions can be taken to eliminate the underlying causes. These could be simple things like introducing or increasing balancing volume or increasing aeration capacity or operating outside the ‘normal’ ranges of operation, on the other hand large scale capital schemes for plant upgrades may be required to ensure that compliance is maintained.

Methodology for microscopic examination

Samples of biomass are collected from the effluent treatment plant; this may be mixed liquor or scrapings from a fixed film process such as a trickling filter or a SAF media. The samples should be examined soon after collection especially for high F/M, low sludge age plants.

The characteristics that are of interest are the floc size and structure; which could be rounded or irregularly shaped, open or diffuse and firmly or weakly held together, the protozoal and metazoal populations, filamentous bacteria (abundance and length), and the clarity of the bulk liquid (space between the flocs) which could be clear or contain individual bacterial cells. These initial characteristics would be determined on a magnification of 100x using a phase contrast microscope. Filamentous identification would be carried out on higher magnification 1000x, to a Type or species level. This may be achieved with the additional use of Neisser, Gram and Indian ink staining where appropriate.

Using these characteristics it is possible to assess the sludge age and the F/M, identify signs of insufficient oxygen and deficiencies of other nutrients as well as noting that shock conditions may have occurred. For example an oxygen deficiency may manifest itself with filament Type 021N or Thiothrix being the most dominant. Shocks might include rapid changes in influent pH and BOD concentration, presence of chemicals or detergents from spillages and changes in temperature. These could lead to flocs being shattered apart and the bulk liquid becoming containing more individual bacterial cells. The exact nature of any shocks can be later verified through site knowledge and operating data.

Other characteristics that might be considered during the analysis are the colour and odour of the sample. A healthy sample would be light to mid brown, although this will depend on the concentration of solids in the sample. Sometimes they can be an unusual colour, for example orange, but be completely typical under the microscope and they might change from one colour to another. A healthy sample will have an earthy aroma while an overloaded one might be more sulphurous in scent. The overall consistency of the sample could be worth noting, this could be watery, thick or stringy. The settlement characteristics might also be commented on; however settlement in small volumes is affected by the walls of the container much more than in clarifiers on site.

Photography of the sample can be used to retain the observations. This can be as simple as using a digital camera or phone down the eye pieces of the microscope or using a system built into the microscope. The pictures can provide evidence to compare to historic samples or information for different analysts completing the examination.

The frequency of analysis should be tailored to the site, for the most part weekly or monthly samples are sufficient for a regular check on the health of the plant. If the analysis is being used to monitor process changes or commissioning of new equipment then this frequency might be increased to daily or a few times a week for the duration of time that works are being undertaken.




Records can be maintained of the observations and if these deteriorate then on site plant alterations can be made, i.e. if filament Type 021N becomes more prevalent then aeration levels may need to be increased. If what is typical for the plant is known, then it can be possible to identify changes from normal and relate them back to operating conditions at the time.

A further use of microscopy can be when biomass is being used to reseed a plant. At this time it would be beneficial to ensure that the mixed liquor to be imported is of good condition and is exhibiting the healthy flocs, filaments, and protozoa prior to delivery to site or if it is being transferred from one tank to another.

Microscopy in practice

Over the last 15 years a wide range of samples have been analysed at Aqua Enviro from both municipal treatment plants and a varied assortment of industrial clients.

Some, particularly for industry, have been undertaken on a regular basis, for example weekly or monthly. The results from which are often fairly predictable and allow a good background to the site to be obtained so that when there are changes can be easily identified and solved before there is a significant impact on treatment.

Others have been when a site is experiencing problems such as foaming, poor settlement and incomplete treatment. Municipal samples tend to have problems with either foaming (which is becoming less common as plant designs are changing) or poor settlement, which unfortunately seems to be increasing in frequency. Foaming tends to be related to the presence of Microthrix parvicella and this is a seasonal problem. While it is almost always present in municipal samples throughout the year, under winter operating conditions it can proliferate producing chocolate mousse foam due the hydrophobic nature of the filament. The second filament causing foaming issues is Nocardia which has a branched structure and this tends to be favoured by longer sludge ages.

For industrial plants, it tends to be settlement that is affected. The most common reasons for this are the presence of filaments such as Type 021N, Type 0041 and Nostocoida limicola in excessive quantities. These either create mesh like structures which reduce the settling velocity of the flocs, or they can prevent the consolidation of smaller flocs into larger ones. Poor floc structure can also be key with small and diffuse flocs being slow to settle in comparison to large, compact flocs. Municipal treatment plants are experiencing more frequent settlement problems often relating to sites which have sludge handling and resultant return liquors. These are affecting the oxygen concentrations leading to filaments which thrive in low oxygen being able to proliferate such as Type 021N or Thiothrix.

A textbook good mixed liquor would be to have rounded compact flocs, with a range of different filaments present along with a range of protozoa and metazoa. However all plants are different and in industry in particular some plants will have poor flocs, a single species of filament present and no protozoa, and for that particular site the final effluent will be of a high quality, with good settlement. If the same characteristics were present at another site it would represent a plant which was failing consent and loosing is sludge blanket. It is about getting the ‘right’ mixed liquor for the plant as opposed to getting ‘textbook’ good mixed liquor.




Table 1: Common filaments, characteristics and causative conditions

Filament Key characteristics CausesType 021N Long filaments with cells

having a barrel like shapeOverloading, low oxygen conditions

Microthrix parvicella Spaghetti tangles like appearance

Municipal effluents, cooler temperatures

Nocardia spp Branched structure Presence of fats oils and greases and longer sludge ages

Type 0041 Square cells often with attached growth

Slight nutrient deficiency, frequently seen in paper mill effluent treatment plants.

Municipal treatment – Filament/Settlement issues

Due to settlement issues on a site with sludge handling facilities, a mixed liquor sample was analysed and revealed that filament Type 021N was present. This particular filament can rapidly proliferate when dissolved oxygen levels are low and when septic conditions are found, as it can metabolise hydrogen sulphide directly. Type 021N can lead to poor settlement and at its worst highly viscous mixed liquor, which in turn affects efficient oxygen transfer further exacerbating the low oxygen conditions.

Figure 1: Bacterial flocs with filament Type 021N extending into the bulk liquid (x100 magnification)

Return liquors from sludge handling can be a rich source of the septicity that this filament species can thrive under. If there is insufficient aeration capacity to drive off the septicity and to treat the BOD of the return liquors the activated sludge can rapidly deteriorate. Sludge samples from outlying sites can be days or in the worst case weeks old when they arrive at sludge handling sites.

In this case there was a mechanical problem with the on-site sludge handling facilities meaning that the sludge was being stored on site and when it was processed it was ‘old’.

Recommendations provided to the client to eliminate this filament was to process the sludge when it was fresher, have additional aeration available, or as a last resort chemical dosing could be utilised to




reduce the length of the filaments, improving settlement. In this circumstance sodium hypochlorite was used to physically damage the filaments and improve the settlement in the short term while the mechanical faults in the sludge handling system were rectified as there was no quick fix solution.

Figure 2: Filament Type 021N that has been damaged by sodium hypochlorite dosing (x1000 magnification)

Municipal treatment – Foaming issues

A mixed liquor sample was analysed from a municipal treatment works. The works was experiencing problems with thick foam being present on the aeration lanes. The sample contained a large amount of Nocardia filament; this filament has a distinctive branched appearance. It is has a positive response to Gram staining and as such is hydrophobic and can lead to foaming and also bulking of the sludge as it prevents compact flocs from forming. Nocardia is typically found in municipal plants with a long sludge age.




Figure 3: Nocardia filament in the bulk liquid and at the floc edges (x1000 magnification)

When speaking to the client, it was revealed that there had been a problem on site where contractors had cracked the waste activated sludge pipe, meaning that there was no sludge surplussing taking place and the sludge age was very long. This linked the foaming problem to the absence of wasting rather than issues with the influent characteristics.

A further sample analysed after repairs had been carried to the pipe and the sludge age had reduced showed that the Nocardia had disappeared from the mixed liquor.

Industrial treatment - Fungal growth

Industrial plants have a wide range of problems which can include overloading, toxicity, nutrient deficiencies and foaming. The causes of these can be varied and tend not to be well documented in the literature due to the unique nature of each industry and treatment technologies in use.

A recent sample of biomass from a SAF contained a thick mat of biological material which was growing on the filter media. Under microscopic examination this was identified as a fungal growth. Fungi tend to grow at pH <6 and when there is a high proportion of easily biodegradable material in the influent, it can outcompete bacteria in cold conditions. Fungi can transfer materials internally more efficiently than bacteria due to the morphology and therefore are particularly able to exploit low oxygen and nutrient conditions.




Figure 4: Fungal mycelium amongst the bacterial floc material (100x magnification)

The observation of the fungus in the sample meant that the site was able to check on the upstream pH balancing which was not performing correctly; this was rectified thus removing the conditions that had allowed the fungus to proliferate. The plant still contained a large amount of fungal matter which needed to be removed from the filter bed. Advice was given on how best to achieve this which included backwashing at the maximal rate to break the fungal filaments and then to wash through into the backflush system for removal for offsite disposal. The problem was able to be remedied without removal of the media or use of chemical dosing.

Industrial treatment - Monocolonies

Samples from an industrial client showed a shift in the populations of filaments and protozoa from those normally observed. There was an increase in mono colonies present, that is, the flocs of bacteria consisted of identical organisms and there were also unusual crystals observed.

Monocolonies, in the frequency present, are a relatively uncommon occurrence and macro and micronutrient analysis were carried out as deficiencies are the most likely cause. It was found that there was a very low level of phosphate within the flocs despite the final effluent containing excess phosphate. It was also found that the pH of the mixed liquor was high and this was leading the precipitation of the phosphate into the crystals.




Figure 5: Flocs with occasional crystals present (x100 magnification)

The client was able to make some changes around the effluent plant, including with the chemical use to carry out the pH correction and they also increased the nutrient dose. Once the pH reduced and the phosphate concentration of the flocs returned to typical levels there was an improvement in the flocs so that they consisted of a consortium of different bacteria.

Conclusion

Activated sludge and other biological treatment systems have problems with the treatment process which can threaten compliance with consents. Microscopic examination can be used to evaluate the condition of activated sludge or the biomass in these systems.

Microscopy can be used to develop a background record of observations under good operating conditions can allow changes in the biomass to be noted and actions taken before a treatment problem develops.

Microscopy can also be very useful when issues with treatment occur, enabling identification of the process conditions that are having a detrimental effect on the final effluent. The information gathered can then be used to ensure that the final effluent consistently achieves the required consent limits using process changes such as varying the F/M and sludge ages and small scale engineered solutions, ahead of chemical dosing and large scale capital schemes which may fall under the scrutiny of the regulatory authorities. Finally it can be used following any changes to operating to confirm that conditions such as overloading or low oxygen conditions have been rectified.



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