17 Sep 2021

By: Pascalle Vermeulen, Mario Pronk, Edward van Dijk

Nereda® Biowatch is a service available for plant performance monitoring. In a Biowatch analysis, we take a look at Nereda sludge through the lens of a microscope. The visual structures in sludge samples can tell us more about the process performance of the reactor. In this blog, we aim to tell you about the use of microscopy in sewage treatment, introduce some theory on biofilms, and explain how we combine these insights in Nereda® Biowatch.

Microscopy in wastewater treatment systems

First of all, let’s talk about method. Scientists have been studying bacteria through microscopes since Antonie van Leeuwenhoek first observed them in 1674. Fittingly enough for the topic of this blog, he was studying a drop of lake water at the time. His notes on these experiments have become quite famous:

"I now saw very plainly that these were little eels, or worms, lying all huddled up together and wriggling just as if you saw, with the naked eye, a whole tubful of little eels and water, with the eels squirming among one another; and the whole water seemed to be alive with these multifarious animalcules.

This was for me, among all the marvels that I have discovered in nature, the most marvelous of all; and I must say, for my part, that no more pleasant sight has every yet come before my eyes that these many thousand of living creatures seen all alive in a little drop of water, moving among one another, each several creature having its own proper motion.”[1]

Since van Leeuwenhoek spotted his little animalcules, many improvements have been made in both microscope technology and in the visual study of microbes. However, this technology was only extensively reported on as useful for wastewater treatment systems around 1975, resulting in a manual for microscopic sludge investigation published in 1983[2] (followed by an updated and expanded version in 2000[3]) by professor Eikelboom. He reported that the quality of flocculent sludge could be studied visually, by examining which type of microbes are present in and around the floc.

This type of analysis has proved very useful for analyzing process performance and stability of conventional activated sludge. The presence of microbes with certain types of morphology (visual shape and form) are indicative for specific problems and solutions. 

To see individual bacteria, which have a diameter of 1-10 micrometer – a hundred to a thousand times smaller than a human hair - a light microscope with a zoom of 100-1000x is usually used. To view bacteria at such high magnifications, a very small drop of sludge (about 0.005 mL) is pressed between two glass slides[4]. Additional techniques can be used to help identify the bacteria, such as chemical staining protocols such as Gram or Neisser staining, or even fluorescent genetic probes. These chemicals color only certain types of microbe. Some images of stained species with different methods (from the Eikelboom (2000) book) are shown below. The captions show the type of staining and the magnification of the image.  

For Nereda® systems, this type of microscopy analysis is much less useful. This is because the bacteria in our treatment plants grow in granules that are much larger and more dense than activated sludge flocs: they can grow to sizes of up to 5 millimeters. If we tried to flatten a granule beneath a glass slide to examine the individual microbes, they would not be distinguishable from one another, but all pressed together. The only microbes we could study with this method would be those growing suspended between the granules (which do not accurately represent the whole system) and microbes in small, diluted samples of crushed granules (which also give a skewed image of the system).

Therefore, for Nereda® Biowatch we use a different method. Instead of a light microscope, we use a stereozoom microscope. We apply lower magnification, only 8-50x, and use 3D samples, not flattened ones. The added benefit is that we can use larger sample sizes, about 5-10 mL instead of 0.005 mL, which makes the analysis more representative. The figure below shows the difference in analysis sample and presents typical images for both analyses.[5]

Instead of inspecting individual microbes and inferring what their presence or absence tells us about performance, we look at the larger structures, the biofilms and flocs as a whole. Biowatch Nereda® is based on the understanding that how and when carbon conversions take place influences the morphology (structure and shape) of the macro structures, the biofilms.

Biofilms

So, what is a biofilm? A biofilm is an aggregate of cells growing together in a matrix of extracellular polymers (usually sugar polymers and proteins). An extremely simplified schematic of this is shown below. Nereda® granules consist of bacteria and other microbes growing together in such a biofilm.

The microbes growing in this matrix produce their own external polymers, so that they remain fixed in one position or remain fixed to each other. A slimy layer in a sink is a biofilm of bacteria that attached there because food will often pass by. The slime is the polymer matrix. The same is true for a slimy layer on a rock in a river, or even dental plaque on your teeth. An extra benefit of growing in a biofilm is the protection it offers: it insulates and protects the microbes inside against external shocks, like drought or shifts in temperature, pH or salinity. In fact, most bacteria on earth grow in some sort of biofilm.

Slime layers are biofilms, so are activated sludge flocs, and so are compact and dense sludge granules. Biofilms can take on many shapes, depending on the available food, the environmental conditions, and the types of organisms.

Morphology of aerobic granular sludge

Growing microbes in biofilms is useful for wastewater treatment. Because the bacteria grow together in dense structures, high levels of dry solids can be attained. A carrier is sometimes used to facilitate the attachment of microbes, such as in IFAS systems. In other cases, like with Nereda® , no carrier is used to make the biofilms grow. Instead, selective pressures are applied that give a benefit to microbes that coagulate together. Other principles that are necessary for granulation are also applied, such as a proper feeding regime. These conditions together stimulate the growth of granular biofilms.

The basic principle of Nereda® Biowatch is that these pressures and conditions, and how effectively they are applied, can be recognized by the outward appearance, or morphology, of the granules and flocs. The Nereda® cycle has all the elements to grow smooth and dense granules. By understanding these elements, microscopic analysis can help to find problems with granulation.   

The image below shows an example of this. The manner in which COD is taken up during the cycle, mostly aerobically or mostly anaerobically, results in different granule morphologies. The arrow colors in the figure show that from left to right, the general image of the sludge progressively deteriorates.

Because we are able to recognize certain types of growth and relate them to elements of the process, we can derive information on the process. Therefore, Nereda® Biowatch is useful for troubleshooting. The structures of the sludge can give indications of what type of conversions are happening. If we couple these insights with in-depth knowledge of the plant, it can help to identify issues.

Another use is in monitoring: the analysis can serve as an early detection analysis. Changes in the process can often be noticed visually in the sludge morphology before the effects of potential issues are seen in effluent quality. Practical examples of this are that Biowatch has helped in troubleshooting to recognize when too many solids were recirculated or when the feeding phase had to be extended.

So, Nereda® Biowatch can be used for monitoring and troubleshooting, and also provides a new and very visual way to “read” the sludge in the reactor. This different method of microscopy compared to studying the individual microbes may not show all the different wriggling multifarious animalcules that have compelled scientists from van Leeuwenhoek on, but they do show us many fascinating sludge structures and provide us with intriguing insights on biofilm morphology in Nereda® systems.

If you are interested in more technical detail on how the analysis is carried out, the scientific theory behind it, what elements of the cycle can be linked to visual aspects of the biomass, and how a Nereda Biowatch analysis is carried out, please have a look at the recording of our webinar on this subject!


[1]https://www.microscope.com/education-center/microscopes-101/history-of-microscopes
[2] Eikelboom, D. H., & Van Buijsen, H. J. J. (1983). Microscopic sludge investigation manual.
[3] Eikelboom, D. H. (2000). Process control of activated sludge plants by microscopic investigation. IWA publishing.
[4] Eikelboom, D. H. (2000). Process control of activated sludge plants by microscopic investigation. IWA publishing.
[5] Light microscopy slide image source: https://coordinatedscience1.wordpress.com/lessons/unit-4-cells/4-3-more-microscope-practice/

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