The phenomenon of granulation of anaerobic sludge

Successful high-rate anaerobic wastewater treatment can only be accomplished when the slowgrowing anaerobic biomass is efficiently held back in the anaerobic treatment system. This biomass retention can be achieved in various ways including immobilization of the organisms on fixed materials and immobilization on mobile support materials: in the latter case, combined with settling of the anaerobic sludge aggregates.

This dissertation focuses on the phenomenon of flocculent anaerobic sludge transferring into highly active well-settling sludge granules under specific conditions. A high level of sludge retention can be achieved with granular anaerobic sludge under conditions of high gas production and turbulence in the digester, and consequently, the anaerobic treatment system accommodating high loading rates. This sludge granulation phenomenon proceeds in treatment systems operated in an upflow mode; the Upflow Anaerobic Sludge Bed (UASB) reactor being the main reactor system supporting this phenomenon. The UASB system is in fact nowadays, the most widely applied high-rate anaerobic treatment system in the world. One of the reasons for the UASB system's popularity is undoubtedly this granulation phenomenon.

The work presented in this thesis is focused on the study of the various operational factors that effect the granulation process. This study was conducted in small-scale UASB reactors using well defined feed stocks. The main objective of this work was to develop useful guidelines for operators of full-scale UASB reactors for cultivating granular sludge in their systems.

Chapter I reviews the present knowledge on relevant factors related to the granulation of anaerobic sludge. Important factors discussed regarding the formation of granular sludge include: the mechanisms governing bacterial adhesion and the production and role of extracellular polymers as "sticking" agents, the role of Ca ²⁺, and the effect of the seed sludge. The ideas of the various researchers are often contradictory, emphasizing the fact that the fundamental mechanism of granulation is a very complex and still only partially understood process.

Considerable emphasis has recently been directed to the characterization of granular sludge, particularly with respect to the chemical and microbiological composition of the granules. The chemical composition varies considerably and depends on the composition of the wastewater. As far as the microbiological composition is concerned, it is commonly agreed that the acetate utilizing methanogen Methanothrix soehngenii is the predominant organism, although various other methanogenic and acetogenic organisms are always present, as well as, depending on the composition of the wastewater, a certain population of acidogenic bacteria. The specific methanogenic activity of the granular sludge is a useful tool for characterizing the sludge. The high methanogenic activities generally found with granular sludges demonstrate their ability to be excellent micro- ecosystems with a good micro-environment for all kinds of organisms, including the syntrophically-growing organisms involved in the anaerobic degradation process.

For the physical characterization of granules, factors of specific importance are size,. size distribution, density, settleability and granule strength.

And finally, presented in Chapter 1 is our hypothesis of the granulation mechanism. In our opinion, the selection pressure plays a key role in the granulation process. It is a result of the hydraulic surface-load and the gas surface-load, and is required for accomplishing the necessary separation of lighter and heavier sludge fractions. Almost all growth should eventually concentrate on or in the heavier particles which can be accomplished by allowing all finely dispersed sludge to wash out of the system. The first generations of granules will be fairly open and fluffy in nature, but as a result of a maturation process the granules will gradually become more compact, and filamentous Methanothrix bacteria will be overgrown by the shortchained Methanothrix cells.

For the proper separation of dispersed sludge and agglomerated sludge, it is important that the sludge bed is kept at a level that leaves an area of free space between the sludge bed and the gas collector.

Chapter 3 describes the experimental results concerning granulation on volatile fatty acid (VFA) mixtures (acetate, propionate and butyrate) with digested sewage sludge as seed material. Various relevant parameters were investigated such as the sludge loading rate, substrate concentration, the effect of Ca ²⁺and NH₄⁺, pH and temperature. The results demonstrate that granulation with a simple VFA substrate proceeds very well. The granules developed on VFA substrates with VFA concentrations below 150 mg/l consisted mainly of filamentous Methanothrix.

In all the granulation experiments, including those discussed in Chapters 4 and 5, independent of the type of substrate used, a very similar pattern of retained sludge amounts in relation to the imposed space load could be observed. Initially, the amount of sludge diminishes as a result of wash-out of finely dispersed sludge ingredients from the system. In this initial phase of the start-up process, to prevent overloading of the system, the space loading rates should not be further increased. As a result of wash-out of inert organic sludge ingredients and the continuous accumulation of new bacterial matter, the specific methanogenic degradation capacity of the sludge (based on overall VSS) will show a distinct continuous increase until a steady state with respect to sludge composition is established.

Generally after a period of 40 to 50 days, sludge granules up to a size of 0.5 mm can be observed in the sludge bed. From then onward, the total amount of sludge in the reactor will again gradually increase, while the space loading rate can usually be increased without further stagnation in the digestion process.

With increasing loading rates, the fraction of granular sludge will increase in the sludge bed.

The importance of imposing and maintaining a proper selection pressure has been demonstrated in the following ways:
- if the system remains underloaded for a long period of time, a bulking type of anaerobic sludge, which is very hard to retain in the reactor, will develop;
- at a given space loading rate, which is determined by the degradation capacity of the sludge present in the reactor, granulation proceeds clearly faster with less concentrated wastewaters.

The influent VFA concentration is also of importance with respect to the ultimate bacterial. composition of the granular sludge. When using a concentrated VFA solution as feed, i.e. 10.000 mg VFA-COD/l, a granular sludge will develop consisting mainly of Methanosarcina rather than Methanothrix. With lower substrate concentrations, Methanosarcina may develop under conditions of continuous overloading. Fluidized bed systems are usually started up under such conditions, however they are kinetically unfavorable to growth of the desired wellattaching Methanothrix organism, and may therefore lead to an unwanted accumulation of less well-attaching Methanosarcina sludge in the system.

The Methanosarcina granules remain very small (Dp < 0.5 mm) and consequently are easily washed out of the reactor. Based on kinetical reasons we recommend maintaining the acetate concentration in the reactor below 150 mg acetate/l, in order to promote the development of Methanothrix granules rather than Methanosarcina granules.

The granulation process can be enhanced significantly by increasing the temperature in the mesophilic range to values near 40 °C. At this temperature however, the process will also become more unstable.

We have found clear evidence indicating that granulation also proceeds well at reduced pH values. At a pH level of 6, start-up and granulation proceeded surprisingly well. Quite interestingly, we also found that the Methanothrix -like organism cultivated in the sludge had a much lower pH optimum than that of the Methanothrix strains described in the literature so far.

Chapter 4 describes the results of experiments investigating the effects of specific additives to the seed sludge. Again, digested sewage sludge was utilized as seed sludge.

In a number of experiments (gently) crushed granular sludge was introduced to the reactor, adding a large number of small granular nuclei to the seed. We observed that with this addition (only a few percent on VSS basis), a distinct enhancement of the granulation occurred.

Moreover, and of great importance, we also observed a clear difference in the type of granule cultivated. On mere digested sewage sludge, granules consist mainly of Methanothrix- like organisms in a filamentous state, whereas in the experiment with digested sewage sludge enriched with a small amount of crushed granular sludge, all of the granules consisted of Methanothrix- like organisms present as short chains of 4 to 6 cells. Both granules exert excellent settling properties and are highly active. Organic loading rates up to 50 kg COD/m ³.day could be very well accommodated in reactors containing 20-25 kg VSS/m ³. The so-called "filamentous" granules have a more open structure, while the first generation granules contain inert support particles originating from the seed sludge. The so-called "rod type" granules are significantly more compact and generally do not contain inert support particles.

Experiments with hydro-anthracite as an additional inert support particle reveal its presence to have a positive effect on the granulation speed. Removal of inert particles from the digested sewage sludge contrarily results in poor granulation. The results of these experiments clearly demonstrate the importance of inert support particles generally present in digested sewage sludge.

We also observed that inert particles with a filamentous nature have a negative effect on the sludge characteristics by reducing the sludge settleability and promoting excessive wash-out of sludge. Seed sludges with a high fibrous material content are therefore not recommended for inoculating the anaerobic system.

The granulation process was also studied on a more complex substrate (Chapter 5). Sucrose was selected for these experiments; its investigations indicating that a stable granular type of sludge could be cultivated on this carbohydrate, and at a faster rate than on VFA substrates.

The experimental results also revealed the importance of imposing proper selection pressure with more complex soluble substrates, in order to avoid the accumulation of fast-growing voluminous and dispersed acidogenic biomass. We wish to emphasize however, that it is not necessary to apply hydraulic retention times lower than the maximum specific growth rate, sometimes considered a prerequisite for the successful start-up of a fluidized bed system.

In Chapter 6 the results of preliminary work on the physical characterization of granular sludge is discussed. Tools for characterization used were:
- a modificated sedimentation balance for the determination of the settling characteristics and particle size distribution;
- an apparatus to measure the resistance against compression forces.

Both methods, combined with a picnometric density measurement, are considered useful tools for the characterization of granular sludge. With the aid of these techniques we were able to demonstrate that a substrate shift from a VFA mixture to a sucrose solution will lead to the formation of weaker and bigger granules. Furthermore, a positive correlation between ash content and granule density could also be demonstrated.

Based on the insights obtained from our investigations, we can conclude that the sludge granulation process, as it occurs in upflow reactors such as the UASB system, originates from:
- the strong tendency of anaerobic organisms to stick together;
- the fact that a complex community is required for the anaerobic degradation of organic substrates, which forces the system to grow in the form of a micro-ecosystem;
- the fact that growth of bacterial matter is stimulated to take place on and in immobilized aggregates (nuclei) present and formed in the sludge, by imposing the proper selection pressure to the system.

We believe that the results of the experiments presented in this thesis provide strong evidence in support of our hypothesis of the granulation phenomenon.

Summarizing the results of the present investigations, the following practical guidelines can be provided for the start-up of full-scale reactors.

I Seed sludge
1. The presence of "proper" carrier materials for bacterial attachment is important for the initiation and stimulation of bacterial aggregation.
2. The specific methanogenic activity of the seed sludge is not the only factor of importance. Thicker types of digested sewage sludge, i.e.>60 kg TSS/m ³, are preferred over thinner types, despite their lower methanogenic activity.
3. The addition of a small amount of (crushed) granular sludge to seed sludge enhances the granulation process.

II The mode of operation of the process
It is essential to sufficiently and continuously remove the lighter sludge fractions from the reactor to retain the heavier sludge ingredients and to promote bacterial growth in/on the heavier sludge ingredients. To achieve this, we recommend the following:
1. Washed out dispersed sludge should not be returned.
2. Apply effluent recycle or dilution at an influent COD of more than 5,000 mg/l.
3. Increase the organic loading rate step-wise, always after at least an 80% reduction in the biodegradable COD has been achieved.
4. Maintain a low acetate concentration (< 200 mg/l).
5. Start with 12-15 kg sludge VSS/m ³with thick seed (>60 kg TSS/m ³) and approximately 6 kg sludge VSS/m ³with thin seed sludge (< 40 kg TSS/m ³).

III Wastewater characteristics
1. A general observation is that the lower the strength of the wastewater the faster the granulation will proceed. The strength however, should be high enough to maintain good conditions for bacterial growth. The minimum COD level is presumably approximately 500 mg/l.
2. Dispersed solids, such as acidogenic biomass and fibrous matter, retard or may even prevent granulation.
3. Granulation will more quickly occur on mainly soluble unacidified wastewaters than on acidified wastewaters.
4. High ion concentrations (e.g. Ca ²⁺, Mg ²⁺) will lead to chemical precipitation (CaCO ₃ , CaHPO ₄ , MgNH ₄ PO ₄ ) resulting in the formation of a granular sludge with a high ashcontent.

IV Environmental factors
1. The optimal temperature for mesophilic treatment is in the range of 30-38 °C and for thermophilic treatment in the range of 50-60 °C.
2. The pH should always be maintained above 6.
3. All essential growth factors such as N, P, S and trace elements (Fe, Ni, Co) should be present in sufficient amounts and in available form.
4. Toxic compounds should not be present at inhibitory concentrations. If they are present, sufficient time should be allowed for bacterial acclimatization.