1. Use the left mouse button to move forward through the show Use the right mouse button to view the slides in normal view, edit or print the slides The following slides are provided by Dr. Vincent O’Flaherty.

2. 2. The role of kinetic parameters in granule formation Growth rates, substrate affinities etc. The granule represents a balanced ecosystem, this is not a straightforward outcome kinetically The growth rates of fermentative organisms are 5-10 times faster than syntrophs and methanogens - so environmental factors will greatly affect the likelihood of a stable community developing - delicate balance

3. The cell yields of methanogens and syntrophs are thus rate limiting for the growth of granules - loading rates and other parameters are based on this However, probably the most important kinetic determinant is the outcome of competition for acetate between two methanogens - Methanosaeta (ex. Methanothrix) sp. and Methanosarcina sp.

4. Acetate Competition and Granulation Methane can be produced via acetate or via H 2 /CO 2 - 70-90% of the biogas produced by anaerobic digesters is via acetate - the key CH 4 source Surprisingly however only two methanogens have ever been discovered that can utilise acetate - Methanosaeta and Methanosarcina

5. Methanosaeta concilli and M. Soehgennii are rod-shaped organisms which tend to grow as filaments

6. Methanosarcina barkeri is coccoid and grows 3-6 times faster than Methanosaeta on acetate, but Methanosaeta is the dominant methanogen in granular sludge (up to 90% of methanogenic cells)

7. Methanosaeta has a much higher substrate affinity and in a balanced anaerobic digester the concentration of acetate will be very low

8. The presence of Methanosaeta is believed to be crucial as the filaments provide a nucleus for granule initiation The role of cell surface characteristics in granule formation Hydrophobicity, electrophoretic mobility, and isoelectric point are used to predict the adhesion of bacteria to surfaces Hydrophobicity measured by water contact angle is the most commonly used - allows estimation of the surface energy of bacteria

9. Work to date suggests that fermentative organisms are hydrophilic ( water contact angle of < 45˚) Most syntrophs and methanogens are hydrophobic - > 45˚ Hydrophobic cells, like oil droplet, tend to stick to each other

10. BUT, by association with hydrophilic cells aggregates which behave hydrophilically can be formed Role of trace elements and nutrients in granule formation Granule formation is optimal when nutrient conditions are ideal for the microorganisms involved

11. Ca 2+ specifically appears to promote granulation up to a concentration of 150 mg/l Role of seed sludge in granule formation Ideally granular sludge should be used as an inoculum, but its expensive (c. € 6000 per load) and transport costs can also be very high Can use local sources like animal manures, sewage sludge which contain high numbers of anaerobic microbes

12. Very important to get the balance of the developed community right - its possible to measure the methanogenic activity of the seed, needs to be high initially Relates to the much slower growth rates of methanogens vs fermentatives If methanogens are not present in high numbers, acidification or souring of the reactor can occur

13. The loading rate to the reactor is normally increased stepwise and each step is only carried out when >80% removal is obtained - keep acetate low (Methanosaeta) Irrespective of the source of seed - the content of Methanosaeta is of crucial importance in achieving granulation

14. Environmental Factors Affecting Granule Formation Can be broken down as follows: Liquid and biogas upflow velocities Substrates Temperature and pH

15. Role of liquid and superficial biogas upflow velocities in granule formation There is a requirement for “selection pressure” to encourage the formation of granules In AD designs like the UASB arises as a result of the liquid and biogas loading rates (m 3 /m 3 /h or m 3 /m 3 /day)

16. Results in washout of non-settling cells or flocs and the retention of granules For UASB start-up loading rates are 0.25- 0.4 m 3 / m 3 /h liquid and 4-7 m 3 /m 3 /day biogas Once stable granular bed is formed, much higher loading rates can be safely applied

17. Role of substrates in granule formation Specific substrates are directly related to the proliferation of certain types of bacteria Energy-rich substrates (e.g. carbohydrates and sugars) give rapid growth of granular sludge

18. Poor granulation and fluffy floc formation are often seen with protein rich wastewaters Temperature has an important bearing on which substrates support granulation - e.g. acetate alone can allow granular growth at 37˚C but not at 55˚C

19. Role of Temperature and pH in granule formation AD operates currently at two main temperature levels 35-40˚C (mesophilic) and 55-60˚C (thermophilic) Reaction proceed faster at high temperatures - so higher loading rates can be applied thermophilically

20. But, a less diverse microbial population develops and the system is much more vulnerable to pH changes and other shocks Also much more expensive to operate (heating) Current research is focused on trying to to operate psychrophilically (<20˚C)

21. Problems with Granulation Operational conditions - not suitable for granulation - pH alterations, toxic shocks Inhibitors H 2 S, NH 3 and toxic chemicals Foaming/floatation - lack of sugars or lots of slowly degraded lipids in the wastewater

22. Current Model of Granule Structure Confocal microscopy, fluorescent DNA probes and microsensors have been the main experimental approaches used to reveal structure/function relationships Granules grown on different substrates have different properties - but a general model can be used to explain common features

26. Anaerobic digestion of sulphate- containing wastewaters Industrial wastewaters can, in general, be effectively treated using anaerobic digestion - produces large quantities of methane which can be burned to generate electricity or for heating - use of combined heat and power plants allows for generation of electricity and heat recovery Normally less than 10% of the biogas produced is required to operate the plant - also produces far less waste biomass than aerobic system = less disposal costs

27. Modern digester design makes the process more attractive - can operate at high rates and therefore smaller, cheaper digester can be used Usual procedure is to have first stage anaerobic and then small activated sludge plant to “polish” the effluent = achieve discharge standards. May need some nutrient removal or other tertiary steps depending on the fate of the effluent

28. Problematic industrial wastewaters Application to industrial wastewaters can occasionally be complicated by microbiological problems - typical example is treatment of sulphate containing wastewaters - examples of the type of negative microbial interactions which can occur in an engineered ecosystem Industrial wastewaters can contain high levels of recalcitrant organic chemicals (e.g. chloroform, carbon tetrachloride etc.), xenobiotic products and side products (insecticides, herbicides, detergents etc.)

29. Can also contain significant quantities of inorganics some of which may be highly toxic e.g. cadmium in tannery wastewater In anaerobic systems the presence of alternative external oxidising agents ( e.g. sulphate SO 4 2- ) can promote the development of a sulphate- reducing rather than a methanogenic population This will result in the channelling of electrons towards the formation of H 2 S not methane

30. SRB and anaerobic digestion Very complex systems - absolute need to fully understand the microbiology in order to control treatment plant operation - i.e on one hand a useable fuel is generated and on the other hand a malodourous atmospheric pollutant is produced under sulphidogenic conditions Presents a challenge to microbiologists because of the complexity of the systems and the technical difficulty in studying them

31. Examples of wastewaters that contain high- levels of sulphate include: 1.Molasses-based fermentation industries - e.g. citric acid production, rum distillery 2.Paper and board production 3.Edible oil refinery Many other industries use sulphuric acid in their processes - leads to sulphate in the ww

32. So what? In the absence of external oxidising agents (sulphate, nitrate, etc.) anaerobic ecosystems are methanogenic - flow of reducing equivalents is directed towards the reduction of CO 2 to CH 4 In the presence of sulphate - the flow may be redirected towards the reduction of sulphate to sulphide by sulphate reducing bacteria (SRB) In other words there is a competition between different microorganisms for substrate

33. What will determine the outcome of competition? Very important to know as on one hand a useable fuel is produced, while on the other hand a toxic, corrosive malodourus compound is produced

34. Bacteria that reduce sulphate to H 2 S are either assimilatory or dissimilatory: 1. Assimilatory Sulphate Reduction: Carried out by many different bacteria - purpose is to reduce sulphate to sulphide prior to uptake of S for assimilation into S-containing proteins etc. No major environmental effect only amount of sulphate needed for bacterial growth is reduced e.g Klebsiella sp. - only reduce 1 mg sulphate for every 200 mg (d.wt.) of cells produced

35. 2. Dissimilatory Sulphate Reduction: Totally different process only carried out by a unique group of bacteria carrying out anaerobic respiration using sulphate as electron acceptor Consequently transform large amounts of sulphate to H 2 S during growth e.g. Desulphovibrio sp. - for every 1 mg of sulphate reduced, only 0.5- 1.0 mg (d.wt) of cells are produced

36. SRB exhibit considerable morphological and nutritional diversity - grouped together only on the basis of carrying out dissimilatory sulphate reduction Widely distributed in the natural environment - include both sporeformers (Desulfomaculum sp.) and non- sporeformers (Desulfovibrio sp.)

37. Can be divided into two broad categories based on their metabolism: 1. Incomplete Oxidisers: carry out incomplete oxidation of organic compounds to acetate, CO 2 and H 2 S - can use a very wide range of starting organics e.g. aliphatic mono- and dicarboxylic acids, alcohols, amino acids, sugars, aromatic compounds etc.

38. 2. Complete oxidisers: Complete oxidation of starting organic substrates to CO 2 and H 2 S - same wide range of substrates, but can also grow on acetate, breaking it down completely to CO 2 Chemolithotrophic species also common - grow on H 2 /CO 2 or on CO very common ability to grow on H 2 very important in certain ecosystems

39. 4H 2 + SO 4 2- + H + -----> 4 H 2 O +HS -  G˚´ = -150KJ/mole Very favourable reaction energetically These species must be able to fix CO 2 - autotrophic

40. SRB very versatile metabolically - in the absence of sulphate in their environment, they can switch from anaerobic respiration to chemoorganotrophic fermentation - energy gain by substrate level phosphorylation only V. important as allows maintainence of SRB in the absence of sulphate

41. What happens during anaerobic treatment of sulphate containing wastewaters? Competition between SRB and other anaerobes for common organic and inorganic substrates - for energy and reducing equivalents Outcome is determined by a number of factors - COD/BOD conc.