granular sludge formation in upflow anaerobic sludge blanket (uasb) reactors

Review Granular Sludge Formation in Upflow Anaerobic Sludge Blanket (UASB) Reactors

Jens E. Schmidt1** and Birgitte K. Ahring'," The Anaerobic Microbiology/Biotechnology Research Group, Institute of

Environmental Science and Engineering, Building 115 and 2Department of Biotechnology, Building 223, The Technical University of Denmark, DK-2800 Lyngby, Denmark

Received December 7, 1994/Accepted September 8, 1995

The state of the art for upflow anaerobic sludge blanket (UASB) reactors is discussed, focusing on the microbiology of immobilized anaerobic bacteria and the mechanism of granule formation. The development of granular sludge is the key factor for successful operation of the UASB reactors. Criteria for determining if granular sludge has developed in a UASB reactor is given based on the densities and diameters ofthe granular sludge. The shape and composition of granular sludge can vary significantly. Granules typically have a spherical form with a diameter from 0.14 to 5 mm. The inorganic mineral content varies from 10 t o 90% of the dry weight of the granules, depending on the wastewater composition etc. The main components of the ash are calcium, potassium, and iron. The extracellular polymers in the granular sludge are important for the structure and maintenance of granules, while the inorganic composition seems to be of less importance. The extracellular polymer content varies between 0.6 and 20% of the volatile suspended solids and consists mainly of protein and polysaccharides. Both Methanosaeta spp. (formerly Methanothrix) and Metha- nosarcina spp. have been identified as important aceticlastic methanogens for the initial granulation and development of granular sludge. Immunological methods have been used to identify other methanogens in the granules. The results have showed that, besides the aceticlastic methanogens Methanosaeta spp. and Methanosarcina spp., hydrogen and formate utilizing bacteria are also present, e.g., Methanobacterium formicicum, Methano- bacterium thermoautotrophicum, and Methanobrevi- bacterspp. Microcolonies of syntrophic bacteria are often observed in the granules, and the significant electron transfer in these microcolonies occurs through interspecies hydrogen transfer. The internal organization of the various groups of bacteria in the granules depends on the wastewater composition and the dominating metabolic pathways in the granules. Internal organization is observed in granules where such an arrangement is beneficial for an optimal degradation of the wastewater. A four- step model is given for the initial development of granular sludge. 0 1996 John Wiley & Sons, Inc. Key words: anaerobic upflow reactors granulation sludge formation methanogen bacteria

INTRODUCTION The concept of the upflow anaerobic sludge blanket (UASB) reactor was developed in the seventies.86 To-

* To whom all correspondence should be addressed.

day the UASB reactor has become the most popular high-rate reactor for anaerobic biological treatment of wastewater, and many UASB reactors are in operation throughout the world. The implementation of the UASB reactor has been very successful and it has been applied to a wide range of industrial and municipal wastewater^.^"'^

In spite of the active research in the area of the UASB reactors, the mechanisms by which granules are formed are not yet well understood. In this article we will review the current knowledge concerning the microbiology of immobilized anaerobic bacteria and present suggested mechanisms of granule formation. For technical reviews we refer to Lettinga et a1.,86 Lettinga and Hulshoff P01,8~*'~ Maat and Habets,88 Lin and Yang,s7 and Hickey et al.67 In these reviews the operation and design of full- scale UASB reactors are described along with modeling of the process, etc.

UASB REACTOR

The advantage of the UASB reactor compared to tradi- tional anaerobic treatment, e.g., the contact process, is the ability to retain high biomass concentrations despite the upflow velocity of the wastewater and the production of biogas. Consequently, the reactor can operate at short hydraulic retention times since the sludge retention time is almost independent of the hydraulic retention time. Successful operation under these conditions requires a highly active biomass with good settling abilities. In UASB reactors, the biomass is retained as aggregates, called granules, formed by the natural self-immobilization of the bacteria, i.e., this kind of immobilization does not employ any supporting material such as Rasch- ing rings or clay. The formation and stability of the granules are essential for successful operation.

The UASB reactor is typically divided into four com- partments: (1) the granular sludge bed, (2) the fluidized zone, ( 3 ) the gas-solids separator, and (4) the settling compartment (Fig. 1). The granular sludge bed is located in the bottom of the reactor. The wastewater is pumped into the bottom of the reactor and passes up

Biotechnology and Bioengineering, Vol. 49, Pp. 229-246 (1996) 0 1996 John Wiley & Sons, Inc. CCC 0006-3592/96/030229-18

4 25 I

.:. - ".. .'. ' . * . '. . .a:: ..". . .,'... ,... . . * . .*. . ,

Figure 1. Schematic representation of a UASB reactor: (1) granular sludge bed; (2) the fluidized zone; (3) gadliquid separator; (4) the settling compartment; (A) influent; (B) effluent; (C) gas outlet.

ward through the granular sludge bed. Here the organic compounds are biologically degraded and biogas is produced. In the top of and just above the granular sludge bed, a fluidized zone develops due to production of the biogas. In this zone further biological degradation can take place. The biogas is separated from the liquid in the gas-liquid separator. Granules with good settling abilities settle back through the fluidized zone to the granular sludge bed, while flocculated and dispersed bacteria wash out of the reactor with the effluent.

GRANULAR SLUDGE

Various types of conglomerates of microbes have been described, such as granules, pellets, flocs, and flocculent sludge. However, there is no clear distinction between the different conglomerates. D ~ l f i n g ~ ~ used the following definitions of flocs, flocculent sludge, pellets, and granular sludge: pellets and granules are conglomerates with a dense structure. After settling, these conglomerates present a well-defined appearance. Flocs and flocculent sludge are conglomerates with a loose structure. After settling, they form one homogeneous macroscopic layer. This gives a good descriptive definition, but gives no guidelines determining the different types of conglomerates.

The diameter of sludge granules varies from 0.14 to 5 mm~8,52,77,79,130,145,160 depending upon the wastewater used, the operational conditions, and the analytical method. Granules cultivated on acidified substrates, such as acetate, are generally smaller than granules

grown on acidogenic substrates, e.g., g l~cose .~*~"~ 79,130,145,160 Th e granules vary widely in shape, depending on the conditions in the r e a ~ t o r ~ ~ , ~ ~ , ~ ' ; but they usually have a spherical form.

Andras et a1.I' have developed a test to characterize the settleability of anaerobic sludge. The test is based on the division of sludge into fractions depending on their resistance to wash-out of a test cylinder with increasing linear flow. A test like this accounts for both the buoyant density, the shape, and the volume of the granules. Granules with high buoyant densities and volume will wash out at higher linear flow rates compared to small granules with low buoyant densities. A similar test has been developed by Beeftink et a1."

The linear liquid flow rate at which a granule with a given volume and buoyant density will be washed out of the reactor can be estimated by Stoke's law. Granules with different volumes and densities can be present in a reactor at a given linear flow rate; both small granules with high densities and larger granules with low and high densities will be present. Reported settling velocities for granular sludge are in the range of 18 to 100 mih, but typical values are between 18 and 50 m/h.'0,12.377,72 Gr anu- lar sludge can therefore be divided into three fractions based on the reported settling velocities: a poor settling fraction, a satisfactorily settling fraction, and a good settling fraction, with settling velocities of up to 20 m/h, from 20 to 50 m/h, and over 50 m/h, respectively. A satisfactory granular sludge contains sludge with its main part in the two last fractions. In Table I values are given for the minimum diameter of granules with a given density, which have a settling velocity of 20 m/h. This table can be used to determine if a granular sludge has been developed in a UASB reactor.

Typical reported buoyant densities of granules are 1.03 to 1.08 g/mL,6* but up to 1.4 g/mL has been re- p ~ r t e d . ~ ~ - ~ * The density of bacteria cells is in the same range,Iz3 indicating that the observed settling abilities of the granules must be due to aggregation of the anaerobic bacteria together with inorganic enclosements.

Inorganic Composition

The inorganic mineral or ash content of granules varies from 10 to 90% of the dry weight of the granules,4,6,8.34.37,42,46-48,S2,72,1 19,130,146,166 d epending upon the wastewater composition, process conditions etc. Ash content of samples taken at various occasions from the same reactor can vary up to 100%.34,37 However, some generalizations can be made. Under mesophilic conditions, granules grown on complex wastewater have a lower ash content4~*~37~72~"9~'30~'6h than granules grown on simple substrates, i.e., acetate, propionate, or butyrate.4,6,47,48,52.130,'36 In addition, the granules grown on complex substrates are often bigger than granules grown on more simple substrates. A study comparing granules from two identical UASB reactors run under the same

230 BIOTECHNOLOGY AND BIOENGINEERING, VOL. 49, NO. 3, FEBRUARY 5, 1996

Table 1. settling velocity of 20 mih estimated using Stoke’s law.”

Minimum effective diameter for spherical granules with a

Diameter (mm) Density ( mgimL) 37°C 55°C

1010 i 020 I030 1040 1050 I060 1070 1080 1090 1100 I200 I300 1400 I 500

1.2 0.8 0.6 0.5 0.4 0.4 0.3 0.3 0.3 0.3 0.2 0.2 0.1 0.1

1.0 0.7 0.5 0.4 0.4 0.3 0.3 0.3 0.3 0.2 0.2 0.1 0.1 0.1

‘’ Stokes law: If Re, < 2:

I f 2 5 RE,, 5 400:

where Re,,, Reynolds number (V$ppD/q): V,, settling velocity (mis); g. gravimetric constant (mi?); D, diameter of granule (m); p, density of water (1000) tkgim’); p,,, density of granule (kglm’); T, viscosity of liquid (37°C : 0.73. 55”C:O.Sl for water) ( k g h s).

operational conditions, but at different temperatures, showed that that ash content was 1.5 times higher in granules from the thermophilic reactor, while the granules were bigger in the mesophilic one.’32 A correlation between the density and the ash content has been found,72 shouing that an increase in density can be ex- plained by an increase in ash content. The difference in shape and density found under various conditions could be due to low porosity of the granules when the density is high. Diffusion limitation will occur if granules with a high ash content increase in size.

The main components of ash content of granules are calcium, potassium, and ir0n.’~5”9. 40,46.48,137*166 Dolfing et a1.j’ reported that 30% of the ash content was FeS, presumably responsible for the black color of the granules. However, it is not evident from the results if FeS was formed before or as a result of incineration during analysis. The stabilizing effect of calcium and potassium precipitates in granular sludge have been confirmed by experiments where calcium and potassium were re- moved from granules by treatment with ethylenedi- aminetetraacetate (EDTA). This led to a decrease in strength and in some cases to disintegration of the g r a n ~ l e s . ~ ~ ~ ~ ~

The precipitation is probably a result of local environmental conditions formed by the bacteria in the granules; i.e., one would expect to find a spatial distribution

in granules. However, no clear distribution of the mineral precipitions within or on the surfaces of the granules has been found. However, phosphorus and sulfur are often found on the surfaces of the granule^.^.^^^^^ Gran- ules from a UASB reactor treating volatile fatty acids were divided into groups according to their color: black, white, and grey. Surprisingly, X-ray analysis showed that the black granules had the lowest S : Fe ratio, indicating that something else could be of importance for the black

No correlation between the ash content and the strength of the granules has been indicating that additional factors can be involved in the stabilization of the granular sludge.

Content of Extracellular Polymers

Extracellular polymers (ECP) can usually be found in abundant quantities in natural systems. Understanding the physical and (bio)chemical characteristics of the extracellular polymeric matrix is important for the understanding of the structure and function of biofilm and granules. Bacterial ECP is defined as polysaccharide- containing structures of bacterial origin lying outside the integral elements of the outer membrane of Gram- negative cells and the peptidoglycan of Gram-positive cell^.^^.^^ ECP is made of organic debris, phages, lysed cells, and other organic material excreted by the microbial cells. It contains polymers of saccharides, proteins, lipids, phenols, and nucleic acids.’42 ECP can have different functions depending on the microorganism. It can trap soluble nutrients, increase pathogenicity, or decrease the susceptibility to phagocytosis. ECP also mediates adhesion of bacteria in natural ecosystem^.^^*^^

Several researchers have shown, using microscopic observations, that the bacteria in granules are surrounded by ECP; and it is generally accepted that the formation of granules is correlated with the production of ECP.34~”~4’~”~”~’oo~118~136 The reported ECP content of granules is between 0.6 and 20% of the volatile suspended solid content,37~54,59~’02.”9.’3” depending on the granular sludge examined, the extraction method, and the analytical method for ECP. The ECP in granules consists mainly of protein and polysaccharides, typically in a ratio of 2 : 1-6 : 1.37,54,59,102.132,138 Lipids have also been o b ~ e v e d . ’ ~ ~ The amounts of neutral sugars in ECP have been found in the relative descending order: glucose, galactose, rhamnose, and m a n n o ~ e . ~ ~ , ’ ~ , ~ ~ The amount of lipids in ECP from granules grown under different conditions was between 0.02 and 0.05% of the volatile suspended solid of the g r a n ~ 1 e s . l ~ ~ The composition of ECP affects the surface properties of the bacterial flocs and the physical properties of the granular s l ~ d g e . ~ ~ . ~ ( ’ ~ Dispersed bacteria are negatively charged and there is electrostatic repulsion between the cells. The production of ECP can change the surface charge

SCHMIDT AND AHRING: GRANULE FORMATION IN UASB REACTORS 231

of the bacteria, resulting in aggregation (Fig. 2). The adsorption of bacteria depends on the surfaces of both cells and the support to which these cells adhere. The composition of ECP is of importance for adsorption due to ECP's influence on the surface charge and energy. Too much ECP can cause a deterioration in floc formation; and, therefore, repulsion can occur (see Granula- tion Process section).

The amount of ECP is affected by the conditions under which the granules are grown. The concentration of ECP is lower in thermophilically grown granules compared to mesophilically grown.'32 The amount of ECP is also affected by the wastewater composition. Shen et al.'38 showed that the amount of carbohydrates extracted from granules increased with the addition of iron and yeast extract to the feed. The opposite effect was seen when iron was absent. Bull et aL19 showed that addition of methanol to the feed improved the start-up performance of a fluidized bed reactor treating synthetic, meat wastewater. The authors showed that an increased C/N ratio stimulated the production of extracellular polysaccharide, resulting in improved bacterial attachment to solid sufaces. A decrease in both the protein and poiysaccharide content in the extracellular material was seen when the feed of a UASB reactor was changed from sugar containing wastewater to a synthetic wastewater containing acetate, propionate, and butyrate. In contrast, the highest amount of lipids was found in the granules from the latter rea~t0r . l~ ' It is not clear whether specific species produce ECP, or several or all species in the granular sludge are able to do so. However, the above results indicate that the production of ECP, in particular polysaccharide production, is restricted in methanogenic and acetogenic systems; acidogenic populations have a greater influence on the production.

The pore size and porosity of the ECP matrix affects cell activity through its regulation of substrate and biogas transport. When the gel of ECP is dense, the pore size and porosity are low, which results in mass transfer resistance. Substrate transport limitations can result in autolysis of the core of the granules, producing hollow granules. The porosity of the granules, thereby, decrease in the interior layers of the granules, possibly as an effect of bacterial lysis, giving an inactive core of rather large Investigations of granules from four

full-scale UASB reactors treating different kinds of wastewaters revealed that the porosity of the granular sludge decreased with increasing granule size. However, no difference in substrate affinity was observed between granules of different sizes, indicating that only part of the biomass present in large granules is biological active.'

Metabolic lnterspecies Transfer

Complete degradation of organic matter to CH4 and CO2 under anaerobic conditions involves a complex in- teraction of several groups of bacteria, all carrying out their part of the overall conversion process. Fermen- tative bacteria hydrolyze biological polymers by means of extracellular enzymes; and the oligomers and mono- mers are further fermented to acetate and other short- chained volatile fatty acids, such as propionate and butyrate, and H2 and C02. Under methanogenic conditions, the short-chained fatty acids other than acetate are oxi- dized to acetate and H2/C02 by H2-producing acetogenic bacteria. Finally, acetate and H2/C02 are con- verted to methane by methanogenic bacteria.

Because of unfavorable thermodynamics, oxidation of propionate and butyrate is only possible if H2 is re- moved efficiently, i.e., a very low hydrogen partial pressure is necessary. Propionate degradation is only possible below a partial pressure of atm H2.'6,38.6' In granules degrading a mixture of acetate, propionate, and butyrate, a clear correlation exists between the degradation rate of propionate or butyrate and the hydrogen partial pressure. A slight increase in the partial pressure of hydrogen results immediately in a decrease in the degradation rate of the two volatile fatty

In anaerobic systems low hydrogen partial pressures can only be achieved by interspecies transfer of molecular hydrogen from hydrogen-producing bacteria to hydrogen-oxidizing methanogens. In granular sludge, microcolonies consisting of syntrophic, propionate, or butyrate degraders and hydrogen-utilizing methanogens have been o b s e r ~ e d . ~ ~ - ~ ' , ~ ~ The degradation of, e.g., propionate and butyrate could take place in these microcolonies. Disintegration of volatile fatty acid degrading granules, and hereby also disintegration of the microcol-

acids. 128.1 29,1 3 1,134

Figure 2. The role of surface charge and the production of ECP in the development of granules.


onies, led to a decrease in the degradation rate of both propionate and butyrate, indicating the importance of microcolonies.128~'29~131~'34 Besides the observed microcolonies in the granules where the degradation of propionate or butyrate could happen, thermodynamic and flux considerations have shown that the most effective degradation of propionate and butyrate will take place in these microcolonies where the distance between the syntrophic bacteria is small.10s,125

Besides hydrogen transfer, formate transfer also has been proposed to play a role in the syntrophic oxidation of fatty acids in flocs or dispersed cultures where the distance between the bacteria is high (more than 10 pm).143146-148 However, recent experiments showed that interspecies formate transfer is of no importance during degradation of propionate and butyrate in granules, perhaps due to the small distance between the bacteria. Electron transfer in the granules occurred only through interspecies hydrogen t r a n ~ f e r ' ~ ~ . ' ~ ' , ' ~ ~ * ' ~ ~ (Fig. 3).

Activity

A specific activity test is used to determine the performance of different specific physiological groups involved in anaerobic digestion.36J44 The specific metabolic activity of granular sludge is measured on defined substrates in batch tests. Specific activity is directly pro- portional to the relative amount of viable bacteria biomass of the trophic group corresponding to the defined substrate used in the batch activity test.36 High methanogenic activity is one of the characteristics of granular sludge and the specific methanogenic activity (SMA) of granules is typically between 0.5 and 2 g COD (chemical oxygen demand) CH4/g VSS (volatile suspended solid)/ day (10-42 mmol CH4/g VVS/day (Table 11). However, the SMA of thermophilic granules has been observed up to 7.1 g COD/g VSS/day (148 mmol CH4/g VSS/day) (Table 11). The specific methanogenic activity of the biomass depends on the energy and carbon source which the granules were grown on. The maximum values are obtained when the test substrate is identical to the

Syntrophic association inside granule

Figure 3. The role of interspecies transfer of hydrogen and formate.

growth substrate or if the test substrate is an important intermediate, e.g., H2.

The activity of granular sludge can be inhibited by high concentrations of fatty acids. Investigations with thermophilic granules degrading a mixture of acetate, propionate, and butyrate showed that propionate can be inhibitory for the degradation of other volatile fatty acids.161-lm Butyrate degradation was immediately re- duced by 50% when propionate was added to a final concentration of 30 mM compared to the maximum activity at 10 mM; and degradation of acetate was immediately inhibited by 50% by adding 25 mM propionate, compared to the maximum degradation rate at 10 mM pr~p iona te . ' ~ ' - ' ~~ Furthermore, propionate degradation was severely inhibited by the addition of 50 mM acetate to the influent of a thermophilic propionate degrading. UASB reactor. Propionate degradation was not affected by 35 mM butyrate to the inf l~ent . '~ '

A mesophilic UASB reactor degradation acetate was completely inhibited when the reactor was subjected to a pulse of lauric acid above 0.5 mM (0.3 g COD/L). However, the addition of an equivalent amount of calcium to the wastewater prevented inhibition.'15 The calcium could reduce the inhibition, probably by chemically binding the lauric acid and thereby preventing the adsorption of the lauric acid to the cell walls. The absorption of long chain fatty acids to the cell wall has been reported to play an important role in the mechanism of i n h i b i t i ~ n . ~ ~

Granular sludge maintains viability without feeding and methanogenic activity can easily be reestablished after the UASB reactor had been shut down for many months.87.139,141

Introduction of New Abilities

Different attempts have been made to introduce new or improved activities to granular sludge. Hendriksen et a1.65,66 have investigated the influence of a supplemental carbon source on the anaerobic dechlorination of pentachlorophenol. The data showed that addition of glucose had a stimulatory effect on dechlorination and at the same time stabilized the process by ensuring the presence of a sufficient amount of active immobilized biomass. In this way it is possible to effectively remove a highly recalcitrant substance in a UASB reactor with conventional granular sludge.

Thiele et al.'46 developed granules with special abilities for wastewater treatment in a UASB reactor with high recirculation rates. These granules are smaller than conventional granules, but have higher COD turnover rates for wastewater treatment.

The best example of engineered granules is the introduction of a de novo bioremediation ability dechlorination, into granular sludge. This was done by inoculation of a pure culture, Desulfomonile tiedjei, to a UASB where the granular sludge did not have the ability to

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dechlorinate. The addition of the pure culture led to immobilization of the bacterium within the granules, demonstrated by the use of antibody probes.2 Wu et al.I6' used a similar approach to introduce dechlorination of pentachlorophenol (PCP) in a granular sludge. The technique has broad application for cleaning wastewater and chemically polluted groundwater. By similar procedures it may be possible to design granules for specific bioremediation needs whenever the microbes performing the job are available.

MICROBIAL COMPOSITION

Bacterial Composition in Granules Determined by Most Probable Number and Isolation Techniques

Counting of the total number of bacteria in granules was done in several ways. Direct counting typically gives a cell concentration of 5.10" to 1.4.1012 cells/mL gran- u l e ~ . ~ , " , ~ ~ . ~ ~ ~ , . ~ ~ The same number of bacteria have been found using staining techniques (Dapi or acridine or- ange)'9 or transmission electron microscopy (TEM)? However, these techniques give no information about the number and the activities of the different trophic groups in the granules.

The most probable number (MPN) technique gives an overview of the number of bacteria in the various trophic groups. Results obtained using this technique on granules grown on different wastewaters and under different temperatures are given in Table 111. D01fing~~ showed that microscopic observations gave up to 50% more methanogens than the MPN technique. However, the MPN method has been used with success to deter-

mine the shift in the microbial populations after changing the feed to a UASB reactor from sugar-containing wastewater to synthetic wastewaters containing ethanol or propionate (Table 111, no. 5-7).j5 Comparing the results of the MPN series and the activities found on different substrates, it was observed that the bacterial count correlated well with the activities, except with acetate as substrate.13'

In some MPN studies cultures were identified: typical methanogens are Methanobrevibacter spp., methanospirillum spp., Methanosaeta spp. (former Meth- anothrix spp.), and Methanosarcina spp39,41,55, and syntrophic bacteria are Syntrophobacter spp., Syntro- phomonas spp., and Pelobacter spp.39,41,55. Sulfate reducing bacteria are also present (Desulfovibrio sp. and De- sulfobulbus ~ p . ~ ~ , ~ * , ' ' ) .

The MPN technique has its limitations because the granules must be disintegrated, destroying the organization of the different species within the granules and, thereby, the interactions between the species. Further- more, all cultivation methods are selective and noncul- tivable strains are not be counted.

Role of Methanosaeta spp. (Former Methanothrix) and Methanosarcina spp.

Methanosarcina and Methanosaeta are important aceticlastic methanogenic species in granules.3~31~53~68~160 Meth- anosarcina spp. usually grow in large aggregates up to 1-3 mm in diameter. These aggregates consist of large numbers of individual cells surrounded by a thick wall. Methanosarcina spp. use several methanogenic substrates, including acetate, methanol, methylamines, and sometimes Hz/C02. Methanosaeta spp. are filamentous organisms which are known to grow only on acetate.

Table 111. MPN methods applied to granules grown under different conditions.

No. bacteria/mL granules Metabolic

group Substrate 1" 2h 3' 4d 5' 6f 78 8 h 9'

Acidogens Glucose 3.6. 10' 1 . 10' 10' 10'" 2. 10lZ 2 . lo8 2 . 108 ND ND Lactate ND 3 . 109 109 ND ND ND ND 6 . 10' ND

Syntrophic Ethanol ND 2. 109 ND ND 2.109 2.1010 2 . 1 0 ~ ND ND Propionate 4.3 . 10' 3 . 10' 10' 107 2.108 3.107 5.106 105 108 Butyrate 2.7. loin 1 . 108 ND 107 ND ND ND ND 105 Valerate ND ND lo8 107 ND ND ND ND ND

Formate 1.3. 10" ND ND ND 2.109 2.108 2.1011 ND ND Acetate 8.5. 10'" 3 . lo' lo9 10* 2. lo8 9 . lo9 5 . lo6 6 . lo7 4 . 10'

Methanogens Hz/COZ 2.3 1012 3.109 loin 109 9.1010 3.109 2.1012 2.108 3.108

a Wu et ai,.'67,'6* synthetic wastewater containing a mixture of volatile fatty acids; mesophilic. Dubourguier et al.,39 starch industry wastewater; mesophilic. Dubourguier et a1.:" synthetic wastewater containing glucose, lactate, acetate, propionate, butyrate, and valerate; mesophilic. Dolfing et al.,37 liquid sugar factory wastewater; mesophilic. Grotenhuis et a1.:' sugar factory wastewater; mesophilic. Grotenhuis et a1.,5' granules from the above reactor after 6 months of adaption to synthetic medium with ethanol; mesophilic.

Fukuzaki et al.,46 synthetic lactate wastewater; mesophilic. g Grotenhuis et al.,55 granules from the above reactor after 6 months of adaption to synthetic medium with propionate; mesophilic.

1 Fukuzaki et a1.,"8 synthetic propionate wastewater; mesophilic.


_ _ _ - - - _-I__--.- Methanosarcina spp. have higher maximum growth rates on acetate than Methanosaeta spp., but K, values on acetate for Methanosaeta spp. are 5-10 times lower than for Methanosarcina spp.74J72 These kinetic data indicate that a selection for granules dominated by Methanosaeta spp. should be favored by low steady-state acetate concentration in the UASB reactor. However, it should be pointed out that Methanosarcina spp., unlike Methano- saeta spp., have the capacity to use several methanogenic substrates; and, therefore, its role in anaerobic reactors may not be solely based on acetate metabolism.

Methanosarcina spp. are easy to detect microscopi- cally due to their distinct morphology and due to their high content of the fluorescent cofactor, F420; Methano- sarcina Spp. fluorescence blue-green when excited by light near 420 nm. Methanosaeta spp. also have a distinc-

Figure 4. Scanning electron micrograph of the surface of an acetate- degrading granule showing the predominant Methanosarcma spp

tive morphology, especially the gas-vacuolated strains; but they do not contain sufficient amounts of F420 to a u t o f l u o r e ~ c e n c e . ~ ~ ~ J ~ ~ , ~ ~ ~ . ~ ~ ~

Direct microscopic counts of methanogenic sludge granules grown on wastewater from a sugar factory revealed that about 20% of the total population consisted of autofluorescent methanogens, and 20-30% of the total population resembled Meihanosaeta

have used microscopic methods to identify Methano- saeta-like organisms as the predominant aceticlastic methanogens in granules. It is important to note that many microorganisms form large filaments resembling Methanosaeta spp. and, therefore, identification of these strains cannot be based solely on microcopic observations. Some workers have also noted the importance of Methanosaeta spp. in determining the development of granules in UASB reactors. Methanosaeta spp. rods ap- pear to provide a network within the granule to which other bacteria become associated. It is the general view- point that Methanosaeta spp. improve granulation and result in more stable reactor performance.68~'60 How- ever, Meihanosaeta spp. have been observed in two morphological form in granules: a rod-shape bacterium in fragments of four to five cells and a filamentous type consisting of long multicellular rod-shaped bacteria.32.6y.145,15y~168,'70 Under mesophilic conditions, the filament type must be avoided because it can lead to bulking sludge during start-up and, hence, to washout of the biomass from the reactor.69-'45'168*170 This phenomenon is often observed under substrate limitation.

Methanosarcina spp. have also been found to predom- inate in stable granules grown under various condi-

vations in our laboratory of granules from several UASB reactors operated under mesophilic and thermophilic conditions, and on various substrates, such as glucose, a mixture of acetate, butyrate, and propionate, and acetate have shown Methanosarcina spp. can be the predominant aceticlastic methanogens (Fig. 4). A thermophilic UASB reactor degrading acetate was

M~~~ investigators22,34,37,46,48,7S,81 ,82,96,101 , I 03,104,106,137,160,166

tions.' ,4,55,68,97.1 14.124,126,127,130,133,136,164 Microscopic obser-

started by applying methods for production of granules dominated by Meihanosaeta spp.? i.e., substrate conversion exceeding 80% and an effluent concentration of less than 0.2 g COD/L. However, the granules developed contained Methanosarcina spp. as the predominant methanogens, and no Methanosaeta spp. were detected. Granules dominated by Methanosarcina spp. can disin- tegrate under certain conditions, such as high cation concentrations (over 30 mM). The change in the morphology from clumps to single cells of Methanosarcina spp. causes disintegration of Meihanosarcina-dominated granu1es.13",'36

Attempts to select for granular sludge dominated with Methanosarcina spp. under mesophilic conditions have been done either by decreasing the pH to 6 (mesophilic pure cultures of Merhanosaeta spp. do not grow below pH 6.6),73,"'7,145 or by applying a rather high steady-state acetate concentration.'60 However, Methanosarcina spp. did not dominate the granules developed after changing the pH14'; and fluffy granules were developed with a high acetate concentration in the effluent."" Forster4' reported that the Methanosarcina : Methanosaeta ratio was not correlated to the concentration of acetate in a UASB reactor, indicating that other factors might influence the selection, such as nutrient and micronutri- ent concentrations and the hydraulic loading.

Microbial Composition Determined Using Immunological Methods

In complex ecosystems such as granules, microscopic identification using morphological criteria will not be a valuable tool for identification of bacteria, as previously discussed. Only presumptive naming can be done based on structural characteristics, shape, cell envelope structure, inclusions, etc. A more direct assessment can be achieved by applying molecular probes, such as mono- clonal or polyclonal antibodies or nucleic acid probes, which can be applied directly to suspensions of disintegrated granules or to thin sections of granules. While


the former approach allows the researcher to count cells and determine the concentration of each type of bacterial subpopulation identifiable with antibody probes, studies with thin histologic sections also provide information on the topography of the same subpopulations within the granules.

The methods for immunological identification have been known for years. The methanogenic bacteria are one of the best studied groups of bacteria, and today we have a comprehensive and detailed understanding of the taxonomic immunology of this group of or- g a n i s m ~ . ~ ~ , ~ ~ . ~ ~ - ~ ' Immunological methods have been used to study methanogenic populations and, thereby, their occurrence in different anaerobic reac- torS.3,9,20,24,2S.7~~,92-94,10S Similar methods have been used to detect methanogens and other anaerobic bacteria in various anaerobic environments.'8,20~ss.'0' Immunologi- cal studies are, however, restricted since the isolation of a specific microorganism is necessary before a probe can be prepared.

The dominant methanogenic subpopulations in granules from some UASB reactors operated at different conditions have been identified (Table IV). No single specific methanogenic population was found to predominant in these investigations. Organisms immunologically related to Methanobacterium formicicum MF, Methanobrevibacter arboriphilus AZ, and Methanobac- terium thermoautotrophicum AH are often predominant hydrogenotrophic methanogens in mesophilic and thermophilic granules, respectively. The data in Table IV also show that aceticlastic methanogens were always present in the investigated granules.

Immunological methods have also been used success- fully to investigate changes in the methanogenic subpopulations under varying operating conditions. Koorneef et aL7' found a wider diversity of methanogenic subpopulations with increased complexity of the substrate fed to the reactor. When changing the temperature from mesophilic to thermophilic conditions, the methanogenic subpopulations changed significantly along with a change in granule structure. The diversity of methanogenic subpopulations was more pronounced before the shift to mesophilic conditions.'s6 The effect of magnesium on the microbial composition of thermophilic granules has also been studied. The complexity of the methanogenic subpopulations decreased with increasing magnesium concentration.136

Only few antisera against other anaerobic bacteria have been made. They have been used to identify bacteria immunologically related to the ethanol-degrading bacterium I'elobacter carbinolicus,ss the propionate- degrading bacterium Syntrophobacter wolinii, the butyrate-degrading bacterium Syntrophomonas wolfei,'oy and the dechlorinating anaerobe Desulfomonile tiedjei2

New molecular methods based on 16s rRNA offer a potential for a better understanding of community composition, structure, and microbial growth of biofilm

Table IV. Predominant methanogens in granules grown under different conditions identified using immunological methods.

Dominant methanogens

Temperature antigenically Substrate ("C) related to References

Wastewater from a sugar factory

Ethanol

Propionate Gelatin

Acetate, propionate, butyrate, glucose, and gelatin

Acetate, propionate, and butyrate

propionate, and butyrate

Wastewater from a sugar factory

Propionate Ethanol Acetateh

Acetate,

Acetate , propionate, and butyrate

propionate, and butyrate'

propionate, and butyrateC

propionate, and butyrateC

Acetate,

Acetate,

Acetate,

32

35

35 35

35

38

55

35

35 35 55

36

46

55

64

MF, AZ,

AZ, TM-1,

MF, Opf MF, JFI,

MF, AZ,

Opf, DH1

MF, DH1

AZ, Opf

JRlm, Opf

ALI, Opf

AZ, Opf, AH

MC3, Opf, AZ, JFl

Opf, AZ MC3, JF1 TM-1, GCI,

AH, S-6 OPf

OPf

Opf, AH

Opf, AH, A 2

I7

17

I1 I1

I1

156

156

55

55 55

136

152

152

152

152

MF, Methanobacterium formicicum MF: JF1, Methanospirillum hungatei (Methanospirillum hungatii) JF1; ALI, Methanobrevihacter smithii ALI; GCI, Methanobacterium thermoautotrophicum GCl; AH, Methanobacterium thermoautotrophicum AH; TM-I, Methanosarcina thermophila TM-1: AZ, Methanobrevibacter arboriphilus (Methano- brevibacter arboriphilicus) AZ; Opf, Methanosaeta concilii (Methano- thrix soehngenii) Opfikon: JRlm, Methanogenium marisnigri JRlm; MC3, Methanosarcina mazei MC3; DH1, Methanobrevibacter arboriphilus DH1; S-6, Methanosarcina mazei S-6.

Adapted from mesophilic to thermophilic conditions. Grown under different magnesium concentrations. Adapted from mesophilic conditions.

and granule^.^^'" Such probes have been used to study interactions between different populations of methanogens and sulfate-reducing bacteria in anaerobic reactors, e.g., biofilm reactors."'-'13 However, 16s rRNA probes have not yet been used on granular sludge.

Structure of Granules Scanning electron microscopy can be used to examine the surface of granules. Cavities and holes are often


observed on the surface of the granules.39.60.78.96.100 The cavities may be channels for transport of gas or substrate (Fig. 5) .

With transmission electron microscopy the internal structure of the granules can be studied. Microcolonies of syntrophic bacteria are often observed37~’9-41~60~63~y6~143 and presumptive identification of these bacteria in microcolonies has been done. The microcolonies include the propionate-degrading bacterium Syntrophobacter, associated with Methanobrevibacter spp.,3y~4’~55~96~109 and the butyrate-degrading bacterium Syntrophomonas with Methanobrevibacter ~pp.”*~’,‘’’ The propionate- producing bacterium Propionibacterium sp. and ethanol-degrading bacterium Pelobacter sp. have also been identified.’y,ss

Some authors claim that the various trophic groups of bacteria are randomly distributed throughout the granule and that an internal organization is not obvi- O U S . ~ ’ . ~ ~ Others have described a more structured organization of the populations in the granules.‘~46~48~5’~,h3~y6 Harada et a1.63 found a fairly distinct localization of bacteria within granules grown on carbohydrate- containing wastewater. In the outer part of the granules hydrolytic and/or acidogenic bacteria were predominant, whereas Methanosaeta-like bacteria dominated in the inner part of the granules. A similar structure was observed in granules grown on lactate or pr~pionate.~‘,~’ Some investigator^^'^^^ found an even more structured pattern in granules grown on glucose or sucrose. The syntrophic bacterial consortia were located between an external, predominantly acidogenic layer, also including hydrogen-consuming bacteria and a core of acetate uti- lizers, creating optimal nutritional conditions for all its members. However, Grotenhuis et al?s found that granules grown on either wastewater from a sugar refinery or ethanol did not show spatial orientation of the bacteria, while granules grown on propionate consisted of two types of clusters. One cluster consisted of bacteria immunologically related to Methanosaera soehngenii. The other cluster consisted of two different bacteria, one immunologically related to Methanobrevibacter arbori-

Figure 5. degrading wastewater from an ice cream factory.

Cavities in the surface of a granule from a UASB reactor

philus A Z and the other was tentative identified as a propionate-oxidizing bacterium not immunologically related to Syntrophobacter wolinii. The two kinds of clusters were distributed randomly throughout the granules.

The internal organization of the various trophic groups in the granules seems to depend on the wastewater composition and the dominating catabolic pathways in the granules. Spatial orientation is often observed in granules grown on complex wastewater, in contrast to granules grown on simple substrates, e.g., a ~ e t a t e . ~ ~ . ~ ~ , ’ ~ ~ In granules grown on complex wastewaters, an internal organization may be beneficial for an optimal degradation of substrates and intermediates. A structured aggregate is a stable metabolic arrangement that creates optimal conditions for all its members. In some studies granules grown on complex wastewaters showed no obvious internal ~ r g a n i z a t i o n . ~ ~ In these systems the complex substrates are probably degraded directly to acetate, with little or no production of other intermediates such as propionate and butyrate.

Macario et al?5 have studied the topography of the methanogenic subpopulations in granules grown on a mixture of acetate, propionate, and butyrate, and adapted from mesophilic to thermophilic conditions. They presented a structural model of these granules, with each subpopulation showing a distinctive distribution pattern. On the surface and inner part of the granules they found colonies of methanogens antigenically rated to Methanobacterium thermoautotrophicum AH; and in the core, clumps of Methanosarcina spp. immunologically related to Methanosarcina thermophila TM-1 were found. Bundles of rods immunologically related to Methanosaeta spp., together with dense lawns of methanogens antigenically related to Methanobrevi- bacter arboriphilus AZ and Methanobrevibacter smithii ALI, and single cells immunologically related to Metha- nosarcina thermophila TM-1, were found between the surface and the core. The presence of Methanobacterium thermoautotrophicum AH in the surface suggests that the H2-using methanogens may consume free hydrogen found in the liquid which then does not need to pene- trate further into the granules. The hydrogen-utilizing bacteria in the middle of the granules remove hydrogen produced by syntrophic bacteria in the granules, permit- ting high metabolic activity of the syntrophic bacteria.

Applying immunological and histologic methods together with isotopic studies, Ahring et al.4 showed that different metabolic pathways dominated different re- gions of thermophilic acetate-utilizing granules. Acetate oxidation occurred in the middle of the granules where the acetate concentration was low, while aceticlastic re- actions dominated at the outer surfaces at higher acetate concentrations.

Further studies using immunological probes on active granules can help in the search for a better understanding of the formation and physiology of granules.


INITIATION AND DEVELOPMENT OF GRANULATION

Granulation Process

Microbial adhesion or granulation, i.e., when a cell at- taches to a surface or another cell, can be defined in terms of energy involved in the formation. A cell can be said to have adhered if it requires energy to remove the cell to its original isolated state.12"

It is important to gain sufficient knowledge on the granulation process to control and regulate the process in UASB systems. Biofilm formation has been investigated intensively, but the granulation process has not be fully studied. However, granular sludge can be char- acterized as a spherical biofilm and similarities between biofilm formation and the granulation process exist.

The initial development of a biofilm or a granule can be divided into four ~ t e p ~ ~ ~ , ~ ~ , ~ ~ ~ , ~ ~ ~ :

1.

2.

3.

4.

transport of cells to the surface of an uncolonized inert material or other cells (in the following called the substratum; Fig. 6); initial reversible adsorption to the substratum by physicochemical forces (Fig. 7); irreversible adhesion of the cells to the substratum by microbial appendages and /or polymers attaching the cell to the substratum (Fig. 7); and multiplication of the cells and development of the granules (Fig. 7).

The cell can be transported to the substratum by any one or a combination of the following mechanisms: diffusion (Brownian motion), advective (convective) transport by fluid flow, gas floatation, sedimentation, or active transport due to flagella, i.e., m ~ t i l i t y ' ~ , ~ ~ , ' ~ ~ (Fig. 6). However, in flowing aquatic systems like the UASB, it seems doubtful that motility would account for any significant transport, except over very small distance~.".'~ The initial adsorption can be achieved through a collision between a microbial cell and the

Active movement

Substratum

Figure 6. The different transport mechanisms for a cell to a substratum.

Figure 7. (a) The reversible association of two bacteria becomes irreversible adhesion. ECP are used to bind the two bacteria to each other. (b) Cell division provides sister cells that are bound within the ECP. (c) Microcolony formation. (d) Granulation is a function of cell division within the microcolonies and new recruitment of bacteria from the liquid.

surface macromolecules. The substratum can be bacterial aggregates present in the sludge, but also inert organic or inorganic materials can function as growth nu- clei. This could be, e.g., precipitations or straw in the sludge.

Initial adsorption is usually a reversible physicochemical process but can lead to immobilization of the bacteria. The adsorption can be described, as a first approxi- mation, by the Derjaguin-Landau-Verwey-Overbeek (DVLO) the~ry . ' ?~ ' "" .~~ This theory can describe the observed change in the reversible cellular adhesion due to changing ionic strength, e.g., Ca2+/Mg2' concentrations, and substratum interfacial physics. According to this theory, three situations are possible between the


cell and the substratum: (1) a weak, reversible attraction when cells are located a certain distance from the substratum (secondary minimum); (2) a repulsion when electrostatic interactions dominate; and (3) a strong irreversible attraction when van der Waals forces are dominating (primary minim~m).~ ' Generally, the initial adsorption takes place in the secondary minimum. The strength of adsorption depends on different physicochemical forces like ionic, dipolar, hydrogen bonds, or hydrophobic interactions. The strength and number of interactions can vary considerably depending on the cell and the substratum."' For adhesion of the bacteria to the substratum more specific bonds are required.

The irreversible adhesion is established by strong bonds between the substratum and the microbial cells via bacterial fimbria, polymers, and other holdfast structures. It is not clear whether the bacteria first adhere reversibly and then produce ECP, or initially make ECP and then adhere irreversibly, i.e., if the genes for fimbria and polymer production are expressed before or after adhesion. lZo

When the bacterium is adhered, colonization has started. The immobilized cells start to divide within the ECP matrix so that the progeny cells are trapped within the biofilm structure. This results in formation of microcolonies of identical cells. The granulation process depends on cell division and recruitment of new bacteria from the liquid phase. The granular matrix can also contain trapped extraneous macromolecules (e.g., precipitates)30 The organization of the bacteria in the granules can ease the transfer of substrates and products.

tion. The granules are developed from a precursor consisting of small aggregates of Methanosaeta spp. and nonmethanogenic bacteria, typically acidogens.1".'21 This is supported by the observation that small aggregates have a high ratio between acidogens and acetogens compared to developed g r a n ~ l e s . 4 ~ ~ ~ ' f ' ~ ~ ' ~ ~ Other investigators have pointed out the importance of ECP- producing bacteria during the initial granulation process.633.1 17,119,122

Effect of Granulation on the Physiology of the Bacteria

Little is known about the physiological changes that takes place in an organism either during or after adsorp- ti011.l~~ However, the physiology of bacteria in the granules is of great importance for the control of granulation and interactions between the granules and the environment. The possible effect of the bacterial cell state on cell activity and resistance to environmental stress is given in Table V. Many factors influence the granulation process. These factors may be intrinsic to the cell: its genetic makeup or its stage in the cell cycle. Other factors could be nutritional or stress. The influence of the abiotic environment on the granulationidisintegra- tion process could be the ionic strength, hydrogen-ion concentration, cation concentration, temperature, mix- ing, etcZ1

CONCLUSIONS

The arrangement may depend on local hydrophobicity, local presence of polymers, or cell geometry.

The development of granular sludge is the key factor for successful operation of UASB reactors. Today it is possible to develop granules on a variety of different

Bacteria Involved in Granulation

Various theories regarding the starting point for granule formation have been proposed. Several research- ers70,96,1 59.160 h ave suggested that a loose structure of filaments of Methanosaeta spp. cells is the precursor for granules. These filaments can function as a nucleation center for further development of the aggregate. Other researchers have suggested that during initial granulation, Methanosaeta spp. colonize the central cavities of Methanosarcina clumps.32 This is supported by observations of small, presumably young granules with a core of Methanosarcina spp.

suggested that the first aggregating bacteria would be those producing acetate. These bacteria will provide Methanosarcina spp. or Methanosaeta spp. with the required substrate. The acetate producers include the fermentative and hydrogen-producing acetogens. The hydrogen-producing acetogens require a syntrophic association with hydrogen using bacteria to degrade substrates such as propionate or butyrate.

Other researchers have pointed out the importance of nonmethanogenic bacteria during the initial granula-

MacLeod et

wastewaters and defined media, but theie have been several reports on lack of granulation on specific wastewaters. Furthermore, some researchers have reported sudden disintegration of granules without any obvious reason.

In recent years, a more pronounced understanding of the microbiology of immobilized anaerobic bacteria and the mechanism of granule formation has been accom- plished. The shape and composition of granular sludge can vary significantly. However, extracellular polymers in the granular sludge are important for the structure and maintenance of the granules, but the inorganic composition seems to be of less importance.

For many years it was the general opinion that the aceticlastic methanogen, Methanosaeta, was critical for the structure and maintenance of methanogenic granules. However, several investigators have found that Methanosarcina spp. can have the same important role in granules. Other bacteria are also important for the granulation process, especially ECP-producing bacteria. Hydrogen-utilizing bacteria together with Hz-producing syntrophic bacteria are observed in microcolonies. The electron transfer in the microcolonies occurs via inter-


Table V. to environmental stress.

Effects of the bacterial state (dispersedlgranular) on cell activities and resistance

Free dispersed Granules Reference

~~

Cell activirie5 Uptake from bulk phase Cross feeding Genetic exchange

Metabolic sequence Environmental stresses

Abiotic Toxic substrates

Thermal stress

Phageivirus

PH

Biotic

Enhanced Depressed

?

Depressed

Sensitive Sensitive Sensitive

Sensitive

Depressed Enhanced Enhancedl

Enhanced depressed

Protected Protected Protected

Protected

5,15,23,33,68,149 41,51,62,68,71,121,123,143 15

41,51,68,71,121,123,158

13 15,68 43

68,99,110,154

species hydrogen transfer, while interspecies formate transfer may not be essential for interspecies electron transfer. The microcolonies give an optimal arrangement of the bacteria within the granules for effective degradation of syntrophic substrates such as propionate or butyrate. EL en greater internal organization has been observed. The granules can be interpreted as a spherical biofilm, and there exist many similarities between biofilm development and granulation. The initial adhesion of granules begins by initial adsorption of bacteria together or adsorption to inert material such as precipitates. Once thc bacterium is adhered, colonization has started. The granulation process depends on cell divi- sions and recruitment of new bacteria from the liquid phase.

Whenever using the UASB reactor for wastewater treatment, it is important to make a preliminary examination of the seed material and wastewater both micro- biologically and chemically. The microbiological analysis should give information about the microbial composition of the granules or sludge used and the ability to degrade the wastewater. The chemical analysis can give information about the wastewater composition, for instance if the necessary nutrients are present. Com- bined with pilot-scale experiments, this can result in the design of an effective anaerobic wastewater treatment system, avoiding sudden unwanted reactor failure.

The introduction of new abilities to granular sludge gives new possibilities for design of better granules, e.g., for bioremediation of contaminated groundwater containing xenobiotics. Also, engineered granules could be created with a higher resistance to the normal variations seen during treatment of wastewaters. Application of this reactor type will probably increase in the next de- cades with the increasing demands for a cleaner environment.

We would like to thank Irini Angelidaki, Hanne Vang Hen- driksen, and lndra M. Mathrani for helpful discussions, and Yang Gong for providing the scanning electron microscopy

picture. This work was supported by funds from the Danish Center of Process Biotechnology and the Nordic Ministe- rial Council.

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granular sludge formation in upflow anaerobic sludge blanket (uasb) reactors

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