Organic Loading Rate OLR

The OLR is related to the amount of "food" available for bacteria growth. In a microbiological sense, the OLR describes the degree of starvation of the microorganisms in a biological system. At a low OLR, microorganisms are subject to nutrient starvation, while a high OLR sustains fast microbial growth (Bitton, 1999). Research efforts have been dedicated to the role of organic loading rate (OLR), which is one of the most important operating parameters in anaerobic granulation process.

Evidence shows that anaerobic granulation can be accomplished by gradually increasing the OLR during the start-up (Hulshoff Pol, 1989; Kosaric et al., 1990; Campos and Anderson, 1992; Tay and Yan, 1996). It is critical to select a reasonably high OLR during start-up, to ensure rapid granulation and a stable treatment process. A simple and practical strategy for rapid start-up of anaerobic granular sludge reactors is to increase the OLR to attain only 80% reduction of biodegradable chemical oxygen demand (COD) with supplementary monitoring of effluent for washout of suspended solids (de Zeeuw, 1988; Fang and Chui, 1993).

An unconventional approach to accelerate start-up and granulation processes in UASB reactors has been developed by stressing the organic loading rate (OLR) without having to reach steady-state conditions (Show et al., 2004). The results indicate that the start-up of reactors could be significantly accelerated under stressed loading conditions. Startup times of the moderately and severely stressed reactors for operating at OLRs of 1 to 16 g COD/l.d ranged from 10 to 80 days and 13 to 90 days, respectively. Comparing with 17 to 120 days needed in the control reactor to reach the same OLRs, the start-up times were shortened by 25 to 41%. The extent of acceleration depends on the level at which the reactor are stressed. Applying stress and the extent of stress level in starting up the reactors did not reduce the reactor loading capacity, as all the reactors reached a similar maximum OLR of 16 g/l.d at the end of operation.

Development of granulation could be accelerated with the unconventional approach of stressed loadings as demonstrated by the results. Under stressed loading conditions, the sludge particles began to form granules earlier in both the stressed reactors after 24 and 30 days of start-up operation. Comparing with the control reactor without applying stress, the times taken to form granule were reduced by 45 and 32% in the severely and moderately stressed units, respectively. The granule formation occurred earlier in the severely stressed reactor than the moderately stressed unit.

While the results obtained had established significant acceleration in start-up and granulation processes, the characteristics of granules developed were greatly influenced by the level of stress exerted. Characterization of bioparticles revealed that the granules developed in the moderately stressed reactor exhibited superior characteristics in terms of settleability, strength, microbial activity and morphology, and granular sludge growth, as compared with both the control reactor operated without stress and the unit which was over-stressed.

Tay and Yan (1996) further proposed that the start-up operation of UASB reactors could be guided by a dimensionless parameter, namely microbial load index (MLI). The MLI is defined by the ratio of OLR applied to specific methanogenic activity (SMA) in terms of gram methane-COD produced by gram VSS per day. An MLI value of around 0.8 was proved appropriate for rapid UASB start-up and microbial granulation. It should be pointed out that the MLI indeed is proportionally related to OLR, i.e. the MLI represents the magnitude of OLR. Large Methanothrix-like species (thrix granules) were cultivated with 1000 to 5000 mg COD/l influent, and small Methanosarcina-like species (sarcina granules) were cultivated with 10,000 mg COD/l influent. The thrix granules with median diameters of 2.5 to 3.4 mm exhibited better settle-ability, higher substrate affinity, and slightly higher bioactivity than the 0.54 mm sarcina granules (Tay and Yan, 1996).

The OLR-associated negative effects have been observed in UASB operation practice. High OLR results in a reduced mechanical strength of granules, i.e. the granules would easily lose their structural integrity, and disintegration would occur (Quarmby and Forster, 1995). Increased biogas production accompanied with high OLR would eventually lead to disintegrated granular sludge being washed out from the reactor. When the best-known Monod model is applied to the UASB system, an increased OLR will raise proportionally the biomass growth rate (Morvai et al., 1992). High growth rate of microorganisms would reduce the strength of three-dimensional structure of microbial community. Such a phenomenon has been observed in biofilm reactors (Liu and Tay, 2001). On the other hand, biogas production is also proportional to the magnitude of the applied OLR. If the applied OLR is too high in the period of start-up of UASB reactor, increased biogas production rate would cause serious hydrodynamic turbulence and further leads to the washout of seed sludge from the reactor, which sometimes is a main reason of unsuccessful startup of UASB reactor. Table 2.1 shows some typical OLR values commonly used during the start-up of anaerobic granulation process, which provides some useful information on the OLR applied for UASB start-up.

Table 2.1. Some OLR values used for rapid start-up of UASB reactors (updated from Morvai et al., 1992)


OLR at start-up

Time required


(kg COD/kg

for granulation

VSS per d)





Hulshoff Pol et al. (1983)



Not observed

Hulshoff Pol et al. (1983)

Brewery wastewater



Wu et al. (1985)

Sucrose wastewater



Wu et al. (1985)

Molasses wastewater



Wu et al. (1987)

Sucrose wastewater



Sierra-Alvarez et al. (1988)




Morvai et al. (1992)

Molasses wastewater



Morvai et al. (1992)

Synthetic wastewater



Campos and Anderson (1992)




Ghangrekar et al. (1996)




Ghangrekar et al. (1996)

Characteristics of Seed Sludge

Theoretically any medium containing the proper bacterial flora can be used as seed sludge for granule cultivation. Common seed materials include manure, fresh water sediments, septic tank sludge, digested sewage sludge, and surplus sludge from anaerobic treatment plants. Apart from its availability and its cost, the quality of a particular seed material can be judged in terms of ash content, the specific methanogenic activity, and the settleability.

Aerobic activated sludge from a sewage treatment plant and primary sludge from an aerobic plant treating textile dyeing wastewater had been used (Wu et al., 1987). It was found that there were sufficient anaerobic nuclei present in the aerobic flocs. All important methanogens seem to be present in aerobic activated sludge. Existing granules can also become seeding alternatives. Quality of seed sludge with respect to specific activity, settleability, and nature of inert fraction are important for anaerobic granulation process.

Two different types of sludges may develop on the same medium depending on the source of the inoculum. Xu and Tay (2002) used methanol-precultured anaerobic sludge to inoculate a UASB reactor. This approach accelerated the formation of embryonic granules in a laboratory-scale UASB reactor. The granulation process reached its postmaturation stage about 15 to 20 days ahead of the control reactor.

In engineering sense, heavy and relatively inactive sludge was preferred over lighter, more active sludge because of expected differences in washout. de Zeeuw (1984) observed two types of sludge washout, i.e. erosion washout and sludge bed expansion washout. Sludge bed erosion washout represents the selective washout on the basis of differences in settleability. Sludge bed expansion washout predominately occurs when using a diluted digested sewage sludge in the treatment of a medium strength wastewater. It is caused by the expansion of the sludge bed as a result of the increased hydraulic and gas loading rates and involves little selection between sludge particles with a difference in settleability. By choosing a concentrated digested sewage sludge as seed the latter type of sludge washout can be avoided.

Although digested sewage sludge is usually used for the start-up of a UASB reactor, various other types of seed sludge can be successfully utilized when granular sludge for seeding is unavailable. Wu et al. (1987) utilized aerobic activated sludge from a sewage treatment plant and primary sludge from an aerobic plant treating textile dyeing waste-water. Apparently, sufficient anaerobic nuclei were present in the aerobic flocs.

Using a MPN technique for counting the methanogens, it was found that aerobic activated sludge contains 108 methanogens/g suspended solids (SS), while in digested sludge, Zeikus (1979) found a number of 108/ml, giving for a 4% (w/v) sludge a figure of 2.5 x 1010/g SS. All the important methanogens seem to be present in aerobic activated sludge. Other seed sludges that have been applied are lotus pond mud (Qi et al., 1985), cow manure (Wiegant, 1986), and primary sewage sludge (Ross, 1984).

The UASB system can also be started-up using existing granules whenever possible. This lends, in general, a decided advantage to the UASB process for start-up, although a successful start-up is not assured simply because granules are available. The inoculation with a large seed amount of granular sludge from a healthy UASB reactor is desirable. However, the availability of granular seed sludge is limited and the expenses for purchase and transportation of the inoculum are expensive. Addition of a small amount of granules to non-granular inoculum was still needed to stimulate the granulation process (Hulshoff Pol et al., 1983). This is probably a consequence of supplying an inoculum of microorganisms, which is responsible for granulation. On the other hand, Hulshoff Pol et al. (1983) reported that the addition of crushed granular methanogenic sludge to digested sewage in a UASB reactor fed with acetate plus propi-onate may give rise to the development of methanogenic sludge granules with a diameter of 1-2 mm. The observation that two different types of sludge developed on the same medium depending on the source of the inoculum, made in parallel experiments indicates that the formation of well settling conglomerates (i.e. granulation and pelletization) initially is a purely biological phenomenon.

The structures of anaerobic granules are closely related to the diversity of microorganisms. El-Mamouni et al. (1997) investigated the influence of four different granulation precursors, syntroph-enriched methanogenic consortia, Methanothrix-enriched, Methanosarcina-enriched nuclei, and acidogenic flocs on the development of anaerobic granules. It was found that granulation proceeded rapidly with syntroph-enriched methanogenic consortia, Methanothrix-enriched and Methanosarcina-enriched nuclei; however, granulation was significantly retarded when acidogenic flocs were used as precursors. The increase rate of granule size was 31 |xm/day for syntroph-seeded granules, 21 |xm/day for Methanothrix-seeded granules, 18 |xm/day for Methanosarcina-seeded granules, and only 7 |xm/day for acidogenic flocs-seeded granules. These results seem to suggest that syntrophs and Methanothrix species would play an important role in the formation of anaerobic granules. In fact, microbial species would differ in their capacity for aggregation, and some species are more competent for aggregation, but some are less under the same operation conditions. It seems certain that anaerobic granulation process can be expedited simply by manipulating the composition of seed sludge. This approach would be very attractive and beneficial to full-scale UASB reactor start-up. However, there is still lack of detail guidelines on which species in seed sludge should be a major component for anaerobic granulation and how to manipulate the species in seed sludge.

Characteristics of Substrate

Characteristics of feed substrate have been considered a key factor influencing the formation, composition, and structure of anaerobic granules. Based on the free energy of oxidation of organic substrate, the substrate can be roughly classified into two categories: high-energy and low-energy feeds. During the UASB start-up period, high-energy carbohydrate feeding can sustain the acidogens and facilitate the formation of extracellular polymers (Liu et al., 2002). Thus, the rapid growth of acidogens due to the presence of high-energy substrate in the influent would facilitate the overall process of sludge granulation in the UASB reactors.

Studies on mesophilic granule formation have shown that varied granular structures may be cultivated on different wastewaters and under different start-up conditions. Filamentous type granules, developed on mainly volatile fatty acid (VFA) feeds tend to be 5 mm in size and mechanically fragile. Those granules contain inert carrier material and are dominated by a highly filamentous form of Methanothrix, presumed to be M. soehngenii. More robust rod-type granules developed on sugar beet or potato processing wastewaters, and they contain no detectable inert carrier and are again dominated by M. soehngenii-like species, but in a much shorter chain-length of up to 3 mm in size (Adebowale and Kiff, 1988). The granules grown on VFA mixture (acetate, propionate, and butyrate)

under mesophilic conditions can be classified into three distinct types according to the predominant acetate utilizing methanogens (Hulshoff Pol et al., 1983; de Zeeuw, 1984; Lettinga et al., 1984): (a) rod-type granules, which are mainly composed of rod-shaped bacteria in fragments of about 4 to 5 cells resembling Methanothrix; (b) filament-type granules, which consist predominantly of long multicellular rod-shaped bacteria; and (c) sarcina type granules, which develop when a high concentration of acetic acid is maintained in the reactor.

Successful formation of very small thermophilic granules (0.2 mm) from a mixture of acetic, propionic, and lactic acids had been reported (Endo and Tohay, 1988), while larger aggregates of 3.0 mm in diameter were obtained by Bochem et al. (1982) in chemostat studies of acetate enrichments. Those granules consisted of densely packed Methanosarcina clusters surrounding a more loosely packed central area, which contained at least two non-methanogenic species.

A trend was observed towards a wider diversity of methanogenic sub-populations paralleling an increase in the complexity of waste composition. At least four distinct micro-colonies were observed in granules treating brewery wastewater (Wu, 1991). One of these micro-colonies was composed of Methanothrix-like rods only, while the other micro-colonies consisted of H2-CO2 utilizing Methanobacterium-like rods in juxtaposition with three different rod-shaped syntrophs (Hickey, 1991). Based on full-scale UASB experiences in treating a variety of different waste-waters, it has been established that granulation of anaerobic sludge takes place in many different types of wastewaters. With a substrate containing 10% sucrose and 90% VFA mixture (acetate plus propionate), granular and flocculent sludge cannot be effectively separated. The granules contained a high fraction of filamentous organisms that were mainly attached to inert support particles. A feed change from a VFA mixture to a carbohydrate solution may lead to problems of flotation and formation of a rather voluminous type of sludge if the granules are cultivated on acidified wastewaters.

Chen and Lun (1993) cultivated three types of anaerobic granules with acetic acid, glucose, and alcoholic stillage, respectively, and found that the properties of three types of granules were significantly different. The anaerobic granules fed with alcoholic stillage have the better physical properties in terms of density, SVI, and intensity. This is probably due to the complexity of the substrate constituents, which leads to an abundant microbial diversity in the granules. It must be realized that the energy containing in the substrate is important for anaerobic granulation, however the complexity of substrate would exert a selection pressure on microbial diversity in anaerobic granules as discussed earlier. Such a selection pressure would influence the formation and microstructure of granules.

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