Journal ofBwtechnology, 17 (1991) 221-232 © 1991Else~er SclencePubhshers B.V 0168-1656/91/$03.50 ADONIS 016816569100063F

221

BIOTEC 00578

Anaerobic upflow sludge blanket reactors: aspects of their microbiology and their chemistry

C.F. Fors te r

School ofCwd Engmeermg~ Btrmmgham Unwerstty, Edgbaston, Btrmmgham, U K

(Received 25 October 1989; revision accepted 1 July 1990)

Summary

The upflow anaerobic sludge blanket reactor is an effective high-rate process for wastewater treatment. However, for optimal performance, the biomass must be in a granular form. This paper discusses the ecological and chemical composition of sludges from several different reactors in relation to granule formation. The results indicate that biopolymers, calcium, phosphorus and the filamentous methanogen, Methanothrix , may play a significant role in the interactions which result in the formation of granules.

Anaerobic treatment; USB; Granulation; Microbiology

Introduction

Biological flocs in aerobic reactors have been an integral part of waste water treatment for most of this century. Over the last decade, however, considerable attention has been focussed on the anaerobic treatment of waste water, in particular, the high-rate treatment of strong industrial wastes. Anaerobic digesters convert the polluting organic matter, which is measured as chemical oxygen demand (COD), into methane and carbon dioxide. The design requirements of these reactors for high organic loading rates (kg CODm -3 d -1) meant that the biomass had to be immobilised and retained within the reactor. In one design, the anaerobic upflow sludge blanket digester (USB) (Fig. 1), the biomass is subjected both to hydraulic

Correspondence to C.F Forster, School of Cwd Engineenng, Umverslty of Birmingham, Edgbaston, Blrmangham B15 2TT, U K

222

~.f Effluent Gas ~ ~-] 6" ~ \ \ l

--Sepo.rat ion

Water Jocket I

~ i Sludge Bed

Feed Distributor Feed

Fig 1 SchemaUc diagram of a USB reactor

shear and to the up flow velocity of the liquid passing through the sludge bed. The sludge must, therefore, form itself into a highly flocculated state if it is to withstand these forces and be retained within the reactor. In a USB digester that ~s performing well, this highly flocculated state takes the form of discrete, compact granules which can be up to 5 mm in diameter (Lettinga et al., 1979). The settlement velocities of these granules are high for biological aggregates, 0.012 m s-m being typical values (Pette and Versprille, 1982). Because of this, considerable attention has been focussed on the criteria necessary for the successful start-up (and granulation) of USB reactors (Hulshoff Pol and Lettlnga, 1986). The conditions most likely to enhance start-up relate to the nature of the seed sludge, the waste water characteris- tics and the operational regime as well as environmental factors (temperature, pH, mixing). However, it does appear that adherence to these "rules" does not neces- sarily guarantee that the resultant biomass is any more than highly flocculent (Alibhai and Forster, 1986a).

Perhaps one reason for this is that the precise nutritional requirements for granulaUon of the bactenal population have not been elucidated, although there is evidence that certain n~neral elements are essential for optimum performance. Adequate quantities of nitrogen and phosphorus are, as might be expected, neces- sary. Souza (1986) suggests that a C O D / N ration below 70 and a C O D / P ration below 350 should suffice. The calcium concentration of the feed has also received particular attention as a factor which appears to influence granulation. Hulshoff Pol et al. (1983) found that influent calcium ion concentrations up to 150 mg 1 1 appeared to promote granulaUon, although no further improvement was observed at higher concentrations. Mahoney et al. (1987) have also reported the stimulation of granulation by feed Ca 2÷ concentrations up to 100 mg 1-1, whilst Alibhai and Forster (1986b) found that both calcium, at 80 mg 1-1, and phosphate, at 192 mg 1-1, improved granulation when added singly; when added together, granules of greater stability were produced.

223

The importance of micronutrients, or trace elements, is also not altogether clear at present, although work has been carried out in this area. Mah et al. (1978) found that growth of a Methanosarcma strain on a synthetic medium was stimulated by the addition of yeast extract and since ashed yeast extract proved almost as stimulatory as the whole extract, it was suggested that the effect was due largely to the addition of minerals. Of these, manganese and cobalt appeared to be significant growth factors. The effect of iron on the utilisation of acetic acid by a mixed methanogenic population has been investigated by Hoban and van den Berg (1979), who reported that the rate of acetic acid conversion was increased by the addition of iron (Fe2+), up to a concentration in excess of 300 mg 1-1. Work by other researchers suggests that iron may be a limiting factor m the growth of the thermophilic bacterium Methanobacterzum thermoautotropicum (Taylor and Pirt, 1977), which has also been found to require nickel, cobalt and molybdenum (Schonheit et al., 1979). The role of nickel in anaerobic processes has aroused much interest recently. There is evidence that methanogenic bacteria possess an essential cofactor, F430, containing a nickel atom in a tetrapyrrole structure (Whitman and Wolfe, 1980). Canovas-Diaz and Howell (1986) have published data which suggest that the presence of nickel may be essential for the satisfactory utilisation of butyric acid by mixed methanogenic cultures. In other studies of the effects of nickel on anaerobic digestion, alone and in combination with other trace elements such as cobalt and molybdenum, the results have indicated that nickel was the most stimulatory of these elements when added singly (Murray and van den Berg, 1981; Speece et al., 1983). The addition of a combination of trace elements produced an even greater effect.

Granule stability also appears to be influenced, at least in part, by the ecology of the granules and by the presence of polymeric material. Although aerobically produced bacterial polymers have received considerable attention, data related either to the formation or to the chemical nature of anaerobic sludge surface polymers is less widely reported in the literature.

Polymer chemistry

A study of a series of different anaerobic sludges obtained from Holland and the UK (mainly the SERC Anaerobic Facility, see Forster, 1988) showed that the yield of extracted polymeric matenal differed significantly depending on the nature of the sludge sample. All the samples, in particular the digested and granular sludges, yielded significantly less ECP than activated sludges. This is compatible with earlier data (Table 1) and may indicate that under anaerobic conditions fewer biopolymers are released, a suggestion supported by Karapanagiotis et al. (1989). Alternatively, Henning Ryssov-Nielsen (1975) showed that, under anaerobic conditions, bacterial biopolymers may be quickly degraded by the bacteria, forming CO 2 and C H 4 a s

byproducts. If true, this could explain why lower concentrations are present in systems where the availability of oxygen is limited. Another feature of the polymers

224

TABLE 1

Comparison of the ECP yields obtained from dlfferent sludges

Source Sludge type ECP yield (% TSS)

Kaff (1978) Activated 6.47 Forster and Clarke (1983) Activated 10.8 KarapanagmtlS et al. (1989) Digested 3 5 Dolflng (1986) Digesting granules 2.0 Morgan et al. (unpubhshed data) Various digesting sludges 2.5 Ross (1984) Digesting granules 4.0

extracted from anaerobic sludges is that, in general, they tend to have higher concentrations of protein (Table 2). The prevalence of protein fractions has also been found previously by Forster (1982) and Karapanagiotis et al. (1989) who quoted protein : carbohydrate ratios of approximately 3 : 1 for digested sludge ECP. The protein:carbohydrate ratios showed a linear relationship (Fig. 2) with the surface charge; the correlaUon coefficient being 0.66 (which was significant at the 95% level). These differences between the gross chemical nature of aerobic and anaerobic biopolymers suggests that in the latter system the different metabolic processes result in the production of polymers which not only are released less readily but are also chemtcally different.

In a study into the physico-chemical and biological characteristics of the blomass from USB reactors, a series of granules, obtained from Dutch digesters, was compared with a non-granular sludge (Alibhai and Forster, 1986a). This latter sample was produced in a laboratory-scale reactor which was treating spent liquors from the production of bakers' yeast. The physico-chemical properties examined in this study were the size, the surface charge, the specific surface area and the stability of the particles (Table 3). In addition, the ash content was also measured. These results show that there is a distinct difference, as would have been expected, between the physico-chemical properties of granular and non-granular sludges. The surface charge and specific surface area of the granular sludges are all significantly

TABLE 2

Companson of the protein-carbohydrate rauos m sludge ECPs

Source Sludge type Protein carbohydrate

Horan and Eccles (1986) Forster and Clarke (1983) Karapanag~otis et al (1989) Forster (1982) Ehhnger et al (1987)

Morgan et al. (unpubhshed data)

Activated 0 16 Acuvated 0 56 D~gested 3.0-3 6 Digested 5 1 Digested

glucose fed 0.61 VFA fed 2 31

Various &gestmg sludges 1 1-2 8

225

0

t~

t - O

o

c

0

t)_

3O \

x

2 \ \ \

\ ~x

\ \ • 2 0

. . . . . - 4 k 0 0 •

1 0

0 5 , ,

\ \

-0'2 - 0 6 - 0 8

Surface charge (meq/g)

I - 1 0

Fig. 2. Relatxonslup between the sludge surface charge and the protein, carbohydrate ratio m the extracted ECP, showing the regression hne and the 95% envelope.

lower than the values obtained for the non-granular sludge, whilst the stability is greater. These results are all compatible with the granules having a compact structure made up of close packed bacteria and the non-granular sludge being a flocculent material with an amorphous structure. Indeed, the value obtained for the specific surface area of F1 is not dissimilar to that of 142 m 2 g - ] reported for activated sludge. However, the results also show that there are differences between the various granular sludges. Thus, G3 and G4 had a much lower stability; they disintegrated after two months on storage at 5°C in distilled water; and G4 had a lower surface charge and higher specific surface area than the other samples. The

TABLE 3

Physlco-chemacal charactenst]cs of sludges from USB reactors (from Ahbhal and Forster, 1986a)

Sludge Size Ash-content Stab]hty Surface charge Specific surface area (mm) (%, w / w ) (months) (mV) (m 2 g- ] )

Granular Dutch, G1 1.6 30 3 Dutch, G2 2.2 21 7 Dutch, G3 1.7 22 0 Dutch, G4 2.6 45 8 Dutch, G5 1.0 24 3

Non-granular Lab-scale, F1 1.9 35 2

> 6 - 2 3 5 76.1 > 6 - 24 2 81.0

2 - 10 5 87 8

2 - 1 4 2 119.1

> 6 - 19 0 115.0

0 03 - 30 0 160 7

226

E

o

0 UJ

100

m /

80 / /

/ o / /

6 0 / / ~ / /

40 ..... -- • 1-

20 • . ~ /

/ I / / I I ' -0 2 -0 4 - 0 6 - 0 8

I - 1 0

Surface charge (meq/g)

Fig 3 Relationship between sludge surface charge and the blopolymer ywld, showing the regression hne and the 95% envelope

low surface charge was attributed to neutrahsatlon by some of the lnorgamc material responsible for the high ash content. However, the high values of the specific surface area of both G4 and G5 are anomalous and quite out of keeping with a close-packed granular structure.

In a separate unpublished study, a relationship between the concentration of surface blopolymers and the sludge surface charge has also been demonstrated (Fig. 3). The correlation coefficient for this relationship was 0.66 which was just signifi- cant at the 95% level. Magara and Nambu (1976) have demonstrated the influence that increased biopolymer synthesis had in increasing the surface charge, measured by increased electrophoretic mobility. High concentrations of anionic surface blo- polymers can consequently be correlated with deteriorating sludge settling char- acteristics because of the influence of floc-repulsion. Bearing this m mind, it is interesting to note that the haghly dispersed samples from the SERC pilot-plant carried relatively high negative charge compared with granular sludge solids and ECP extracted from the granules.

Microbiology

In a comparative study into the ecology of the bmmass from a range of upflow sludge blanket reactors, Methanothnx was found to be present in all the stable granules that were exanuned, whereas in loosely aggregated non-granular sludges, the dominant species was a thin filamentous strain thought to be either Methano- bactertum bryantn or Methanosptnllum hungatel (Ahbhai and Forster, 1986a). Other workers have also noted the importance of Methanothnx in determining the morphological development of the biomass in UASB reactors. Similar results have

227

been reported by Wei-Min et al. (1985) although these workers found that, in addition to Methanothrtx, Methanosarcma and Methanobacterlum formtctum were dominant in granular UASB sludges.

The ecology of the biomass in anaerobic reactors is not only important in determining the initiation and the stability of the immobilisation processes, but it is also a significant factor in defining the rate at which acidification and methanogene- sis take place. Lettinga et al. (1985), for example, have suggested that granules dominated by Methanosarcma are an undesirable development in USB reactors because of the low activity of this species at low acetate concentrations. Hulshoff Pol et al. (1983) have also noted differences in the activity of USB granules which were dominated by different microbial species; "rod-type" granules having a slightly lower activity than "filamentous" granules. Ecological selection within the biomass of an anaerobic reactor can be influenced by a variety of factors. Two obviously influential aspects are the hydrauhc (both liquid and gaseous) loading rates and the nutrient concentrations. During the start-up phase, the former wdl select those species which can form aggregates with higher settling velocities than dispersed species. Since the "long rod and filament" group have frequently been found in granules, it must be assumed that the species within this group have the ability to initiate biomass lmmobilisation. The main nutrient involved in the selection of methanogens is acetate and it has previously been estabhshed that the growth of Methanosarcma will be promoted at high acetate concentrations, rather than that of Methanothrtx. However, an examination of the start-up of the USB reactor at the SERC Anaerobic Facility showed that the Methanothrtx: Methano-

Fig 4. Electron rmcrograph of a sucrose-fed granule surface showang a diverse ecology.

228

Fig 5 Electron-rmcrograph of an acetate-fed granule surface showing a dorrunatlon by Methanothnx sp

s a r c l n a ratio showed no relationship to the concentration of acetate in the USB reactor. Also, m a separate laboratory-scale study, a change in the carbonaceous component of the feed from sucrose to sodium acetate resulted in a major alteration in the mlcrohiology of the granules; from a diverse ecology of rods, cocci and filaments (Fig. 4) to one which was donunated by a filamentous species whose morphology was apparently identical to that of M e t h a n o t h r t x (Fig. 5). This same laboratory study showed that whilst the granule morphology could change, the granule stabihty did not appear to alter significantly.

Metal composition

Electron dispersive X-ray analyses (EDAX) were used to examine the elemental composition of the surfaces of granules (Alibhai and Forster, 1986a). Sectioned granules were also examined. The results obtained for the Dutch granules (see Table 3) showed that there were distinct differences in the elemental make-up (Table 4). The analysis of the Gl-core gave no information about its composition relative to the components of other regions of the granule. However, the G5-core contained high levels of calcium which were not found elsewhere in the granule. On the basis of the results reported in Table 4, it seems reasonable to suggest that precipitated or msoluble lnorgamc compounds, in particular sulphides and silicates, are an integral part of the sludge structures. Sulphur (possibly existing as sulphide) also is present in the flocculent sludges produced in the laboratory USB reactors (Table 4). These

TABLE 4

Dominant elements in USB granules as detertmned by EDAX (Ahbhai and Forster 1986a)

229

Sludge Dormnant elements

G1 G2 G3 (34 G5 F1

Potassium; alumtraum a, sulphur s~hcon a

Potassmm; iron; sulphur Potassium; sulphur Potassmm, sulphur Calcmm b; potassmm; sulphur slhcon Potassmm, sulphur; sihcon

a Only at the granule surface. b Only at the granule core

results could be interpreted in either of two ways; that sulphides are not related to granule formation and structure at all, or, that there is a sequential progression from dispersed to flocculent to granular sludge with insoluble sulphides acting as nuclei for the process. Until a better understanding of the granulation process is achieved, this question will remain unresolved.

The EDAX technique was also used to examine the elemental composition of the polymers extracted from two USB sludges (one obtained from Aberdeen, the other from Holland). These polymers were ashed prior to analysis. In both cases, the dominant elements were calcium and phosphorus (e.g. Fig. 6). However, the molar ratios of these elements were different. In the ECP obtained from the Aberdeen granules, the C a : P ratio was 1:1 .2 whereas in that originating from the Dutch granules it was 1 : 3.6.

ABERDEEN GPANU LES , r

100~0 ' /

~_'~0

70

~_ 5O

40

20

/

0 NA Id?- AL P S CL K CA FE

ELEMENTS

Fig. 6. Typical EDAX spectrum for ashexi ECP.

230

Conclusions

Although the USB process demands that, tf optimal performance is to be achieved, the sludge must be fully granulated, the tdeal chemacal and rmcroblal composit ion of granules is yet to be determined. It may be that there is no unique ecology which defines a stable granule However, current reformation does suggest that certain species (particularly Methanothrlx and possibly Methanosarcma) may play a significant role in granule formation. What is not known is whether this role is one associated with microbial attachment or with the production of polymers which could act as a matrix for the granule structure. It is also not clear whether lnorgamc material has any particular function either as the nuclei for granules or as agents for cross-linking between polymers. It can be seen, therefore, that the understanding of granule formation is far from being complete and that a consider- able amount of work wall be necessary before a full biotechnologlcal control of granule formation (and stability) is possible.

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