Bioresource Technology 59 (1997) 37-43

1997 Elsevier Science Limited All rights reserved. Printed in Great Britain 0960-8524/97 $17.00 ELSEVIER

PII:S0960-8524(96)00134-4

AN INVESTIGATION INTO MICROBIAL REMOVAL OF FATS, OILS AND GREASES

N. G. Wakelin & C. F. Forster*
School of Civil Engineering, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK (Received 13 August 1996; revised version received 14 September 1996; accepted 19 September 1996)
Abstract

Fast-food restaurants generate grease-containing wastewaters for which there is, currently, no acceptable treatment technology. The development of microbial cultures for use in a bioreactor could, therefore, provide effective treatment of these wastewaters. Thus, the growth of a range of pure and mixed cultures was examined using vegetable oils, lard and 'grease' from a fast-food restaurant grease-trap. The pure cultures were Acinetobacter sp., Rhodococcus rubra, Nocardia amarae and Microthrix parvicella and these were compared with a mixed culture isolated from a greasetrap, MC1, and with activated sludge. The effectiveness of these cultures was assessed in terms of their greaseremoval efficiency, the biomass production and yield coefficients. Acinetobacter was the most effective of the pure cultures, typically removing 60-65% of the fatty material whose initial concentration had been 8 g/l. The effectiveness of the mixed culture, MC1, was variable, with the removal efficiency ranging from 29% for rapeseed oil to 73% for the restaurant grease. The activated sludge gave a more consistent removal, which was generally better than 90%. However, there was a lag phase of about 1 day in every case. Acclimatised activated-sludge did not exhibit a lag phase and also achieved a high ( > 90%) removal efficiency. The absence of a lag phase resulted in faster growth and fat removal 1997 Elsevier Science Ltd. All rights reserved. Key words: Fats, oils, grease, biodegradation, fast foods, removal efficiency.
INTRODUCTION

Fats and oils are essentially triglycerides consisting of straight-chain fatty acids attached, as esters, to glycerol. The component fatty acids of edible fats and oils vary considerably. They can differ in chain length, may be saturated or unsaturated, and may contain an odd or even number of carbon atoms. The term 'grease', as commonly used, includes fats, *Author to whom correspondence should be addressed. 37

oils, waxes and other related constituents found in wastewater. However, in this paper, the term 'FOG' has been used to represent, collectively, the vegetable and animal fats which could be associated with the wastewater from a fast-food restaurant. Aerobic FOG removal by microorganisms has been documented by several workers (Nunn, 1986; Ratledge, 1992). The initial attack on triglycerides by microorganisms is extracellular and involves the hydrolysis of the ester bonds by lipolytic, hydrolytic enzymes (lipases) which remove the fatty acids from the glycerol molecules of the triglycerides. Lipases can be highly specific (Shimada et al., 1992) and, therefore, attack triglycerides containing specific fatty acids. Alternatively, they can be totally nonspecific (Anon, 1993a) and attack triglycerides containing different fatty acids. After entry into the cell, fatty acids are either catabolised or directly incorporated into complex lipids. The main pathway for the oxidation of fatty acids involves repetition of a sequence of reactions, which results in the removal of two carbon atoms as acetyl-CoA with each repetition of the sequence: beta-oxidation. Tan and Gill (1985; 1987) studied FOG removal by different microorganisms in batch-growth studies, and reported that removal could be significantly affected by the substrate specificity of the induced extracellular lipases, the physical and chemical characteristics of the substrate, and the pH of the culture medium. The fast-food industry is one which has extensive problems related to the treatment of grease-containing wastewater, generated by its daily kitchen activities. The fatty acid composition of the effluent grease will depend on the menu, and the types of cooking fats and oils used in the kitchens. Despite the existence of diverse products, such as greasetraps and biological/nutrient supplements, available to fast-food restaurants for tackling grease-related problems, none is considered by the restaurant managers to deliver an adequate performance. This would suggest that the operators of conventional grease-traps and those using biological/nutrient supplements should be highly receptive to any com-

38

N. G. Wakelin, C. E Forster

mercial development of microbial cultures for use in a bioreactor for the treatment of fast-food-restaurant wastewater. Various microorganisms have been used for the treatment of grease-containing restaurant wastewater (Anon, 1993b; 1994a) and other grease-containing wastewaters (Ansenne et al., 1992; Anon, 1994b). The aim of this research was, therefore, t o identify a pure or mixed culture capable of effective FOG removal in batch-growth by conversion of freefloating FOG from a two-phase into a single-phase liquor. The most effective pure- or mixed-culture could then be used for future bioreactor studies for the treatment of fast-food-restaurant wastewaters.

METHODS Microbial inocula

Four pure cultures were used as microbial inocula for these batch growth studies: Rhodococcus rubra (Pollution Research Unit, UMIST), Acinetobacter sp. (NCIMB 11742), Nocardia amarae (NCIMB 11222) and Microthrix parviceUa (School of Civil Engineering, University of Birmingham). In addition, two mixed cultures, culture MC1 (Viridian Bioprocessing Ltd, Thanet Way, Whitstable, Kent) and activated sludge were used. The reasons for selecting these bacteria were that several workers have suggested that appreciable amounts of grease in the influent raw wastewater may be responsible for promoting the selection of specific filamentous actinomycetes, Rhodococcus sp., Nocardia amarae and Microthrix parvicella, as the dominant microorganisms in activated sludge and the subsequent formation of stable, viscous foams in the surface of activated-sludge aeration-tanks at sewage treatment works (Greenfield et al., 1985; Goddard & Forster, 1987). The use of Acinetobacter sp. for grease removal from wastewater has also been documented (Anon, 1994b). The cultures were stored on slopes with media specific for their growth (Table 1). Inocula for the growth studies were developed in specific liquid

media (200 ml) (Table 1) in conical flasks (250 ml). After an asceptic inoculation, these flasks were incubated on an orbital shaker (105 + 2 rpm) at 28 + IC for 3 days. Culture MC1 was an unidentified mixed culture which had been isolated by staff at Viridian Bioprocessing Ltd from grease-trap residues obtained from a fast-food restaurant. It was used by Viridian Bioprocessing Ltd as a culture for treating fat-rich wastewaters and was donated to Birmingham as a culture in treated wastewater. The activated sludge was obtained from the aeration tank at a local sewage-treatment works, where the raw wastewater contained an element of greasy material. Culture MC1 (100 ml) and activated sludge were initially centrifuged (2355 g) for 30 s, and the individual supernatants were removed. The pellets of biological solids were then resuspended to their original volumes in quarter-strength Ringer's solution. This procedure was repeated two more times, in order to remove all traces of residual substrate carbon from the inocula.
Preliminary growth studies

Initially, the performance of Rhodococcus rubra, Acinetobacter sp., Nocardia amarae and Microthrix parvicella was examined using different concentrations of corn oil (4, 8 and 12 g/l) as sole carbon sources. The results were used to select the FOG concentration for use in subsequent bioreactor inoculum selection studies.
Bioreactor inoculum selection studies

The performance of Rhodococcus rubra, Acinetobacter sp., Nocardia amarae, Microthrix parvicella, culture MC1 and activated sludge using corn oil, olive oil, linseed oil, coconut oil, rapeseed oil and fast-food-restaurant grease was examined. In addition, batch-growth studies involving sunflower oil, castor oil and lard were carried out using only the mixed cultures. The cultures, obtained at the end of the activated sludge studies, described as 'acclimatised activated sludge', were subsequently reinoculated into fresh culture media, in order to

Table 1. Composition of the growth media used in the preparation of inoculum shake-flasks for preliminary and bioreactor-inoculum selection studies

Species
R. rubra Acinetobacter sp. N. amarae M. parvicella

Source Khan & Forster (1988) Milligan (I 992) Baumann et al. (1988)

Component Czapek Dox medium + Yeast extract Glucose + Yeast extract Fructose + Yeast extract + Peptone Tween 80 + Lab-lemco powder + Peptone

Concentration

(~)
2 10 5 ! 5 I0 5 3 3

33.4

Microbial treatment of fatty wastewater

39

examine whether an improved performance with respect to growth and F O G removal could be achieved. All these tests used a FOG concentration of 8 g/1. Conical flasks (250 ml), containing culture medium (200 ml), were prepared, and a magnetic flea was added to each flask to ensure that representative samples of free-floating biomass and FOG could be removed from the culture media. The growth media used in all the studies consisted of M9 salts (27.2 ml/1 Solution A+2.72 ml/l Solution B+0.27 ml/! Solution C; Table 2), to which the F O G component was added. This was supplemented with ammonium sulphate to maintain an excess of nitrogen so that all the FOG-carbon could be utilised. The flasks were autoclaved at 121C and 15 psi pressure for 20 min prior to inoculation. The selection of the F O G substrates was based on the fact that each substrate provided a different proportion of saturated, unsaturated and polyunsaturated fatty acids. Grease-trap residue, supplied by a local branch of a major fast-food restaurant chain in Birmingham, was initially melted at 80C for 3 h, and the upper hydrophobic grease-layer, subsequently referred to as 'fast-food-restaurant grease' (FFRG), was removed for use in the batch-growth studies. The shake flasks were inoculated (5%, v/v) with the pure and mixed cultures: pure cultures in their growth media or the mixed cultures in Ringer's solution. The flasks were then placed on an orbital shaker (105 + 2 rpm) 28 + 1 C for 7 or 8 days. When lard was used, the temperature was raised to 30+ IC. The performance of the different bacteria was evaluated by taking samples (10 ml) periodically and measuring the total suspended solids concentrations (TSS). With the bioreactor-inoculum selection studies, TSS and FOG concentrations were measured. Representative sampling was ensured by placing the shake flask, containing a magnetic flea, onto a magnetic stirrer pad (Baird & Tatlock, Chad-

well Heath, Essex). The stirrer speed was set at 80% of its maximum rate.
Analyses FOG and TSS concentrations were measured from the same sample. In this way, FOG could be removed from the sample prior to the TSS analysis. FOG concentrations were measured using the standard partition-gravimetric method with 1,1,2-trichlorotrifluroethane (5520D; Greenberg et al., 1992). TSS concentrations were measured using the standard gravimetric method (2540 D; Greenberg et al., 1992). During FOG analysis, the hydrophobic phase from the separatory funnel was passed through a filter paper, therefore removing any hydrophobic microorganisms from the sample. At this point, the filter paper was transferred to another buchner apparatus, where the remainder of the contents of the separatory funnel, the hydrophilic phase, was filtered, therefore removing the remaining hydrophilic microorganisms from the sample. Assuming that the FOG-containing hydrophobic phase left a negligible residue of FOG on the filter paper, the gain in weight of the filter paper would be due to the presence of suspended solids in the sample. Control tests for the measurement of TSS with 1,1,2-trichlorotrifluoroethane showed that there was a negligible FOG residue [of < 1 % (w/w): n = 10] on the filter paper for samples containing _<100 g/l FOG, and there was a negligible reduction in the TSS concentration [of <3%: n = 10] for samples containing a variety of hydrophobic and hydrophilic microorganisms. RESULTS AND DISCUSSION

The effectiveness of any trial was assessed in terms of the maximum TSS yield (g/l), the FOG removal (%) and the yield coefficient (the mass of cells produced per unit mass of FOG removed).

Table 2. Composition of the component solutions for the M9 salts solution

Component Solution A (Miller, 1972) Solution B (Miller, 1972) Solution C (Bauchop & Elsden, 1960) Na2HPO4 KH2PO4 NaC1 MgSOa-7H20 CaCI2 MgO CaCO3 FeSOa'7H20 ZnSO4'7H20 MnSO4"4H20 CuSO4'5H20 COSO4"7H20 HsBO~ HCI

Concentration 6{1 g/I 30 g/I 5 g/1 0.1 M 0.01 M 10.75 g/l 2.0 g/I 4.5 g/l 1.44 g/I 1.12 g/I {).25 g/l 0.28 g/I 0.06 g/l 51.3 ml/I

40

N. G. Wakefin, C. E Forster Acinetobacter sp. had a significantly higher growth than the other species at all corn oil concentrations, with M. parvicella showing the worst growth. Although FOG removal was not measured during these growth studies, a possible explanation for the growth could be connected with the presence of a more effective lipase system in Acinetobacter sp., resulting in more corn oil being removed and metabolised, and hence an enhanced growth and higher TSS-yield values compared to the other pure cultures. The results from these preliminary studies suggested that 8 g/l would provide a suitable FOG concentration for use in subsequent bioreactorinoculum selection studies. Bioreactor-inoculum selection studies A comparison of the performances of the pure test cultures, using different FOG substrates, is shown in Fig. 2 and Table 3. A comparison of the TSS yield values (Fig. 2) with those obtained for the preliminary studies (Fig. 1) shows that, in relative terms, they were very similar. FOG removal by the filamentous species was generally poor, ranging from 17.5% for R. rubra/olive oil to 38.1% for N. amarae/coconut oil. The exception was the removal of the FFRG by M. parvicella. The biomass-yield values were also low, as were the yield-coefficient values. The results obtained with R. rubra could possibly be explained by the work of Khan and Forster (1991), who reported that fatty acids < C14:0, whether present as added substrate or as catabolites, were inhibitory to
3.5

The mean recovery of free-floating FOG substrates on day 0 of the bioreactor inoculum selection studies was 95.60+4.29% (n = 5), whilst the mean variability of replicate TSS yield, FOG removal and yield-coefficient values was + 4% (n = 5) for hydrophobic microorganisms and + 2 % ( n = 5 ) for hydrophilic microorganisms.
Preliminary studies Rhodococcus rubra has been shown to be hydrophobic in nature (Khan et al., 1991), as has N. amarae (Greenfield et al., 1985). This hydrophobicity resulted in R. rubra and N. amarae growing in intimate association with the various FOG substrates, resulting in the formation of agglomerations or 'balls' of biomass and FOG at the surface of the culture media. In contrast, Acinetobacter sp., M. parvicella, culture MC1, activated sludge and acclimatised activated sludge appeared to be hydrophilic cultures, which grew in association with the aqueous phase of the culture media. In this initial screening, performance was judged purely in terms of biomass productivity. The results (Fig. 1) showed that the growth of R. rubra, N. amarae and M. parvicella was proportional to the initial corn oil concentration. With the Acinetobacter sp., however, a concentration of 8g/1 gave the highest yield. This could imply that there was some degree of substrate inhibition by corn oil at 12 g/l, but, although this concept is supported by earlier. work (Ratledge, 1992), further work would be required to determine why only Acinetobacter behaved in this way.
3.0

3.0

2.5 2.5

2.0

ol

2.0

-o
4~

.o
m

> 1.5
th V)

E ._o
m 1.0

E o

1.G

1.0

0.5 0.S

4 8 12 C o r n oil c o n c e n t r a t i o n ( g / I )

Corn

Olive

Linseed

Coconut Rapeseed

FFRG

Fig. 1. Comparison of the biomass yields produced with corn oil as the sole carbon source,

Fig. 2. Comparison of the biomass yields produced with different FOG substrates.

Microbial treatment o f fatty wastewater

41

Table 3. Comparison of the performances of the pure cultures with different FOG substrates

FOG removal (%)

Acinet. N. am. M. par. R. rub. Acinet.

Yield coefficient (g/g)

N. am. M. pal: R. rub.

Corn Olive Linseed Coconut Rape FFRG

63.8 50.9 67.5 60.6 64.4 65.0

35.0 19.4 28.3 38.1 37.5 26.3

26.9 25.5 27.4 25.0 28.8 83.8

24.4 17.5 33. l 31.3 23.8 22.5

0.60 0.61 0.51 0.67 0.67 0.41

0.23 0.58 0.48 0.46 0.52 0.36

0.19 0.28 0.18 0.15 (I.22 0.35

0.52 (/.96 0.28 0.45 0.62 0.26

Acinet. = Acinetobacter sp. N. am. = Nocardia amarae. M. par. = Microthrix parvicella. R. rub. = Rhodococcus rubra. R. rubra. However, this explanation could not be applied to the growth of R. rubra on coconut oil,

which contained a high proportion of fatty acids < C14:0 (86.87%). The performance of M. parvicella on FFRG was exceptional, in comparison to that observed on the other FOG substrates. This difference in performance would suggest that some performance-enhancing chemical components were intrinsic to fast-food-restaurant grease. The low yield coefficient values for M. parvicella implied that a low proportion of the FOG substrate was incorporated into the growth of M. parvicella. The use of hydrophobic species, such as R. rubra and N. a m a r a e , could, because of the biomass 'bailing', create severe operational problems during future bioreactor studies for the treatment of fastfood-restaurant wastewater. Thus, care would need to be taken over the bioreactor configuration used for the treatment process. The overall performance of A c i n e t o b a c t e r sp. was generally good, with FOG removal values ranging from 51 to 67%. The biomass-yield figures and the yield-coefficient values were also, in general, better than the values obtained with the filamentous species. Overall, the results were consistent with earlier work (Anon, 1994b), and, therefore, confirmed the ability of A c i n e t o b a c t e r sp. to remove FOG substrates efficiently. A comparison of the performance of the mixed cultures: MC1, activated sludge and acclimatised activated sludge on different FOG substrates, is provided in Fig. 3 and Table 4. A typical comparison of the way the performance of the mixed cultures varied with time is shown in Fig. 4, using activated sludge and acclimatised activated sludge with olive oil as the example. Activated sludge achieved a better performance than MC1 which, in turn, was better than the pure cultures. The exception was the performance of activated sludge with castor oil as the substrate. In comparison to the results with the other FOG substrates, a relatively low biomass yield (3.63 g/i) and FOG removal value (65.38%) was obtained. A possible reason for this could have been the high viscosity of castor oil, which, despite the orbital shaking, could have been less effectively dispersed in

the culture medium than the other FOG substrates. However, the overall performance with activated sludge indicated that it was the most effective culture for treating FOG. This result is consistent with earlier work (Mulligan & Sheridan, 1975; Hrudey, 1981; 1982), which reported the ability of activated sludge to remove FOG substrates efficiently. Such high performance would promote the use of activated sludge as a microbial inoculum for future bioreactor studies for the treatment of fast-food-restaurant wastewater. The production of single-phase culture media (with the exception of the castor oil) suggested that the use of activated sludge would be conducive to grease-deposit-free sewer pipes and blockage-free restaurant floor-drains. The growth curves for activated sludge were characterised by a significant lag phase (ca. 1 day) and

E
O

Corn

Olive

Linseed

Coconut Rapeseed FFRG

Fig. 3. Comparison of the biomass yields produced by mixed cultures with different FOG substrates.

42

N. G. Wakelin, C. E Forster
Table 4. Comparison of the performances of the mixed cultures with different FOG substrates

FOG removal (%) MC1 Corn Olive Linseed Coconut Rape FFRG Sunflower Castor Lard 35.0 53.8 66.3 71.9 28.8 72.5 58.8 48.8 61.8 AS 97.5 96.9 97.9 87.4 96.0 98.0 97.5 65.4 95.8 AAS 97.0 95.4 98.5 95.5 97.3 95.6 99.6 52.4 93.6 MC1 0.44 0.69 0.61 0.64 0.75 0.47 0.69 0.58 0.39

Yield coefficient (g/g) AS 0.80 0.80 0.77 0.68 0.75 0.57 0.78 0.69 0.57 AAS 0.90 0.88 0.73 0.55 0.71 0.66 0.86 0.51 0.64

AS = Activated sludge. AAS = Acclimatised activated sludge. this was reflected by a minimal FOG removal during this period (Fig. 4). Lag phases have been observed by several workers for the growth of activated sludge on FOG substrates (Loehr & Roth, 1968; Malaney & Gerhold, 1969; Kramer, 1971). Lag phases have also been observed for the growth of various microbial cultures on different FOG substrates (Tan & Gill, 1987; Del Rio et al., 1990), but no significant lag phases were observed during the growth of the pure cultures in this current work. This implied that substantial changes in cell metabolism were not necessary for the effective utilisation of the various FOG substrates.
z Biomass [activated sludge] Oil

removal [activated sludge] removal [acclimatised activated sludge] [acclimatised activated sludge]
-8

-. ~,--Oil

-- o - - Biomass

:r

~s
C 0

$
o

~4
C

4~

g3

31
0

Acclimatised activated sludge also showed a high performance and the general pattern of growth and FOG removal was similar to that achieved with unacclimatised sludge (Fig. 4). Again, the exception was with castor oil. With this substrate, the performance was considerably lower for the acclimatised sludge than for the unacclimatised activated sludge. The reasons for this are unclear. The main difference between the two types of sludge was that growth of acclimatised sludge did not exhibit lag phases, and, as a result, growth and FOG removal proceeded at a faster rate. Acclimatisation of activated sludge to FOG substrates has been reported by several workers (Loehr & Roth, 1968; Novak & Kraus, 1973). Any activated sludge is a consortium of bacteria which can be dominated by different species. This domination can be dictated by the substrate. Therefore, the absence of a lag phase would suggest that there has been a selection of active lipase-producing species. It is suggested that the differences in the overall performances of the various cultures could be because of differences in the lipase systems. However, no correlation could be found between the individual performances for any microbial culture and the fatty acid composition of the FOG substrates. This could imply that the lipases produced by the various cultures were non-specific in nature. However, further work would be required to substantiate such a theory and provide possible reasons behind individual results. These results are in contrast to earlier work (Jonsson, 1976; Andersson, 1980; Shimada et al., 1992), which reported that differences in FOG hydrolysis could be attributed to differences in the substrate specificity of the extracellular lipases produced by the microorganisms. CONCLUSIONS

"&..

...............

0
0 1

~
2 3 Time

0
4 S 6 7

(days)

Fig. 4. Comparison of the performance of activated sludge and acclimatised activated sludge in the degradation of olive oil.

The use of a mixed culture such as activated sludge, particularly when it has been acclimatised to the FOG substrate, offers the best option for the treatment of fat-rich wastewaters. This is fortunate, since a pure culture would not be ideally suited to use in

Microbial treatment of fatty wastewater

43

any commercial, non-sterile, open bioreactor being used for the treatment of fast-food-restaurant wastewaters. There would be a high cost associated with the production of a pure culture and severe problems in maintaining the integrity of the culture in the reactor. Contamination of the sludge by opportunist strains, with the evolution of stable, mixed populations, would be unavoidable. The selection of a microbial culture on the basis of performance does not assess whether the biomass can tolerate perturbations, tolerate shock loadings of substrate or cleaning reagents or withstand periods of non-optimal growth conditions, for example, oxygen tension, pH and temperature.

ACKNOWLEDGEMENTS

This research was carried out as part of a BBSRCDTI LINK programme between the School of Civil Engineering and Viridian Bioprocessing Ltd, Whitstable, Kent, and the authors gratefully acknowledge the support given by Viridian Bioprocessing Ltd. The authors would also like to acknowledge the cooperation of the managers and staff at the Birmingham fast-food restaurant when a supply of grease-trap residue was required.

REFERENCES

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dida rugosa growing on olive oil. Biotech. Letters, 12, 835-838. Goddard, A. J. & Forster, C. F. (1987), A further examination into the problem of stable foams in activated sludge plants. Microbios, 50, 29-42. Greenberg, A. E., Clesceri, L. S. & Eaton, A. D. (eds) (1992). Standard Methods for the Examination of Water and Wastewater, 18th edn. American Public Health Association, American Water Works Association and Water Environment Federation, Washington, DC, USA, Greenfield, P. F., Pettigrew, A. E., Blackall, L. L. & Hayward, A. C. (1985). Actinomycete scum problems in activated sludge plants, Report No. 10. Department of Chemical Engineering, University of Queensland, Australia. Hrudey, S. E. (1981). Activated sludge response to emulsified lipid loading. Water Res., 15, 361-373. Hrudey, S. E. (1982). Factors limiting emulsified lipid treatment capacity of activated sludge. J. Wat. Pollut. Control Fed., 54, 1207-1214. Jonsson, U. (1976). Rates of hydrolysis of olive oil, soybean oil and linseed oil by Saccharomw;opsis lipolytica and Micrococcus caseolyticus. Chemie Mikrobiologie Technologie der Lebensmittel, 4, 139-146. Khan, A. R. & Forster, C. F. (1988). Biosurfactant production by Rhodococcus rubra. Environ. TechnoL Letters, 9, 1349-1360. Khan, A. R. & Forster, C. F. (1991). Aspects of the nutrition and the growth of Rhodococcus rubra in relation to the formation of stable foams. Environ. Technol., 12. 271-277. Khan, A. R., Kocianova, E. & Forster, C. F. (1991). Activated sludge characteristics in relation to stable foam formation. J. Chem. Tech. Biotechnol., 52, 383-392. Kramer, G. R. (1971). Hydrolysis of lipids in wastewatcr. J. Sanit. Eng. Div. ASCE, 97, 731-744. Loehr, R. C. & Roth, J. C. (1968). Aerobic degradation of long-chain fatty acid salts. J. Wat. Pollut. Control Fed., 40, R385-R403. Malaney, G. W. & Gerhold, R. M. (1969). Structural determinants in the oxidation of aliphatic compounds by activated sludge. J. Wat. Pollut. Control Fed., 41, R18R33. Miller, J. H. (1972). Experiments in Mok~cular Genetics. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York, USA. Milligan, W. A. (1992). Bacterial degradation of thin oil films on inland waters, M. Phil. Thesis, School of Chemical Engineering, Birmingham University, UK. Mulligan, T. J. & Sheridan, R. P. (1975). Treatment of high strength fatty acid derivative wastewatcrs. Proceedings of 30th Industrial Waste Cot~['erence, Purdue University, pp. 997-1004. Novak, J. T. & Kraus, D. L. (1973). Degradation of long chain fatty acids by activated sludge. War. Res., 7, 843-851. Nunn, W. D. (1986). A molecular view of fatty acid catabolism in Escherichia co6. Microbiol. Rev., 50, 179-192. Ratledge, C. (1992). Microbial oxidations of fatty alcohols and fatty acids. J. Chem. Tech. Biotechnol., 55, 399-400. Shimada, Y., Sugihara, A., Nagao, T. & Tominaga, Y. (1992). Induction of Geotrichum candidum lipase by long-chain fatty acids. J. Ferment. Bioeng., 74, 77-81). Tan, K. H. & Gill, C. O. (1985). Batch growth of Saccharomycopsis lipolytica on animal fats. Appl. Microb. Biotechnol., 21,292-298. Tan, K. H. & Gill, C. O. (1987). Utilization of substrates during batch growth of Pseudomonas .fluoreseens on olive oil, lard, and mutton tallow. Appl. Microb. Biotechnol., 26, 443-446.