j o u r n a l of MEMBRANE SCIENCE

ELSEVIER Journal of Membrane Science 119 (1996) 9-16

An enzymatic approach to the cleaning of ultrafiltration membranes fouled in abattoir effluent

A. M a a r t e n s a,* p. S w a r t
a

E.P. J a c o b s b

a Department of Biochemistry, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa b Institute for Polymer Science, University of Stellenbosch, Private Bag X1, Matieland, 7602, South Africa

Received 18 July 1995; accepted 20 December 1995

Abstract
Membrane fouling severely curtails the economical and practical implementation of ultrafiltration for the purification of biologically related process streams such as abattoir effluent (Jacobs, WRC Report no. K5/362, 1991, Pretoria, South Africa [1]). Mechanical and chemical removal of foulants usually lead to membrane damage and additional pollution. Enzymes, specific for the degradation of proteins and lipids, were tested as key components of biological cleaning regimes for membranes fouled in abattoir effluent. Fouling of polysulphone membranes was assessed as previously described by Maartens et al. (J. Membrane Sci., 119 (1996) 1 [2]) and optimal enzyme concentrations and incubation times were determined for the different preparations. The ability of each cleaning agent to remove adsorbed protein and lipid material, as well as their ability to restore the water-contact angle and the pure-water flux of the fouled membranes, were determined and compared. These variables were also used to compare the cleaning efficiency of enzymatic cleaning agents with conventional chemical agents under optimal conditions. The enzymes and enzyme detergent mixtures were effective cleaning agents and the pure-water flux of statically fouled membranes could be restored by treatment with these agents.
Keywords: Ultrafiltration; Membrane fouling; Cleaning; Abattoir effluent; Enzymatic cleaning

1. Introduction
When ultrafiltration (UF) membrane systems are used for the treatment of biological process effluents the main aim is to obtain the required water purification as economical as possible at acceptable flux rates. The realisation of this goal in the direct treatment of effluents is, however, severely hampered by membrane fouling [3]. The fouling problem has been previously approached from a number of angles * Corresponding author.

which included: the optimisation of flow conditions, pre-treatment of the effluent, the production of membranes with reduced adsorptive properties [3-7], the optimisation of operational factors [8] and the use of high-quality rinse-water [9]. All these methods yielded moderately satisfactory results but at a relatively high cost. An alternative approach to the fouling problem would be to reduce pre-treatment to minimum acceptable levels and to introduce extensive, but simple, membrane-cleaning protocols. Membranes are normally cleaned by a combination of mechanical a n d / o r [9,10] chemical [11]

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A. Maartens et al. / Journal of Membrane Science 119 (1996) 9-16

a n d / o r biological methods using alkalis [9,12,13], acids [9], surface-active agents [11,12], sequestering agents [6], disinfectants [6] and enzymes [1,11,14-16] as cleaning agents. Biological cleaning can be broadly described as the use of cleaning mixtures which contain bioactive agents (micro-organisms or enzymes) to enhance the removal of foulants. Substrate specificity and environmental friendliness have lead to an increase in the use of enzymes and enzyme detergent mixtures in cleaning mixtures for household and industrial use. Detergent or cleaning mixtures that contain enzymes have, however, not previously been used on a large scale for the cleaning of UF membranes fouled by biological process effluents. Enzymes are ideal cleaning agents for this purpose as they are highly specific for the reactions they catalyse and the substrates with which they interact. In addition, enzymes act under mild conditions of pH, temperature and ionic strength and will not damage the membrane surface. A number of factors have, up to now, prohibited the large-scale use of enzymes for cleaning membranes. Extension of membrane technology to effluent treatment of biologically related processes, as well as the increasing pressure on the environment has, however, compelled researchers to re-examine enzymes as possible cleaning agents for fouled UF membranes [1,14,15] and for the prevention of fouling by the adsorption of enzymes onto membranes [ 15,16]. This paper describes the use of enzymes and enzyme detergent mixtures for the removal of foulants from polysulphone membranes (PSM) statically fouled in real abattoir effluent. The nature and identity of the major membrane foulants present in abattoir effluent were previously determined and it was found that lipids and proteins are the major foulants in this effluent that adsorb onto PSM under these conditions [2]. It was also shown that adsorption alone can cause flux reduction as well as changes to the membrane surface character [2]. Statically fouled membranes were treated with different enzymes and enzyme/detergent mixtures to determine if these cleaning agents could restore pure-water flux. Four variables were used to evaluate the efficiency of the cleaning mixtures; the potential of a cleaning agent to remove protein and lipid material (potential foulants in abattoir effluent) adsorbed onto membranes (by analysis of the protein and lipid

concentrations before and after cleaning) as well as the ability of a cleaning mixture to restore the water-contact angle and pure-water flux of the fouled membranes.

2. Experimental
2.1. Materials
Enzymes used in cleaning experiments were obtained from Sigma Chemical Co., St. Louis, USA, unless otherwise mentioned, and included: protease A (type XXIII from Aspergillus oryzae (a serine protease with an optimal activity (3.5 units/mg protein) at pH 7.5 and 37C); lipase A (a crude porcine type II pancreatic lipase for the hydrolysis of triglycerides, with an optimal activity (37 units/rag protein using triacetin and 128 units/mg protein using olive oil) at pH 7.5 and 37C. The commercial agents, Alkazyme and Zymex ~ (formulated cleaning agents with proteolytic activity), were obtained from Syndachem Sales Ltd., Milnerton, South Africa. A 3% aqueous solution of Alkazyme:Zymex (1:1) was used at room temperature according to the manufacturer's specifications. Detergents used were sodium dodecyl sulphate (SDS) (2% and 4% solutions ( m / v ) in distilled water at 37C) and Triton X100 (0.1% solution ( m / v ) in distilled water at 37C). To create an artificial oil-water interface lipase A (1 m g / m l ) was dissolved in a 0.1% Triton X100 Clark and Lubs solution (pH 7.5). Reagents and membranes for the determination of the cleaning efficiency, were used as previously described [2].

2.2. Membrane fouling

Flat sheet PSM, cast from a solution of Udel P3500 polysulphone in N-methyl 2-pyrrolidone, were statically fouled in abattoir effluent for a period of 8 h as previously described [2]. After fouling, the membranes were washed thoroughly with tap water, placed in distilled water and preserved with sodium azide (5 mg/1) at 4C until cleaning experiments and analyses were done. All experiments were replicated 4 times and all final values reported are the mathematical means.

A. Maartens et al. / Journal of Membrane Science 119 (1996) 9 - 1 6

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2.3. Cleaning methods

Fouled membranes were cut into 3 0 10 cm strips, washed in the Clark and Lubs buffer (pH 7.5), and incubated in a 600 ml solution containing the cleaning agent or mixtures of cleaning agents. Cleaning agents were used at the optimal pH, and temperature to ensure maximum efficiency. After incubation the membranes were removed from the cleaning solutions, washed thoroughly with distilled water to remove all excess materials, and stored in distilled water at 4C until further analyses. Cleaned as well as reference membranes were analysed for protein and lipid content, contact angle changes and purewater flux changes as previously described [2]. The following reference membranes were used; an 8 h fouled membrane and a fouled membrane incubated only in the Clark and Lubs (pH 7.5) buffer for 1 h.

3. Results

The effect of increasing concentrations of protease A and lipase A on the removal of proteins adsorbed on to PSM fouled in abattoir effluent, is shown in Fig. 1. The protein content of fouled membranes decreased by 80% and an increase in protease A concentration beyond an enzyme concentration of 3 m g / m l , yielded no further significant
50

improvement in protein removal. Treatment with lipase A and a lipase A:Triton X100 mixture resulted in a 66% and 90% reduction in adsorbed protein concentration respectively. No significant increase in protein removal was observed at enzyme concentrations beyond 1 m g / m l . As expected, protease A was more effective in protein removal than lipase A alone, but when lipase A was combined with Triton X100, the combination yielded better results. The effect of incubation time on protein removal by the different enzyme preparations is shown in Fig. 2. Maximum protein removal from the fouled membranes was obtained after 60 min incubation with lipase A while an incubation period of 90 min yielded the best results for protease A and the lipase A:Triton X100 mixture (Fig. 2). The effects of enzyme concentration and varying incubation times of the different enzymatic cleaning agents on the lipid contents of fouled PSM are also represented in Fig. 1 and Fig. 2. The amount of adsorbed lipid material decreased with an increase in protease A and lipase A concentrations. It is interesting to note that protease A was more effective in lipid removal than lipase A alone at the higher enzyme concentrations. In combination with Triton X100 (0.1%), lipase A, however, yielded optimal lipid removal at a concentration of 2 mg enzyme/ml. Of the three preparations used, the lipase A:Triton X100 mixture was the most effective for lipid removal. The time course
350 300 I

I --" 40
e.

30

250

"-~

..,.%
200

2o

2
:::t.

150 0 0 i 1 i 2 100 3

Enzyme concentration (mg/ml)

Fig. I. Effect of enzyme concentration on protein and lipid removal from fouled PSM. Enzymes evaluated were: protease A ( ), lipase A ( ) and a lipase A:(0,1%) Triton X100 mixture (O). Incubation time was 60 min, at pH 7.5 and 37C.

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A. Maartens et al. / Journal of Membrane Science 119 (1996) 9-16

50 I --" 40

340 320?

3o

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

300

280 E 260 240;~

20
m lO ~o
:::L

220 ~
0 ~ ' 200

30

60

90

Incubation time (min)

Fig. 2. Effect of incubation time on protein and lipid removal from fouled PSM. Enzymes evaluated were: protease A ( ) , lipase A ( ) and a lipase Triton X100 mixture ( 0 ) . Concentration used was 1 m g / m l at pH 7.5 and 37C.

study shown in Fig. 2 shows that the lipase A alone requires a longer incubation time (90 min) to be most effective. After 90 min incubation with protease A the lipid content of the membrane, however, increased slightly. The lipase A:Triton X100 mixture gave maximum lipid removal after 60 min and 40% of the absorbed lipid material was removed, compared to approximately 25% removal with the other two solutions.
80

Changes in the water-contact angle of fouled membranes, after treatment with different concentrations of enzyme and enzyme detergent preparations and a time course study, are shown in Fig. 3 and Fig. 4. Increases in the lipase A and protease A concentrations produced lower contact angle values which agreed well with the decrease in lipid content observed for the same treatment (see Fig. 1). The membranes treated with the lipase A:Triton X100
1,600 ......................................... 1,400
_

70 _~ 60

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1,200
1,000

,.,
50 o 40 30 i 0 i 1 i 2 / ,.. .,J .........

J.------- 800
- 600 400

=x

0 3

Enzyme concentration (mg/ml)

Fig. 3. Effect of enzyme concentration on the contact angle and pure-water flux of fouled PSM. Enzymes evaluated were: protease A ( ) , lipase A ( 0 ) and a lipase A:(0.1%) Triton X100 mixture ( 0 ) . Incubation time was 60 min at pH 7.5 and 37C.

A. Maartens et al. / Journal of Membrane Science 119 (1996) 9 - 1 6

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80
/

1,400

40
30 I , ,

30

60

"0 90

Incubation time (rain)

Fig. 4. Effect of enzyme incubation time on the contact angle and pure-water flux of fouled PSM. Enzymes evaluated were: protease A ( ), lipase A ( , ) and a lipase A:(0.1%) Triton X100 mixture ( 0 ) . Concentration used was 1 m g / m l at pH 7.5 and 37C.

mixture, showed the greatest decrease in contact angle. The effect of incubation time with the different cleaning agents, on the contact angle of the fouled membranes showed the same pattern as lipid removal. Protease A treatment between 60 to 90 min resulted in an increase in lipid content with a concomitant increase in the contact angle. The pure-water flux through fouled PSM, treated with different concentrations of protease A and lipase A, is shown in Fig. 3. The pure-water flux increased with a decrease in lipid concentration and

a corresponding decrease in the contact angle of the fouled membranes. The lipase:Triton X100 mixture gave the best overall pure-water flux improvement. Although maximum flux recovery, by all cleaning agents, was achieved with enzyme concentrations of 3 m g / m l , this flux value was not significantly higher than the increase obtained by treatment with a 1 m g / m l enzyme concentration. The effect of incubation time on the pure-water flux through fouled PSM is shown in Fig. 4. A rapid flux increase was obtained after only 30 rain of incubation with the

Table 1 Comparison of the cleaning efficiency of different membrane cleaning protocols using the following four variables: removal of proteins and lipids, changes in water contact angle as well as the effect on the membrane surface and permeability character. Values presented are the mathematical mean of at least four determinations _ SD Cleaning treatment Unfouled membrane Fouled for 8 h Buffer 60 min SDS (0.2%) 60 min SDS (0.4%) 60 min Triton X100 (0.1%) 60 min Lipase A (3 m g / m l ) 60 min Protease A (3 m g / m l ) 60 min Alkazyme:Zymex (1 : 1) 3% solution, 60 min Lipase A:Triton X100 (3 mg/ml:0.1%) 60 min Lipase A:Triton X100 (1 mg/ml:0.1%) 60 min, followed by Protease A (1 m g / m l ) 60 rain Protein reduction (%) 0 2.62 51.33 66.84 76.48 57.37 85.80 95.90 94.74 90.08 Lipid reduction (%) 0 0.70 + 4.5 50.5 _ 5.15 56.50 5- 1.9 32.50 5- 1.5 31.90 + 1.2 55.0 5- 4.12 57.40 + 2.1 59.00 _+ 2.7 64.30 + 2.5 Contact angle (C) 63.36 _+ 1.6 74.5 -4- 2.5 74 + 2.08 73.24 :t: 4.1 65.53 + 3.9 49.21 + 2.9 50.15 _ _ _3.1 48.37 5- 1.4 41.84 _ _ _2.1 36.25 + 4.1 31.3 5- 2.05 Pure-water flux ( l / m 2 h) 829.16 + 20 233.3 + 17.2 332.18 + 14 332.23 + 5.1 493.72 + 18 681.3 5- 16.1 720.3 _ 15 800.20 -5- 14 1001.3 5- 20 1370 + 22.1 1382.7 + 17

5-- 2.37 + 9.5 + 7.10 + 7.10 5- 4,74 _+ 4.74 5- 1.24 5- 1.20 _+ 4.74

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A. Maartens et al. / Journal ~f Membrane Science 119 (1996) 9-16

different agents, after which there was no further significant increase in flux. For protease A, in fact, the flux decreased slightly between incubation times of 60 to 90 rain indicating possible re-adsorption of lipids (see also Fig. 2).

3.1. Comparison of enzymatic cleaning regimes with commercial and conventional cleaning agents
Table 1 shows the effect of different cleaning agents on the protein and lipid removal as well as the changes observed in the water contact angle and pure-water flux of PSM fouled in abattoir effluent. The different cleaning mixtures and conditions used are indicated in the table. The combination of lipase A:Triton X100 with subsequent treatment with protease A yielded the best reduction in adsorbed lipid as well as the biggest increase in pure-water flux. There was statistically no significant difference in protein removal between the commercial preparation, the lipase A:Triton X100 mixture and the lipase A:Triton X100 protease A combination.

4. Discussion

Only a few direct references to the use of proteases (protein breakdown) and lipases (lipid breakdown) for cleaning fouled UF membranes could be found in the literature [1,14]. Proteins and lipids present in abattoir effluent represent a very broad spectrum of both these classes of compounds. Protease A and lipase A were selected for our studies as these two enzymes exhibit a relatively broad spectrum of activity towards protein and triacylglyceride hydrolysis respectively. The activity of these enzymes are normally measured in units. For the protease A used in this study one unit of enzyme is defined as the amount of enzyme that will hydrolyse casein to produce colour (by Folin-Ciocalteu reagent) equivalent to 1 mmol (181 mg) of tyrosine per rain at a pH of 7.5 at 37C. For lipase A one enzyme unit will hydrolyse 1.0 microequivalent of fatty acid from triglyceride in 1 h at a pH of 7.5 at 37C. As mentioned earlier, however, the type of proteins and lipids adsorbed onto the PSM fouled in abattoir effluent, are unknown and therefore the opti-

real enzyme concentrations and contact time with the fouled membranes had to be determined. Lipase activity can be increased by the addition of Triton X100. The detergent creates an oil-water interface which greatly enhances the activity of the enzyme [17]. In addition Triton X100 interacts with foulants causing a less dense foulant layer that is more accessible to the cleaning enzymes. The positive effect of the addition of 0.1% Triton X100 on the ability of the lipase A preparation to remove lipids from the fouled PSM, was clearly demonstrated in this study. Treatment of fouled membranes with the lipase A:Triton X100 mixture also very effectively removed proteins. In addition lipase A combined with Triton X100 lowered the enzyme concentration required, and reduced the incubation time needed, for more efficient cleaning. A fact that could be of great importance in the future practical application of these types of cleaning regimes. Detergents such as SDS or Triton X100 used alone as cleaning agents also removed lipid material. The detergents alone were, however, significantly less effective than when used in conjunction with enzymes, a phenomenon also observed by Coolber et al. [14]. An interesting aspect of this investigation was the fact that the protease A had the ability to remove a significant amount of lipid material from the fouled membranes. Protease A has no lipase activity, as determined by the method of Miles et al. [18], and it can therefore be assumed that lipids were liberated into the solution due to the disturbance of lipid-protein interactions by the degradation of protein "anchors". Support for this hypothesis comes from the fact that the lipid content of fouled membranes, treated with protease A, increased after 60 min of incubation indicating re-adsorption. Lipid re-adsorption can also explain the subsequent increase in the contact angle and pure-water flux decrease, observed for these membranes at incubation times longer than 60 rain. These findings emphasise the importance of using real effluent instead of model foulants containing only lipid or protein material, as we have indicated previously that protein-lipid interactions could be a major factor in membrane fouling [2]. The lipase A:Triton X100 mixture was the most effective protein removal mixture tested. Protein removal by the lipase A alone or in conjunction with

A. Maartens et al. / Journal of Membrane Science 119 (1996) 9 - 1 6

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the detergent, could also be attributed to the removal of anchoring lipids. Re-adsorption of protein did, however, not occur and a possible explanation for this phenomenon is the limited protease activity associated with the lipase A preparation used in these experiments [19]. It is important to note that adsorbed protein material could be completely removed from the fouled membranes. In contrast not even the most effective cleaning method used in this study, could remove all the lipids. Approximately 64% of the absorbed lipid material was removed from the membrane by the lipase A:Triton X100 mixture. This phenomenon may be attributed to the fact that lipids can penetrate into membrane pores and could not be reached by the higher-molecular-mass enzyme molecules. Information obtained from data such as lipid and protein content as well as contact angle values are extremely useful in quantifying fouling, determining the nature of foulants and evaluating cleaning regimes for PSM used in the treatment of streams of biological origin. Flux recovery is, however, the ultimate test for the assessment of the effectiveness of cleaning techniques. Of the cleaning mixtures used in this study the commercially available preparation, Alkazyme:Zymex, and the lipase A:Triton X100 mixture alone, and in conjunction with protease A, gave the best flux recovery (Table 1). The flux recoveries for these three cleaning mixtures were all significantly higher than 100% when compared with the flux through a new unfouled membrane. The lower flux of the unfouled control can be explained by the fact that the flux through these membranes was measured before any membrane swelling occurred. An explanation for the higher pure-water flux after membrane cleaning with an enzyme/detergent mixture, is that the surfactant included in the cleaning protocol, changes the surface morphology of the membrane to a more homogeneously permeable surface [20]. Unused membranes were incubated with 0.1% Triton X100 in the buffer (pH 7.5) at 37C as controls to test this hypothesis. Pure-water flux and contact angles values obtained were 1200 + 20 1 / m 2 h and 3 5 2.14 respectively. This finding is supported by previous observations of initial flux improvements of 20-40% after membranes had been treated with non-ionic surfactants [20-23]. The contact angle values obtained after cleaning with only

the detergent emphasised the effect of detergents on the membrane surface. Results obtained in this investigation show that enzymes can be used effectively as cleaning agents in biological effluent streams. It is, however, important that the effluent as well as the fouling agents in the effluent should be as well characterised and identified as possible, in order to facilitate the correct choice of enzymes or enzyme/detergent mixtures.

Acknowledgements
We would like to thank the Water Research Commission for financial support and Mrs. C.A. de Villiers for technical support. We are also thankful to the Maitland abattoir and Syndachem Sales for their contributions.

References
[1] E.P. Jacobs, Cleaning of PES tubular UF membranes: an abattoir case-study, WRC Report no. K5/362, 1991, Pretoria, South Africa. [2] A. Maartens, P. Swart and E.P. Jacobs, Characterisation techniques for organic foulants adsorbed onto flat-sheet UF membranes used in abattoir effluent, J. Membrane Sci., 119 (1996) 1-8. [3] G. Spencer and R. Thomas, Fouling, cleaning and rejuvenation of formed-in-place membranes, Food Technol. Des., (1991) 98-99. [4] K.F. Eckner and E.A. Zottola, Effects of temperature and pH during membrane concentration of skim milk on fouling and cleaning efficiency, Milchwissenschaft, 48(4) (1993) 187191. [5] R.H. Sedath, D.R. Taylor and N.N. Li, Reduced fouling of UF membranes via surface fluorination, Sep. Sci. Technol., 28(1/3) (1993) 255-269. [6] H.C. Flemming, Biofouling in water treatment, Proc. Int. Workshop Biofouling Biocorrosion, Stuttgart, September, 1990, pp. 13-14. [7] A.G. Fane, C.J.D. Fell and A. Suki, The effect of pH and ionic environment on the UF of protein solutions with retentive membranes, J. Membrane Sci., 16 (1983) 195-210. [8] H. Bauser, H. Chmiel, N. Stroh and E. Walitza, Interfacial effects with microflltration membranes, J. Membrane Sci., 11 (1982) 321-332. [9] S. Bragulla and K. Lintner, Basics of cleaning and disinfection for UF, reverse osmosis and electrodialysis plants, Alimenta, 5 (1986) 197-203. [10] B.T. Croll, Membrane technology: The way forward?, J. Iwem., 6 (1992) 121-129. [11] G. Tr~tgard, Membrane cleaning, Desalination, 71 (1989) 325-335.

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[12] K. Kim, P. Sun, V. Chen, D.E. Wiley and A.G. Fane, The cleaning of UF membranes fouled by protein, J, Membrane Sci., 80 (1993) 241-249. [13] H.F, Bohner and R.L. Bradley, Effective cleaning and sanitizing of polysulphone UF membrane systems, J. Dairy Sci., 75 (1992) 718-724. [14] T. Coolbear, C. Monk., K. Peek, H.W. Morgan and L. Daniel, Laboratory-scale investigations into the use of extremely thermophilic proteinases for cleaning UF membranes fouled during whey processing, J. Membrane Sci., 67 (1992) 93-101. [15] J. Chen, L. Wang and Z. Zhu, Preparation of enzyme immobilized membranes and their self-cleaning and anti-fouling abilities in protein separations, Desalination, 86 (1992) 301315, [16] J.A. Howell and O. Velicangil, Theoretical considerations of membrane fouling and its treatment with immobilized enzymes for protein UF, J. Appl. Polym. Sci., 27 (1982) 21-32. [17] W. Norde, Adsorption of proteins from solution at the solid-liquid interface, Adv. Colloid Interface Sci., 25 (1986) 267-340.

[18] R.J. Miles, E.L.T. Siu, C. Carrington, A.C. Richardson, B.V. Smith and R.G. Price, The detection of lipase activity in bacteria using novel chromogenic substrates, FEMS Microbiol. Lett., 90 (1992) 283-288. [19] A.R. Subramanian and G. Kalnitsky, The major alkaline proteinase of Aspergillus oryzae, Aspergillopeptidase B. I. Isolation in homogeneous form, Biochemistry, 3(12) (1964) 1861-1876. [20] A.G. Fane, C.J.D. Fell and K.J. Kim, The effect of surfactant pretreatment on the ultrafiltration of proteins, Desalination, 53 (1985) 37-55. [21] V. Chen, A.G. Fane and C.J.D. Fell, The use of anionic surfactants for reducing fouling of ultrafiltration membranes: their effects and optimization, J. Membrane Sci., 67 (1992) 249-261. [22] K.J. Kim, A.G. Fane and C.J.D. Fell, The performance of ultrafiltration membranes pretreated by polymers, Desalination, 70 (1988) 229-249. [23] L.E.S. Brink and D.J. Romijn, Reducing the protein fouling of polysulphone surfaces and polysulphone ultrafiltration membranes: optimization of the type of presorbed layer, Desalination, 78 (1990) 209-233.