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Effective Control of Microbial Populations in Polysulfone Ultrafiltration Membrane System1
Effective Control of Microbial Populations in Polysulfone Ultrafiltration Membrane Systems1 H. F. BOHNER and R. L. BRADLEY University of Wisconsin Madison 53706 ABSTRACT
indicato~s used are sight, smell, and final product qualIty (1, 2, 8). These indicators will detect only heavily contaminated UP membranes but cannot indicate sanitary conditions. More recent work emphasizes direct observation of the membranes by microbiological assay and scanning electron microscopy (SEM) (12). The failure of common cleaning procedures and sanitizers to clean sufficiently and sanitize membranes leads to the use of new cleaning procedures and sanitizers (11). Chlorine dioxide has a long history as a sanitizer (13). An acidified solution of sodium chlorite, developing chlorine dioxide, has been proposed as a sanitizer for UP systems. A solution of chlorine dioxide produced by acidification of sodium chlorite is effective against bacteria of concem to public health (5). The purpose of this study was to determine the efficacy of a new sanitizer, which contains chloride di~xide in acidic solution. Efficacy was determmed by the number of surviving microorganisms at the various stages of cleaning and sanitation and by direct observation of the membrane by SEM.
Sanitizers currently used in the food industry are not efficient in destroying bacterial populations in polysulfone UP membrane systems. A new sanitizer composition that releases chlorous acid and chlorine dioxide from sodium chlorite at pH 2.7 was evaluated. PolysuIfone UP membranes were soiled for 2.5 h by circulating and concentrating Cheddar cheese whey and skim milk. A cleaning regimen was established whereby acid and caustic cleaning solutions were circulated to clean the UP membrane system. Restoring permeate flux to initial values did not indicate that the system was effectively cleaned. The UP system was sanitized by recycling sanitizer solutions. Stainless steel and membrane surfaces were examined by swabbing to determine bacterial populations and sections of membranes were removed for examination using a scanning electron microscope. The new sanitizer appeared to control microbial populations effectively in UP membrane systems. (Key words: microbial populations, polysulfone ultrafiltration membrane systems, sanitizers)
MATERIALS AND METHODS EqUipment
INTRODUCTION
. Ult,rafiltrati~n h~s a wide range of applications m the dairy mdustty. Although considerable research has been directed toward new products and processes, cleaning and sanitation has received much less attention. Many researchers consider UP membranes clean when permeate flux is restored. Other
Received November 15. 1989. f-ccepted March 29, 1990. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by Alcide Corp., Norwalk, CT.
1990 J Dairy Sci 73:2309--2317
The UP system consisted of a 38o-L jacketed vat with agitator and thermometer followed by a prefilter, centrifugal pump, and two membranes (Desalination Systems, Inc., Escondido, CA) in parallel with valves and pressure gauges before and after the membranes (friClover, Inc., Kenosha, WI). Spiral-wound, polysulfone UP membranes, the most wide~pread design for UP membranes in the dairy mdustry, was used. The membrane pore size was given by the membrane manufacturer in form of molecular weight cutoff and was 10,000 to 15,000 Da Soiling conditions that exist during the continuous processing of whey by UF were simulated by returning the permeate to the feed tank to keep the composition of
2309
2310
BOHNER AND BRADLEY
the feed material constant. Diverting the penneate to drain was done to increase solids concentration of the feed and consequently enhance soiling of the membrane. For recycling of cleaning solutions and water, permeate and retentate streams were returned to the feed tank; for flushing operations, both were diverted to drain.
CONDITIONS
PROCEDURE
TIME/pH
OAT A COLLECTED
3B:Jl
TS, pH,
Fa~,
SFC
Flu~
20'1
R::CYCLED
TS. pH. Flu
~at.
SPC
TS, pH, Fat. SPC Flwx
FI~t
38D L 1,Oh
Soiling Procedure
acid 1.8 ca~s:ic 11.8
Separated Cheddar cheese whey and raw skim milk were obtained immediately before use from a local dairy and the University of Wisconsin-Madison dairy plant, respectively. The soiling procedure consisted of recycling 380 L of whey or skim milk for 2.0 h and concentrating the products for an additional .5 h. Cooling water was circulated in the jacket of the vat to maintain temperature at 38 to 43"C. pH; percentage of total solids, protein, and milk fat; and numbers of bacteria were determined for product in the tank and permeate stream initially, during recycling, and at the start and end of concentration according to Standard Methods for the Examination of Dairy Products (10). Moisture determination was done in an atmospheric oven, protein by the Kjeldahl method, fat by the Mojonnier method, and numbers of bacteria by standard plate count (10). A feed rate of 95 L/min per membrane with an average pressure differential across the membrane of 300 kPa was selected for soiling.
VOLUME
l_O~
RECYCLE)
ac,dl,8 ca~s:>c
RECYCLED
".8 380 L
FI'JX, Swabs
lQn:27
DVERNIGHT
380 l
Fru~,
10 min RECYCLEJ
Flu), Swabs
Swabs
Figure 1. Outline of soiling with whey and skim milk, cleaning, and data collection. TS = Total solids; SPC = standard plate count.
hydroxide solution at .1% (wt/vol). Oeaning was finished with a water flush. In the case UP membrane soiled from skim milk, the acid cleaning step followed the caustic cleaning step to avoid precipitation of milk residues in the membrane. sanitizer
CleanIng Proceclure
A flow diagram of the cleaning procedure is given in Figure 1. For all cleaning and sanitation operations, a valve setting was selected that resulted in a high feed rate of 160 L/min per membrane (the manufacturer recommends a minimum of 95 L/min per membrane and 140 kPa). Average pressure differential across the membrane was 400 kPa Following soiling, the UP system was flushed with water (209 L, 4O"C) to remove loosely held soil. If soiling was with whey, the UP system was then cleaned by recycling a 1: 1 blend of nitric and phosphoric acid at 43"C and pH 1.8 for 1 h followed by a water flush and recycling of a sodium hydroxide solution at 43"C and pH 11.8 for 1 h. Abcor Ultraclean II, a surfactant preparation (Koch Membrane Systems, Inc., Wilmington, MA) was added to the sodium Journal of Daily Science Vol. 73,
No.9, 1990
The sanitizer evaluated consisted of two components: an aqueous sodium chlorite solution, ''base''; and a second component, "activator", was lactic acid and a surfactant. One part base was diluted with 200 parts water and then one part activator was added. The use temperature was 4O"C. This mixture yielded pH 2.7 in tap water (350 ppm hardness). Acidification of sodium chlorite produced chlorous acid (reaction I): NaO(h + H+
= HO(h
+ Na+
which decayed in various reactions (6). The most important product is chlorine dioxide (reaction 11):
2311
MICROBIAL CONTROL IN ULTRAFIL1RAll0N SYSlEMS
TABLE 1. Composition of whey during UF.
Processing step Initial
Total solids, %
6.4
pH
6.4
Fat. % Total plate count, log
.08
cfu/ml
5.3
5.4 6.4
o
3.3
Start of concentration
Bnd of concentration
W
P
W
P
6.4 6.0 .07 6.2
5.4 6.0 0 4.0
7.9 6.0
5.8 6.0 0 4.4
.10
6.7
-vi = Whey. bp = Pcnneate.
Chlorate and chloride are produced in minor quantities. Disproportionation of chlorous acid depends on the concentration of sodium chlorite, pH, temperature, and chloride ions, which have catalytic properties (4). Chlorine dioxide formation in the sanitizer was measured in a Beckman DU-6 spectrophotometer (Beckman Instruments, Inc., Philadelphia, PA) at 405 DOl. Chlorine dioxide standards were prepared according to Standard Methods for the Examina-
tion of Water and Wastewater (9). sanitation
Sanitizer base (.5 L) was added to 100 L of water (40"C). Under agitation, .5 L of activator was added and the mixture left to react for 1 h. This sanitizer solution was then recycled for 30 min. The system was flushed with water and held overnight with a 50 ppm Antibac B (dichloroisocyanurate) solution (Diversey-Wyandotte, Wyandotte, MI). Data Collection
Permeate flux was determined for unsoiled membranes and after each step of cleaning and sanitation. Values for flux were determined at 13, 27, and 43·C, and lines of best fit were calculated using linear regression. Microbial populations of stainless steel and membrane surfaces were determined by the swab contact method using sterile cotton swabs (10). In each trial, 50-cm2 sections on right and left front faceplates, rear faceplates, sidewalls, and permeate discharge tubes were swabbed. Membranes were removed from the housing and four 5O-cm2 surfaces of membrane were swabbed. The number of microorganisms per
square centimeter at each of the tested locations within the UF system was calculated by dividing the sum of colony forming units found by the amount of surface area swabbed. In some experiments, 5O-cm2 sections of polysulfone membrane material were placed inside the membrane and then the membrane was carefully rerolled before soiling. These sections were removed after soiling, cleaning, and sanitation. Each 5O-cm2 section was placed in a petri dish and overplated with standard methods agar. All agar plates were incubated for 48 h at 32"C. ScannIng Electron MIcroscope Procedures
Membranes were carefully unwound at different stages of soiling and cleaning, and sections were cut with a sterile razor blade. Sections were removed in the middle of the entire membrane sheet and 30.5 cm in from the edge. Membrane sections for examination by SEM were placed on filter paper in a petri dish and dried at 32"C for 24 h. The dried sections were mounted on specimen holders and evenly coated with a about 1 nm platinum in a model B 370 EM-Microsputter coater (Ion Tech LTD., Teddington, Middlesex 1Wl1 OLT, England). A Hitachi S-900 field emission scanning electron microscope (Hitachi, Ltd., Tokyo), using acceleration voltages from 2.5 to 5 kV, was used at magnifications of up to 50,OOOX. Magnification and accelerating voltage are shown in the data line of each individual picture. RESULTS AND DISCUSSION
Whey composition throughout UP is given in Table 1. Values of total solids, protein, and Journal of Dairy Science Vol. 73,
No.9, 1990
2312
BOHNER
AND
BRADLEY
70 - , - - - - , - - - - , - - - - - - - - , - - - - - - - - , - - - - ,
'00+----.---60 +---+---+---+--_..+-------1
#.
UnSOIled new membrane
-------1
80
X ::J
Li '2 50 +---+---+----+--1'=--.....-1------1 '13
--..
W
::;
w a.
'"'
.3 40 ;..
~
([
:':~
60
W
20
+---+---+-----:'---II"------.t+-----1
;":;
§
d:
30 +---+----,'1--/--.----1-----1
20 +----j-ht---if---+-
.13C .27 C ... 43 C
10 +--,--+-z....,..-+---.--+--..,.-+----,----I o 400 100 300 200 500 Press ure IkPa)
Figure 2. Permeate flux for new membrane with water.
milk fat in the retentate remained unchanged until concentration and increased during concentration. Microbial growth during the whole soiling procedure is indicated by increasing nwnbers of colony forming units and decreasing pH. Water permeate flux of a new polysulfone membrane is given in Figure 2. Flux at 4O"C and 300 kPa pressure differential across the membrane at the different stages is given in Figure 3. Abcor Ultraclean II and the sanitizer both contain surfactants, which apparently adhere to the membrane (14); thus, permeate flux decreased to about 65% of the flux determined with water and new membranes. These surfactants are slowly removed by prolonged flushing with water and also are removed by whey and skim milk. Flux with whey did not change when membranes were cleaned with solutions containing surfactant and compared to the flux value determined on new membranes. Due to adhering surfactant, decreased flux did not indicate an unsanitary condition of the membrane. Following the use of cleaners or sanitizers that contained surfactants, the first part of the next product through the system· was contaminated with surfactant and was discarded (3). This Journal of Dairy Science Vol. 73.
Ftgnre 3. Flux at 4O'C and 300 kPa at stages of process.
No.9. 1990
same practice should be followed in industrial settings. Fouling reduced permeate flux. Water flushing and chemical cleaning steps increased permeate flux to the level of unsoiled, surfactant-<:ontaminated membranes. Sanitizing did not alter flux. The absotption maximum of chlorine dioxide in aqueous solution is at 359 om. The
"\\
I
I
, I
I
I
i
I I
\
\ I
\ \ \ 200
I I
\./ 300
I
~ \
\
\
~\
\ 400
500
600
700
Wavelength (nm)
Figure 4. Absorption spectra for chloriDe dioxide (dashed line) and chlorine dioxide containing sanitizer (full line).
2313
MICROBIAL CONTROL IN ULTRAFILTRATION SYSTEMS
20
16
Q
1---./
•
Q u~ ~
E
B
-J
,
"" :~
&0
+-----
~
+--------1-
1-
~; "-0
/
'-'
8:
li
~
./
12
.-- .-.l__ L-~----
@~
•
.~
~~
•
acid cleaned
I
•
I 5
6
time (h)
.. caustic cleaned
... chlorine dox,de sanitized
• sanitized wilh 50 ppm Anlibac B overnight
•
Front laceplales and sidewalls
•
Permeate ILbes
•
Rear faceplates ar\d sidewa'is
~
Membra". surlaces
FiguIe 6. Percentage of acceptable surfaces after stages of the cleaning and sanitation process of whey soiled UP membranes determined by Ibe swabbing teclmique.
Figure 5. ChloriDe dioxide measured in the sanitizer as influeoced by lime aft« activation at 4O·C.
sanitizer always contains a high amount of chlorite and chlorous acid, which both have significant absorption at the absotption maximum of chlorine dioxide. Absorbance spectra for pure chlorine dioxide and the sanitizer are shown in Figure 4. Absorbance at 405 nm was chosen for the subsequent measurement of chlorine dioxide, because at this wavelength the interference of chlorite and chlorous acid was reportedly lower than at the absorption maximum of chlorine dioxide (13). The concentration of chlorine dioxide during the first 6 h after activation is illustrated in Figure 5. For sanitizing the UP system, an activation time of 1 h was selected. About 15 ppm of chlorine dioxide was used, because chlorine dioxide has about seven times the bactericidal effect of hypochlorite (7). Thus, 15 ppm chlorine dioxide would approximately equal a 100 ppm solution of sodium hypochlorite. The number of bacteria fOWld on membranes soiled with whey decreased with cleaning and sanitation. Membrane and stainless steel surfaces with less than 1 cfu/cm2 were considered acceptable. The percentage of acceptable surfaces after the respective cleaning and sanitation steps of whey soiled UP membranes are in Figure 6. Acid and caustic cleaning steps reduced the number of bacteria considerably. However. chlorine dioxide sanitizer treatment rendered membranes free from detectable or-
ganisms. Twenty-four sections (three trials with eight sections each) of polysulfone UP membrane material plated in standard methods agar showed no microbial growth. Holding the system overnight in 50 ppm Antibac B solution further reduced the number of microorganisms fOWld on some surfaces. This can be explained by the design of the UP system. Antitelescoping devices prevent effective circulation of solutions at front and rear faceplates and rear sidewalls. Cleaning of the rear faceplates proved to be especially troublesome, as indicated by the high number of inadequately cleaned surfaces. The UP system was similarly cleaned and sanitized after soiling with raw skim milk. In two trials involving swabbing of 24 stainless steel surfaces, 8 membrane surfaces, and plating 8 membrane sections, all surfaces were acceptable; most had no detectable microorganisms. Scanning Electron Microscopy
Considering only flux data and the results of microbiological assays, the membranes appeared to be effectively cleaned and sanitized. Under visual inspection, when the membranes were partially unrolled, each appeared soiled with dust-like particles. Scanning electron microscopy showed this contamination on the microbiologically clean membranes. Figure 7 shows membranes after soiling with whey and after the full cleaning cycle, but before sanitation. Evident are dust particles, Journal of Dairy Science Vol. 73,
No.9, 1990
2314
BOHNER AND BRADLEY
Figure 7. Deposits on the membrane following soiliDg with whey and cleaning. a) Overview (dotted bar = 30~) of deposits scattered on tbe membrane surfll(:C. Dust particles (D) and numerous bacteria (B). b) Bacteria (B) embedded in a protein layer, granular protein (0) and channels in tbe protein layer (C) (dotted bar = 2~). c) Details (dotted bar = 600 om) of channels (C) in the protein layer (1..) and granular protein (0).
Journal of Dairy Science Vol. 73,
No.9, 1990
MICROBIAL CONTROL IN UL1RAFILTRATION SYSTEMS
2315
Figure 8. Deposits on the membrane following soiling with skim miJk, cleaning and sanitation. a) Overview (dotted bar (D) and numerous bacteria (B). b) Bacteria (B) embedded in a protein layer, granular protein (0), and channels in the protein layer (C) (dotted bar = 3 1JDl). c) Details (dotted bar = 600 um) of channels (C) in the protein layer (L) and granular protein (0).
= 30 1JDl) of deposits scattered on the membrane surface. Dust particles
Journal of Dairy Science Vol. 73,
No.9, 1990
2316
BOHNER AND BRADLEY
Figme 9. Deposits on the membrane following soiling with whey. cleaning and sanitation. a) Overview (dotted bar = 30 of deposits scattered on the membrane surface. Dust particles (0) and numerous bacteria (8). b) Bacteria (8) embedded in a protein layer, granular protein (0), and channels in the protein layer (C) (dotted bar 2 ~). c) Details (dotted bar 670 nm) of channels (C) in the protein layer (L) and granular protein (0). ~)
=
=
Journal of Dairy Science Vol. 73.
No.9. 1990
MICROBIAL CONTROL IN ULTRAFILTRATION SYSTEMS
bacteria embedded in a protein layer, and granular protein and channels in the smooth protein layer caused by the rinsing and cleaning solutions. Due to these channels, flux is restored to values, indicative of a clean membrane, even though the membrane is still heavily contaminated. Similar smooth and granular protein deposits and bacteria on UF membranes after cleaning have been reported (11).
Figure 8 shows pictures of the membrane taken after soiling with skim milk, cleaning, and sanitation. High numbers of bacteria and considerable prote·.laceous material are visible. Figure 9 is from the polysulfone membrane after soiling with whey, cleaning, and sanitation. The surface still is contaminated with particles, protein, and bacteria Scanning electron microscopy, however, cannot distinguish dead from viable bacteria Scanning electron microscopy shows that cleaning was incomplete, but data from swabbing and membrane pieces suggest that bacteria were destroyed. CONCLUSION
Considering only the microbiological data and flux, chlorine dioxide as a sanitizer appeared to be efficient in controlling microbial populations in spiral-wound UF systems. The sanitizer is powerful enough to apparently destroy bacteria within protein deposits; however, sterile itlth does not constitute cleanliness. Improvements in the design of UF systems and new membrane materials may provide the answer to removal of entrapped deposits. ACKNOWLEDGMENTS
The SEM pictures by Mark W. Tengowski of the Integrated Microscopy Resource (IMR)
2317
in Madison, WI. The IMR in Madison is
funded as an NllI Biomedical Research Technology Resource (RR570). REFERENCES 1 Beaton, N. C. 1979. Ultraf"lltration and reverse osmosis in the daily industry - An introduction to sanitary considerations. J. Food Prot. 42:584. 2 Cheryan, M. 1986. VItrafiltrationbandbook. Technomic Publ. Co., Inc., Lancaster, PA. 3 Dunsmore, D. G. 1983. The incidence and implications of residues of detergents and sanitizers in daily products. Residue Rev. 86:1. 4 Gordon, G., R. G. Kieffer, and D. H. Rosenblatt. 1972. The chemistry of chlorine dioxide. Prog. Inorg. Chem. Vol. 15, S. J. Lippard, ed. Wiley Interscience, New York, NY. 5 Harakeh, S., A. Dlescas, and A. Malin. 1988. Inactivation of bacteria by purogene. J. Appl. Bacteriol. 64:459. 6 Hefti, H. 1956. Uber den Zerfall von Natriumchlorit in Bleichlosungen. Textilrundschau 11:82. 7 Lillard, H. S. 1979. Levels of chlorine and chlorine dioxide of equivalent bacteriocidal effect in poultry processing water. J. Food Sci. 44:1594. 8 Parkin, M. F., and K. R. Marshall. 1976. The cleaning of tubular cellulose acetate ultrafiltration membranes. N .Z. 1. Dairy Sci. Technol. 11:107. 9 Rand, M. C., ed. 1976. Standard methods for the examination of water and wastewater. 14th ed. Am. Publ. Health Assoc. Inc., Washington, DC. 10 Richardson, G. A., ed. 1985. Standard methods for the examination of milk and milk products, 15th ed. Am. Publ. Health Assoc., Washington, DC. 11 Smith, K. E., and R. L. Bradley. 1986. Ineffective cleaning of polysulfone ultraf"lltration membrane systems and corrosion by bisulfite used as a sanitizer. J. Dairy Sci. 69:1232. 12 Smith, K. E., and R. L. Bradley. 1988. Evaluation of three different cleaners recommended for ultrafiltration systems by direct observations of commercial-scale spiral-wound ultrafiltration membranes. 1. Food Prot. 51: 89. 13 Synan, J. F., J. D. MacMahon, and G. P. Vincent. 1944. Chlorine dioxide - a development in treatment of potable water. Water Works Sewage 91:423. 14 Tragardh, G. 1989. Membrane cleaning. Desalination 71:325.
Journal of Dairy Science Vol. 73,
No.9, 1990
indicato~s used are sight, smell, and final product qualIty (1, 2, 8). These indicators will detect only heavily contaminated UP membranes but cannot indicate sanitary conditions. More recent work emphasizes direct observation of the membranes by microbiological assay and scanning electron microscopy (SEM) (12). The failure of common cleaning procedures and sanitizers to clean sufficiently and sanitize membranes leads to the use of new cleaning procedures and sanitizers (11). Chlorine dioxide has a long history as a sanitizer (13). An acidified solution of sodium chlorite, developing chlorine dioxide, has been proposed as a sanitizer for UP systems. A solution of chlorine dioxide produced by acidification of sodium chlorite is effective against bacteria of concem to public health (5). The purpose of this study was to determine the efficacy of a new sanitizer, which contains chloride di~xide in acidic solution. Efficacy was determmed by the number of surviving microorganisms at the various stages of cleaning and sanitation and by direct observation of the membrane by SEM.
Sanitizers currently used in the food industry are not efficient in destroying bacterial populations in polysulfone UP membrane systems. A new sanitizer composition that releases chlorous acid and chlorine dioxide from sodium chlorite at pH 2.7 was evaluated. PolysuIfone UP membranes were soiled for 2.5 h by circulating and concentrating Cheddar cheese whey and skim milk. A cleaning regimen was established whereby acid and caustic cleaning solutions were circulated to clean the UP membrane system. Restoring permeate flux to initial values did not indicate that the system was effectively cleaned. The UP system was sanitized by recycling sanitizer solutions. Stainless steel and membrane surfaces were examined by swabbing to determine bacterial populations and sections of membranes were removed for examination using a scanning electron microscope. The new sanitizer appeared to control microbial populations effectively in UP membrane systems. (Key words: microbial populations, polysulfone ultrafiltration membrane systems, sanitizers)
MATERIALS AND METHODS EqUipment
INTRODUCTION
. Ult,rafiltrati~n h~s a wide range of applications m the dairy mdustty. Although considerable research has been directed toward new products and processes, cleaning and sanitation has received much less attention. Many researchers consider UP membranes clean when permeate flux is restored. Other
Received November 15. 1989. f-ccepted March 29, 1990. Research supported by the College of Agricultural and Life Sciences, University of Wisconsin-Madison and by Alcide Corp., Norwalk, CT.
1990 J Dairy Sci 73:2309--2317
The UP system consisted of a 38o-L jacketed vat with agitator and thermometer followed by a prefilter, centrifugal pump, and two membranes (Desalination Systems, Inc., Escondido, CA) in parallel with valves and pressure gauges before and after the membranes (friClover, Inc., Kenosha, WI). Spiral-wound, polysulfone UP membranes, the most wide~pread design for UP membranes in the dairy mdustry, was used. The membrane pore size was given by the membrane manufacturer in form of molecular weight cutoff and was 10,000 to 15,000 Da Soiling conditions that exist during the continuous processing of whey by UF were simulated by returning the permeate to the feed tank to keep the composition of
2309
2310
BOHNER AND BRADLEY
the feed material constant. Diverting the penneate to drain was done to increase solids concentration of the feed and consequently enhance soiling of the membrane. For recycling of cleaning solutions and water, permeate and retentate streams were returned to the feed tank; for flushing operations, both were diverted to drain.
CONDITIONS
PROCEDURE
TIME/pH
OAT A COLLECTED
3B:Jl
TS, pH,
Fa~,
SFC
Flu~
20'1
R::CYCLED
TS. pH. Flu
~at.
SPC
TS, pH, Fat. SPC Flwx
FI~t
38D L 1,Oh
Soiling Procedure
acid 1.8 ca~s:ic 11.8
Separated Cheddar cheese whey and raw skim milk were obtained immediately before use from a local dairy and the University of Wisconsin-Madison dairy plant, respectively. The soiling procedure consisted of recycling 380 L of whey or skim milk for 2.0 h and concentrating the products for an additional .5 h. Cooling water was circulated in the jacket of the vat to maintain temperature at 38 to 43"C. pH; percentage of total solids, protein, and milk fat; and numbers of bacteria were determined for product in the tank and permeate stream initially, during recycling, and at the start and end of concentration according to Standard Methods for the Examination of Dairy Products (10). Moisture determination was done in an atmospheric oven, protein by the Kjeldahl method, fat by the Mojonnier method, and numbers of bacteria by standard plate count (10). A feed rate of 95 L/min per membrane with an average pressure differential across the membrane of 300 kPa was selected for soiling.
VOLUME
l_O~
RECYCLE)
ac,dl,8 ca~s:>c
RECYCLED
".8 380 L
FI'JX, Swabs
lQn:27
DVERNIGHT
380 l
Fru~,
10 min RECYCLEJ
Flu), Swabs
Swabs
Figure 1. Outline of soiling with whey and skim milk, cleaning, and data collection. TS = Total solids; SPC = standard plate count.
hydroxide solution at .1% (wt/vol). Oeaning was finished with a water flush. In the case UP membrane soiled from skim milk, the acid cleaning step followed the caustic cleaning step to avoid precipitation of milk residues in the membrane. sanitizer
CleanIng Proceclure
A flow diagram of the cleaning procedure is given in Figure 1. For all cleaning and sanitation operations, a valve setting was selected that resulted in a high feed rate of 160 L/min per membrane (the manufacturer recommends a minimum of 95 L/min per membrane and 140 kPa). Average pressure differential across the membrane was 400 kPa Following soiling, the UP system was flushed with water (209 L, 4O"C) to remove loosely held soil. If soiling was with whey, the UP system was then cleaned by recycling a 1: 1 blend of nitric and phosphoric acid at 43"C and pH 1.8 for 1 h followed by a water flush and recycling of a sodium hydroxide solution at 43"C and pH 11.8 for 1 h. Abcor Ultraclean II, a surfactant preparation (Koch Membrane Systems, Inc., Wilmington, MA) was added to the sodium Journal of Daily Science Vol. 73,
No.9, 1990
The sanitizer evaluated consisted of two components: an aqueous sodium chlorite solution, ''base''; and a second component, "activator", was lactic acid and a surfactant. One part base was diluted with 200 parts water and then one part activator was added. The use temperature was 4O"C. This mixture yielded pH 2.7 in tap water (350 ppm hardness). Acidification of sodium chlorite produced chlorous acid (reaction I): NaO(h + H+
= HO(h
+ Na+
which decayed in various reactions (6). The most important product is chlorine dioxide (reaction 11):
2311
MICROBIAL CONTROL IN ULTRAFIL1RAll0N SYSlEMS
TABLE 1. Composition of whey during UF.
Processing step Initial
Total solids, %
6.4
pH
6.4
Fat. % Total plate count, log
.08
cfu/ml
5.3
5.4 6.4
o
3.3
Start of concentration
Bnd of concentration
W
P
W
P
6.4 6.0 .07 6.2
5.4 6.0 0 4.0
7.9 6.0
5.8 6.0 0 4.4
.10
6.7
-vi = Whey. bp = Pcnneate.
Chlorate and chloride are produced in minor quantities. Disproportionation of chlorous acid depends on the concentration of sodium chlorite, pH, temperature, and chloride ions, which have catalytic properties (4). Chlorine dioxide formation in the sanitizer was measured in a Beckman DU-6 spectrophotometer (Beckman Instruments, Inc., Philadelphia, PA) at 405 DOl. Chlorine dioxide standards were prepared according to Standard Methods for the Examina-
tion of Water and Wastewater (9). sanitation
Sanitizer base (.5 L) was added to 100 L of water (40"C). Under agitation, .5 L of activator was added and the mixture left to react for 1 h. This sanitizer solution was then recycled for 30 min. The system was flushed with water and held overnight with a 50 ppm Antibac B (dichloroisocyanurate) solution (Diversey-Wyandotte, Wyandotte, MI). Data Collection
Permeate flux was determined for unsoiled membranes and after each step of cleaning and sanitation. Values for flux were determined at 13, 27, and 43·C, and lines of best fit were calculated using linear regression. Microbial populations of stainless steel and membrane surfaces were determined by the swab contact method using sterile cotton swabs (10). In each trial, 50-cm2 sections on right and left front faceplates, rear faceplates, sidewalls, and permeate discharge tubes were swabbed. Membranes were removed from the housing and four 5O-cm2 surfaces of membrane were swabbed. The number of microorganisms per
square centimeter at each of the tested locations within the UF system was calculated by dividing the sum of colony forming units found by the amount of surface area swabbed. In some experiments, 5O-cm2 sections of polysulfone membrane material were placed inside the membrane and then the membrane was carefully rerolled before soiling. These sections were removed after soiling, cleaning, and sanitation. Each 5O-cm2 section was placed in a petri dish and overplated with standard methods agar. All agar plates were incubated for 48 h at 32"C. ScannIng Electron MIcroscope Procedures
Membranes were carefully unwound at different stages of soiling and cleaning, and sections were cut with a sterile razor blade. Sections were removed in the middle of the entire membrane sheet and 30.5 cm in from the edge. Membrane sections for examination by SEM were placed on filter paper in a petri dish and dried at 32"C for 24 h. The dried sections were mounted on specimen holders and evenly coated with a about 1 nm platinum in a model B 370 EM-Microsputter coater (Ion Tech LTD., Teddington, Middlesex 1Wl1 OLT, England). A Hitachi S-900 field emission scanning electron microscope (Hitachi, Ltd., Tokyo), using acceleration voltages from 2.5 to 5 kV, was used at magnifications of up to 50,OOOX. Magnification and accelerating voltage are shown in the data line of each individual picture. RESULTS AND DISCUSSION
Whey composition throughout UP is given in Table 1. Values of total solids, protein, and Journal of Dairy Science Vol. 73,
No.9, 1990
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70 - , - - - - , - - - - , - - - - - - - - , - - - - - - - - , - - - - ,
'00+----.---60 +---+---+---+--_..+-------1
#.
UnSOIled new membrane
-------1
80
X ::J
Li '2 50 +---+---+----+--1'=--.....-1------1 '13
--..
W
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20
+---+---+-----:'---II"------.t+-----1
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20 +----j-ht---if---+-
.13C .27 C ... 43 C
10 +--,--+-z....,..-+---.--+--..,.-+----,----I o 400 100 300 200 500 Press ure IkPa)
Figure 2. Permeate flux for new membrane with water.
milk fat in the retentate remained unchanged until concentration and increased during concentration. Microbial growth during the whole soiling procedure is indicated by increasing nwnbers of colony forming units and decreasing pH. Water permeate flux of a new polysulfone membrane is given in Figure 2. Flux at 4O"C and 300 kPa pressure differential across the membrane at the different stages is given in Figure 3. Abcor Ultraclean II and the sanitizer both contain surfactants, which apparently adhere to the membrane (14); thus, permeate flux decreased to about 65% of the flux determined with water and new membranes. These surfactants are slowly removed by prolonged flushing with water and also are removed by whey and skim milk. Flux with whey did not change when membranes were cleaned with solutions containing surfactant and compared to the flux value determined on new membranes. Due to adhering surfactant, decreased flux did not indicate an unsanitary condition of the membrane. Following the use of cleaners or sanitizers that contained surfactants, the first part of the next product through the system· was contaminated with surfactant and was discarded (3). This Journal of Dairy Science Vol. 73.
Ftgnre 3. Flux at 4O'C and 300 kPa at stages of process.
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same practice should be followed in industrial settings. Fouling reduced permeate flux. Water flushing and chemical cleaning steps increased permeate flux to the level of unsoiled, surfactant-<:ontaminated membranes. Sanitizing did not alter flux. The absotption maximum of chlorine dioxide in aqueous solution is at 359 om. The
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I I
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\./ 300
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\ 400
500
600
700
Wavelength (nm)
Figure 4. Absorption spectra for chloriDe dioxide (dashed line) and chlorine dioxide containing sanitizer (full line).
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MICROBIAL CONTROL IN ULTRAFILTRATION SYSTEMS
20
16
Q
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•
Q u~ ~
E
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,
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~
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~
./
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.-- .-.l__ L-~----
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acid cleaned
I
•
I 5
6
time (h)
.. caustic cleaned
... chlorine dox,de sanitized
• sanitized wilh 50 ppm Anlibac B overnight
•
Front laceplales and sidewalls
•
Permeate ILbes
•
Rear faceplates ar\d sidewa'is
~
Membra". surlaces
FiguIe 6. Percentage of acceptable surfaces after stages of the cleaning and sanitation process of whey soiled UP membranes determined by Ibe swabbing teclmique.
Figure 5. ChloriDe dioxide measured in the sanitizer as influeoced by lime aft« activation at 4O·C.
sanitizer always contains a high amount of chlorite and chlorous acid, which both have significant absorption at the absotption maximum of chlorine dioxide. Absorbance spectra for pure chlorine dioxide and the sanitizer are shown in Figure 4. Absorbance at 405 nm was chosen for the subsequent measurement of chlorine dioxide, because at this wavelength the interference of chlorite and chlorous acid was reportedly lower than at the absorption maximum of chlorine dioxide (13). The concentration of chlorine dioxide during the first 6 h after activation is illustrated in Figure 5. For sanitizing the UP system, an activation time of 1 h was selected. About 15 ppm of chlorine dioxide was used, because chlorine dioxide has about seven times the bactericidal effect of hypochlorite (7). Thus, 15 ppm chlorine dioxide would approximately equal a 100 ppm solution of sodium hypochlorite. The number of bacteria fOWld on membranes soiled with whey decreased with cleaning and sanitation. Membrane and stainless steel surfaces with less than 1 cfu/cm2 were considered acceptable. The percentage of acceptable surfaces after the respective cleaning and sanitation steps of whey soiled UP membranes are in Figure 6. Acid and caustic cleaning steps reduced the number of bacteria considerably. However. chlorine dioxide sanitizer treatment rendered membranes free from detectable or-
ganisms. Twenty-four sections (three trials with eight sections each) of polysulfone UP membrane material plated in standard methods agar showed no microbial growth. Holding the system overnight in 50 ppm Antibac B solution further reduced the number of microorganisms fOWld on some surfaces. This can be explained by the design of the UP system. Antitelescoping devices prevent effective circulation of solutions at front and rear faceplates and rear sidewalls. Cleaning of the rear faceplates proved to be especially troublesome, as indicated by the high number of inadequately cleaned surfaces. The UP system was similarly cleaned and sanitized after soiling with raw skim milk. In two trials involving swabbing of 24 stainless steel surfaces, 8 membrane surfaces, and plating 8 membrane sections, all surfaces were acceptable; most had no detectable microorganisms. Scanning Electron Microscopy
Considering only flux data and the results of microbiological assays, the membranes appeared to be effectively cleaned and sanitized. Under visual inspection, when the membranes were partially unrolled, each appeared soiled with dust-like particles. Scanning electron microscopy showed this contamination on the microbiologically clean membranes. Figure 7 shows membranes after soiling with whey and after the full cleaning cycle, but before sanitation. Evident are dust particles, Journal of Dairy Science Vol. 73,
No.9, 1990
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BOHNER AND BRADLEY
Figure 7. Deposits on the membrane following soiliDg with whey and cleaning. a) Overview (dotted bar = 30~) of deposits scattered on tbe membrane surfll(:C. Dust particles (D) and numerous bacteria (B). b) Bacteria (B) embedded in a protein layer, granular protein (0) and channels in tbe protein layer (C) (dotted bar = 2~). c) Details (dotted bar = 600 om) of channels (C) in the protein layer (1..) and granular protein (0).
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MICROBIAL CONTROL IN UL1RAFILTRATION SYSTEMS
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Figure 8. Deposits on the membrane following soiling with skim miJk, cleaning and sanitation. a) Overview (dotted bar (D) and numerous bacteria (B). b) Bacteria (B) embedded in a protein layer, granular protein (0), and channels in the protein layer (C) (dotted bar = 3 1JDl). c) Details (dotted bar = 600 um) of channels (C) in the protein layer (L) and granular protein (0).
= 30 1JDl) of deposits scattered on the membrane surface. Dust particles
Journal of Dairy Science Vol. 73,
No.9, 1990
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BOHNER AND BRADLEY
Figme 9. Deposits on the membrane following soiling with whey. cleaning and sanitation. a) Overview (dotted bar = 30 of deposits scattered on the membrane surface. Dust particles (0) and numerous bacteria (8). b) Bacteria (8) embedded in a protein layer, granular protein (0), and channels in the protein layer (C) (dotted bar 2 ~). c) Details (dotted bar 670 nm) of channels (C) in the protein layer (L) and granular protein (0). ~)
=
=
Journal of Dairy Science Vol. 73.
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MICROBIAL CONTROL IN ULTRAFILTRATION SYSTEMS
bacteria embedded in a protein layer, and granular protein and channels in the smooth protein layer caused by the rinsing and cleaning solutions. Due to these channels, flux is restored to values, indicative of a clean membrane, even though the membrane is still heavily contaminated. Similar smooth and granular protein deposits and bacteria on UF membranes after cleaning have been reported (11).
Figure 8 shows pictures of the membrane taken after soiling with skim milk, cleaning, and sanitation. High numbers of bacteria and considerable prote·.laceous material are visible. Figure 9 is from the polysulfone membrane after soiling with whey, cleaning, and sanitation. The surface still is contaminated with particles, protein, and bacteria Scanning electron microscopy, however, cannot distinguish dead from viable bacteria Scanning electron microscopy shows that cleaning was incomplete, but data from swabbing and membrane pieces suggest that bacteria were destroyed. CONCLUSION
Considering only the microbiological data and flux, chlorine dioxide as a sanitizer appeared to be efficient in controlling microbial populations in spiral-wound UF systems. The sanitizer is powerful enough to apparently destroy bacteria within protein deposits; however, sterile itlth does not constitute cleanliness. Improvements in the design of UF systems and new membrane materials may provide the answer to removal of entrapped deposits. ACKNOWLEDGMENTS
The SEM pictures by Mark W. Tengowski of the Integrated Microscopy Resource (IMR)
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in Madison, WI. The IMR in Madison is
funded as an NllI Biomedical Research Technology Resource (RR570). REFERENCES 1 Beaton, N. C. 1979. Ultraf"lltration and reverse osmosis in the daily industry - An introduction to sanitary considerations. J. Food Prot. 42:584. 2 Cheryan, M. 1986. VItrafiltrationbandbook. Technomic Publ. Co., Inc., Lancaster, PA. 3 Dunsmore, D. G. 1983. The incidence and implications of residues of detergents and sanitizers in daily products. Residue Rev. 86:1. 4 Gordon, G., R. G. Kieffer, and D. H. Rosenblatt. 1972. The chemistry of chlorine dioxide. Prog. Inorg. Chem. Vol. 15, S. J. Lippard, ed. Wiley Interscience, New York, NY. 5 Harakeh, S., A. Dlescas, and A. Malin. 1988. Inactivation of bacteria by purogene. J. Appl. Bacteriol. 64:459. 6 Hefti, H. 1956. Uber den Zerfall von Natriumchlorit in Bleichlosungen. Textilrundschau 11:82. 7 Lillard, H. S. 1979. Levels of chlorine and chlorine dioxide of equivalent bacteriocidal effect in poultry processing water. J. Food Sci. 44:1594. 8 Parkin, M. F., and K. R. Marshall. 1976. The cleaning of tubular cellulose acetate ultrafiltration membranes. N .Z. 1. Dairy Sci. Technol. 11:107. 9 Rand, M. C., ed. 1976. Standard methods for the examination of water and wastewater. 14th ed. Am. Publ. Health Assoc. Inc., Washington, DC. 10 Richardson, G. A., ed. 1985. Standard methods for the examination of milk and milk products, 15th ed. Am. Publ. Health Assoc., Washington, DC. 11 Smith, K. E., and R. L. Bradley. 1986. Ineffective cleaning of polysulfone ultraf"lltration membrane systems and corrosion by bisulfite used as a sanitizer. J. Dairy Sci. 69:1232. 12 Smith, K. E., and R. L. Bradley. 1988. Evaluation of three different cleaners recommended for ultrafiltration systems by direct observations of commercial-scale spiral-wound ultrafiltration membranes. 1. Food Prot. 51: 89. 13 Synan, J. F., J. D. MacMahon, and G. P. Vincent. 1944. Chlorine dioxide - a development in treatment of potable water. Water Works Sewage 91:423. 14 Tragardh, G. 1989. Membrane cleaning. Desalination 71:325.
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