Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to...

1

J. Dairy Sci. 96 :1–10http://dx.doi.org/ 10.3168/jds.2013-7012 © American Dairy Science Association®, 2013 .

ABSTRACT

This experiment evaluates the effectiveness of individ-ual steps of a clean-in-place protocol against the biofilm constitutive microflora isolated from the biofilms devel-oped on whey reverse-osmosis membranes, aged 2 to 14 mo, under industrial processing conditions. The isolates used for the in vitro resistance studies included species of Bacillus, Enterococcus, Streptococcus, Staphylococcus, Micrococcus, Aeromonas, Corynebacterium, Pseudomo-nas, Klebsiella, and Escherichia. The 6 cleaning steps (alkali, surfactant, acid, enzyme, a second surfactant, and sanitizer treatment) revealed resistance of isolates in both planktonic and biofilm-embedded cell states. The most effective step was the acid treatment, which resulted in 4.54 to 7.90 and 2.09 to 5.02 log reductions of the planktonic and biofilm-embedded cells, respec-tively. Although the sanitizer step causing a reduction of 4.91 to 8.33 log in the case of planktonic cells, it was less effective against the biofilm-embedded cells, resulting in a reduction of 0.59 to 1.64 log. Bacillusspp. showed the highest resistance in both planktonic, as well as embedded cell states. Key words: biofilm , reverse osmosis , membrane , cleaning

INTRODUCTION

Previous studies in our laboratory demonstrated the presence of bacterial biofilms on whey reverse-osmosis (RO) membranes obtained at 2-mo intervals during a total of 14 mo of whey-processing operations. These multispecies biofilms had, on average, about 5.0 log counts that constituted diverse bacterial spe-cies, including Enterococcus, Staphylococcus, Klebsiella, Escherichia, Corynebacterium, Pseudomonas, Bacillus,

Micrococcus, Streptococcus, and Aeromonas (Biswas et al., 2010; Hassan et al., 2010; Anand et al., 2012). These biofilms developed on RO membranes despite regular clean-in-place (CIP) protocols followed by the whey processing plant, carried out under dynamic flow conditions [around 300–400 psi (2,068.43–2,757.90 kPa) and 80–100 GPM (302.83–378.54 L/min)]. A typical membrane-cleaning process includes the application of alkaline solution, acids, metal chelating agents, surfactants, and enzymes (Tragardh, 1989; Moham-madi et al., 2002). Chelating agents bind metal ions from the complex organic molecules, with increased effectiveness of cleaning (Hong and Elimelech, 1997), whereas surfactants remove the foulants by solubiliz-ing macromolecules by forming micelles around them (Rosen, 1987). Addition of enzymes provided enhanced cleaning efficiency by breaking proteinaceous materials and polymeric foulants (Sutherland, 1995). Resistance of biofilm-embedded bacteria to cleaning processes has also been previously reported to be different from that of their planktonic counterparts (Sternberg et al., 1999; Loo et al., 2000; Stewart and Costerton, 2001; Donlan and Costerton, 2002; Keevil, 2002; Sauer et al., 2002; Chmielewski and Frank, 2003; Shi and Zhu, 2009). These enhanced resistances of biofilm-entrapped cells are due to their different transcriptional programs (Asad and Opal, 2008) and their complex distribution (Stoodley et al., 2002). To inactivate these biofilm-em-bedded cells, it is essential for the sanitizer to penetrate first the surrounding polysaccharide material. Bacterial cells attached to biofilms were reported to be about 1,000 times more resistant to antimicrobial stress than free-flowing bacteria of the same species. Studies have also indicated that selection of high persisters within biofilm matrices may also be responsible for the recalci-trance to antimicrobials (Lewis, 2010). This resistance capacity was shown to be dependent on the type of organism and the antimicrobial system (Lewis, 2001; Mah and O’Toole, 2001; Stewart, 2002). The high toler-ance of mature biofilms to chlorine-based sanitizers and antimicrobials was reported to be due to their lower penetration into the biofilm matrix (Gilbert et al.,

Resistance of the constitutive microflora of biofilms formed on whey reverse-osmosis membranes to individual cleaning steps of a typical clean-in-place protocol Sanjeev Anand 1 and Diwakar Singh 2 Midwest Dairy Foods Research Center, Dairy Science Department, South Dakota State University, Brookings 57006

Received May 8, 2013. Accepted June 9, 2013. 1 Corresponding author: [email protected] 2 Current affiliation: Jerome Cheese Company, a division of Davisco

Foods International Inc., Jerome, ID 83338.

2 ANAND AND SINGH

Journal of Dairy Science Vol. 96 No. 10, 2013

1990; Marshall, 1992; Stewart, 2002). The other related factors are the biofilm maturation stage (Drenkard, 2003), lower growth rate, and the associated changes in the cell physiology (Stewart, 2002; Shah et al., 2006). Chlorine transport was shown to improve due to weak-ening of the biomass at the periphery of cell clusters by increased fluid shearing (Davison et al., 2010). Corbin et al. (2011) concluded that the in vitro time for ac-tivity against biofilm was much longer than diffusive penetration time, using an in vitro oral biofilm model. In addition, microbial activity was enhanced with bio-film formation, facilitating a protective shield against environmental stresses such as desiccation, starvation, or the presence of heavy metals.

The presence of multispecies biofilms on whey RO membranes observed in our previous study (Anand et al., 2012) led us to hypothesize the enhanced resistance of constitutive microflora to typical CIP protocols as the membranes age. The present study evaluates the ef-fectiveness of individual cleaning steps of CIP protocols against both planktonic and biofilm-embedded cells. Although no set standards exist for logarithmic reduc-tions, attempts were made to simulate the biofilms developed under industrial conditions by achieving em-bedded cell levels up to 5.0 log using membrane biofilm isolates. These resistant isolates were obtained from membrane biofilm consortia of 2- to 14-mo-old used

membrane cartridges. This study also helps identify the most effective cleaning steps of a typical CIP protocol.

MATERIALS AND METHODS

Source of Bacterial Isolates

Used whey RO membranes were aseptically collected from a commercial dairy plant at intervals of 2, 4, 6, 8, 10, 12, and 14 mo (Anand et al., 2012). Standard microbiological methods were used to isolate the con-stitutive microflora of biofilms (Wehr and Frank, 2004). For further identification to genus level, the isolates were biotyped using microbiological culturing tech-niques and biochemical identification protocols in the Veterinary Science Department, South Dakota State University (Brookings). A total of 26 isolates, belong-ing to 10 genera, were finally selected (Table 1) and termed biofilm isolates in this experiment. The isolates were coded as 3 digits to represent consortium, age of membrane cartridge, and isolate number, respectively.

Maintenance of the Isolates

The bacterial isolates, as listed in Table 1, were stored in Cryovials (CRYO/B; Copan Diagnostics Inc., Murrieta, CA) at −80°C (−112°F) in a deep freezer

Table 1. Biofilm constitutive microflora on the retentate side of whey reverse-osmosis (RO) membranes

Membrane consortium

Membrane age (mo)

Number of isolates Isolate number

1 2 3 1.2.1 Enterococcus spp.1.2.2 Staphylococcus spp.1.2.3 Micrococcus spp.

2 4 4 2.4.1 Enterococcus spp.2.4.2 Klebsiella spp.2.4.3 Bacillus spp.2.4.4 Corynebacterium spp.

3 6 3 3.6.1 Enterococcus spp.3.6.2 Aeromonas spp.3.6.3 Bacillus spp.

4 8 6 4.8.1 Enterococcus spp.4.8.2 Staphylococcus spp.4.8.3 Bacillus spp.4.8.4 Corynebacterium spp.4.8.5 Escherichia coli4.8.6 Pseudomonas spp.

5 10 3 5.10.1 Streptococcus spp.5.10.2 Staphylococcus spp.5.10.3 Bacillus spp.

6 12 3 6.12.1 E. coli6.12.2 Klebsiella spp.6.12.3 Bacillus spp.

7 14 4 7.14.1 Enterococcus spp.7.14.2 Staphylococcus spp.7.14.3 E. coli7.14.4 Klebsiella spp.


RESISTANCE OF MEMBRANE BIOFILMS TO CLEAN-IN-PLACE PROTOCOL 3

(NuAire Inc., Plymouth, MN). Cryobeads of isolates were activated by propagation in 10 mL of brain-heart infusion broth (Difco Laboratories Inc., Franklin Lakes, NJ) tubes and incubated aerobically at 37°C (98.6°F) for 24 h. The Gram-staining technique was used to check the purity of the activated cultures.

Preparation of Chemical Solutions as per the CIP Protocol

A CIP protocol similar to the one being followed in the dairy plant was adopted during the cleaning experi-ments. The protocol included 6 cleaning steps based on the application of alkali, surfactant, acid, enzyme, a second surfactant, and periodic use of a sanitizer, under the conditions described in Table 2. All chemicals were obtained from the dairy plant and tested in accordance with the cleaning process being used in the day-to-day operations of the dairy plant. The routine membrane-cleaning process was conducted at a temperature of 50°C (122°F), whereas the sanitizer treatment was performed at 21.1°C (70°F). The concentrated chemical solutions obtained from the dairy plant were diluted with distilled water to maintain the pH as per the rec-ommended CIP protocol (Table 2).

Planktonic Cell Resistance

Cell Preparation. Cultures were activated by in-dividually transferring 3 times in the brain-heart infu-sion broth and overnight incubation at 37°C (98.6°F). The broth cultures were centrifuged (2, 900 × g) for 15 min and the cell pellets washed twice with sterile PBS (pH 7.0) before being suspended in sterile PBS. The required amount of bacterial suspension was added to sterile phosphate buffer to obtain a final concentration of log 7 to 8. This bacterial suspension was serially diluted using 9.0 mL of sterile PBS and plated on plate count agar (PCA) (Difco Laboratories Inc.); viable counts were later enumerated and expressed as log10 colony-forming units per milliliter.

Planktonic Cell Survivability Against Cleaning Steps. Planktonic cells of all 26 isolates were tested for

resistance under static conditions against the cleaning protocol (Table 2). For this evaluation, individually suspended culture isolates, with average counts of 7.0 log cfu/mL each, were spiked with different chemical solutions and vortexed for 1 min. To validate inocu-lation rates, untreated culture isolates were plated on the PCA to estimate viable counts; these were termed pretreatment counts.

The isolates mixed with chemical solutions were kept in a water bath to maintain the cleaning temperatures as per the CIP protocol mentioned in Table 2. Before plating on the PCA to enumerate viable cells, treated cells were cooled down to room temperature with a continuous flow of tap water and serially diluted using sterile phosphate buffer. To estimate the effectiveness of the treatment, logarithmic reductions were cal-culated based on pre- and posttreatment counts. All experiments were conducted in duplicate and repeated 3 times, resulting in a total of 6 replicates for each isolate.

Resistance of Biofilm-Embedded Cells

Development of In Vitro Biofilms. Single-species in vitro membrane biofilms were developed under static conditions to evaluate the CIP protocol as individual cleaning steps (Table 2). An unused spiral-wound RO membrane cartridge obtained from the dairy plant was sliced using an electric cutter (powered hand saw; Black & Decker Corp., Towson, MD). Aseptic conditions were maintained throughout by wiping the electric cutter and surrounding areas with 70% ethyl alcohol. From these slices, membrane pieces of 3 × 3 cm2 were cut to develop static biofilms. The whey growth medium used to develop membrane biofilms was sterilized using a Stericup, a vacuum-driven disposable filtration system having 0.22-μm pore size (Millipore Corp., Billerica, MA). Filtered sterile whey was stored until further use in the Millipore bottle itself at −20°C (−4°F) in a deep freezer (Frigidaire, Augusta, GA).

All individual isolates (Table 1) were activated and propagated as mentioned above. Membrane pieces of 3 × 3 cm2 were sanitized with a 0.5% solution of hy-

Table 2. Cleaning steps of the clean-in-place (CIP) protocol

Step TreatmentTime (min)

Temperature [°C (°F)]

Target pH

Step 1 Alkali rinse 12 50 (122) 11.2Step 2 Surfactant 1 30 50 (122) 11.2Step 3 Acid 30 50 (122) 2.1Step 4 Enzyme 45 50 (122) 10.75Step 5 Surfactant 2 10 50 (122) 11.2Step 6 Sanitizer 1 21.1 (70) 3.5

4 ANAND AND SINGH


drogen peroxide and kept in a large Petri dish (150 × 15 mm2). About 25 mL of sterile whey, as prepared above, was poured into the Petri dish. Another 25-mL aliquot of sterile whey was mixed with the activated culture isolate, vortexed, and added to the Petri dish. The final culture suspension was kept at an inoculation rate of approximately log10 7.0. Incubations to develop the biofilms were at 37°C (98.6°) for 12 and 24 h.

Biofilm-Embedded Cell Survivability Against Cleaning Steps. The 12- and 24-h-old membrane bio-films, as developed above using single-culture isolates, were treated with individual and sequential cleaning steps of the CIP protocol (Table 2) to observe their resistance. Two membrane pieces (3 × 3 cm2) with bio-films were randomly selected and the estimated biofilm counts were designated as pretreatment counts. The remaining membrane pieces with biofilms were treated in duplicate against each of the 6 individual chemical cleaning steps of the CIP protocol (Table 2). The treat-ed membrane pieces were rinsed with neutralized PBS [PBS with 0.5% polysorbate (Tween 80) plus 0.07% soy lecithin] to neutralize any chemical residues before bacterial enumeration using the swab technique. The swabs were taken on the predefined area of the mem-brane samples to recover the biofilms and the cells were dislodged in to the diluent by vigorous vortexing. The recovery efficiency was checked by repeat swabs and scanning electron microscopy of the swabbed surfaces for any remaining cells. Such studies were conducted while initially standardizing the protocols (Avadhanula, 2011). The surviving membrane biofilm-embedded cells were designated as posttreatment counts. The logarith-mic reductions were calculated by comparing posttreat-ment counts with pretreatment counts for both 12- and 24-h-old biofilms.

Statistical Analysis

Data were analyzed by ANOVA using the general linear models procedure of SAS statistical analysis soft-ware package (SAS Institute, 1999) and means were compared using the Tukey test. Differences in all ex-periments were considered significant at P < 0.05.

RESULTS AND DISCUSSION

Evaluation of Individual Cleaning Steps Against the Planktonic Cells

Presence of membrane biofilms despite regular clean-ing had previously suggested that these biofilm isolates were resistant against the cleaning protocol followed in that dairy plant (Anand et al., 2012). The biofilm constitutive microorganisms belonged to the genera

Enterococcus, Staphylococcus, Bacillus, Klebsiella, Esch-erichia, Corynebacterium, Micrococcus, Aeromonas, Pseudomonas, and Streptococcus. In the current study, all isolates were treated against the existing CIP chemi-cal treatment steps in planktonic cell states.

Initial attachment of planktonic cells to contact surfaces is known to result in their greater resistance against regular cleaning processes. Surviving microflora, thus, get firmly attached to the membrane surface and gradually develop into resistant mature biofilms (Lewis, 2010). It was, therefore, considered important to first evaluate the effectiveness of CIP chemicals against the planktonic cells. The pretreatment counts and log10 reduction values for all isolates of different consortia in a planktonic state are presented in Table 3. The results obtained from this study for planktonic cells revealed variations in the effectiveness of CIP treat-ment steps against different bacterial isolates. Sanitizer and acid treatment steps resulted in greater reductions among all the chemicals used for cleaning. Overall, the sanitizer treatment (step 6) resulted in 4.91 to 8.33 log reduction of the isolates in a planktonic cell state. All isolates of Enterococcus, Staphylococcus, Escherichia coli, Micrococcus, Pseudomonas, and Streptococcus were completely inactivated in their planktonic state by the sanitizer treatment (step 6). However, resistance was observed for the isolates of Bacillus, Klebsiella, Cory-nebacterium, and Aeromonas. The next-most-effective cleaning step across all isolates was the acid treatment (step 3), which resulted in 4.54 to 7.90 log reduction. It was revealed that the sanitizer treatment (step 6) was more effective against the planktonic cell state com-pared with the acid cleaning step. All other cleaning steps, such as alkali, surfactant 1, enzyme, and surfac-tant 2, resulted in only 2 to 3 log reduction against the planktonic state.

Among the isolates, Bacillus spp. were found to be most resistant to the sanitizer treatment (step 6), which reduced their counts by 4.91 to 5.65 log compared with the other bacteria, which showed much higher loga-rithmic reductions after this step. Similarly, the acid treatment (step 3) also resulted in only 4.54 to 5.13 log reduction for different Bacillus isolates. The lower reduction observed for the 5 Bacillus isolates against both acid and sanitizer treatments when compared with the rest established their higher resistance pattern. It was thus concluded that although all the isolates of the biofilm consortia showed sensitivity toward acid and sanitizer treatment steps, the isolates of Bacillus were generally resistant. Previous researchers have also identified the resistance of Bacillus species against dis-infectants and cleaning agents (Grönholm et al., 1999).

A unique part of the study was to obtain isolates from membranes of different ages, varying from 2 to



Tab

le 3

. Pos

ttre

atm

ent

redu

ctio

ns o

f m

embr

ane

biof

ilm i

sola

tes

(log

10 c

fu/m

L;

mea

n ±

SD

of

3 re

plic

ates

) in

a p

lank

toni

c st

ate

trea

ted

agai

nst

differ

ent

clea

n-in

-pla

ce (

CIP

) ch

emic

als1

unde

r st

atic

con

dition

s

Mic

roor

gani

sms

Isol

ate

num

ber

Pre

trea

tmen

t

coun

t

CIP

cle

anin

g st

ep

Alk

ali

Surf

acta

nt 1

Aci

dE

nzym

eSu

rfac

tant

2Sa

nitize

r

Ent

eroc

occu

s sp

p.1.

2.1

8.33

± 0

.51

2.22

± 0

.58A

,b2.

45 ±

0.5

2A,b

7.90

± 0

.39A

,a3.

03 ±

0.4

6A,b

2.83

± 0

.54A

,b8.

33 ±

0.5

1A,a

2.4.

17.

73 ±

0.4

51.

94 ±

0.4

6A,b

2.04

± 0

.38A

,b7.

25 ±

0.3

1A,a

2.44

± 0

.15A

B,b

2.34

± 0

.24A

,b7.

73 ±

0.4

5A,a

3.6.

17.

88 ±

0.7

21.

84 ±

0.4

5A,b

1.98

± 0

.43A

,b7.

29 ±

0.6

4A,a

2.31

± 0

.41A

B,b

2.28

± 0

.47A

,b7.

88 ±

0.7

2A,a

4.8.

17.

95 ±

0.3

11.

65 ±

0.2

7A,c

1.76

± 0

.27A

,c7.

14 ±

0.1

3A,b

2.14

± 0

.22B

,c2.

14 ±

0.2

8A,c

7.95

± 0

.31A

,a

7.14

.17.

87 ±

0.3

02.

03 ±

0.3

4A,b

2.43

± 0

.36A

,b7.

36 ±

0.1

3A,a

2.60

± 0

.29A

B,b

2.61

± 0

.24A

,b7.

87 ±

0.3

0A,a

Stap

hylo

cocc

us s

pp.

1.2.

28.

02 ±

0.6

22.

10 ±

0.4

9A,b

2.04

± 0

.53A

,b7.

37 ±

0.4

2A,a

1.81

± 0

.31A

B,b

2.03

± 0

.40A

,b8.

02 ±

0.6

2A,a

4.8.

28.

05 ±

0.3

82.

00 ±

0.4

5A,b

1.82

± 0

.39A

,b7.

29 ±

0.2

2A,a

1.64

± 0

.21A

B,b

1.80

± 0

.34A

,b8.

05 ±

0.3

8A,a

5.10

.27.

44 ±

0.3

91.

57 ±

0.4

4A,b

1.45

± 0

.26A

,b6.

60 ±

0.2

6A,a

1.44

± 0

.20B

,b1.

41 ±

0.3

4A,b

7.44

± 0

.39A

,a

7.14

.27.

69 ±

0.5

22.

21 ±

0.3

3A,b

2.11

± 0

.14A

,b7.

29 ±

0.2

2A,a

2.10

± 0

.15A

,b2.

20 ±

0.1

1A,b

7.69

± 0

.52A

,a

Bac

illus

spp

.2.

4.3

7.35

± 0

.39

2.97

± 0

.48A

,b3.

02 ±

0.2

3A,b

5.13

± 0

.63A

,a2.

92 ±

0.1

6A,b

3.10

± 0

.49A

,b5.

65 ±

0.5

2A,a

3.6.

37.

40 ±

0.4

12.

83 ±

0.4

4A,b

2.81

± 0

.15A

B,b

5.05

± 0

.63A

,a2.

79 ±

0.1

7A,b

3.06

± 0

.52A

,b5.

50 ±

0.3

8A,a

4.8.

37.

19 ±

0.0

92.

54 ±

0.3

2A,b

2.54

± 0

.06B

C,b

4.75

± 0

.44A

,a2.

48 ±

0.0

6B,b

2.82

± 0

.34A

,b5.

09 ±

0.4

5A,a

5.10

.37.

13 ±

0.1

22.

35 ±

0.2

4A,b

2.43

± 0

.04C

,b4.

56 ±

0.3

5A,a

2.32

± 0

.02B

,b2.

59 ±

0.3

7A,b

4.91

± 0

.25A

,a

6.12

.37.

25 ±

0.0

72.

30 ±

0.2

7A,b

2.37

± 0

.06C

,b4.

54 ±

0.4

5A,a

2.30

± 0

.04B

,b2.

47 ±

0.3

3A,b

4.92

± 0

.39A

,a

Kle

bsie

lla s

pp.

2.4.

28.

44 ±

0.3

33.

44 ±

0.3

4A,b

3.51

± 0

.29A

,b7.

81 ±

0.0

6A,a

3.66

± 0

.47A

,b3.

93 ±

0.2

8A,b

8.11

± 0

.29A

,a

6.12

.28.

42 ±

0.2

63.

07 ±

0.3

1A,b

3.37

± 0

.25A

,b7.

63 ±

0.1

0A,a

3.50

± 0

.42A

,b3.

67 ±

0.3

0A,b

8.06

± 0

.38A

,a

7.14

.48.

36 ±

0.2

53.

42 ±

0.2

2A,b

3.65

± 0

.13A

,b7.

82 ±

0.1

9A,a

3.70

± 0

.41A

,b4.

28 ±

0.5

1A,b

8.16

± 0

.42A

,a

Esc

heri

chia

col

i4.

8.5

8.26

± 0

.04

2.98

± 0

.26A

,b3.

23 ±

0.1

7A,b

7.64

± 0

.27A

,a3.

00 ±

0.3

5A,b

3.33

± 0

.13A

,b8.

26 ±

0.0

4A,a

6.12

.18.

32 ±

0.2

62.

75 ±

0.2

2A,b

2.88

± 0

.20A

,b7.

74 ±

0.3

5A,a

2.80

± 0

.29A

,b3.

17 ±

0.1

0A,b

8.32

± 0

.26A

,a

7.14

.38.

01 ±

0.2

33.

04 ±

0.1

9A,b

3.18

± 0

.18A

,b7.

49 ±

0.4

4A,a

3.09

± 0

.06A

,b3.

28 ±

0.1

9A,b

8.01

± 0

.23A

,a

Cor

yneb

acte

rium

spp

.2.

4.4

8.00

± 0

.28

2.05

± 0

.05A

,b1.

99 ±

0.2

1A,b

6.72

± 0

.35A

,a2.

09 ±

0.1

5A,b

2.13

± 0

.25A

,b7.

03 ±

0.1

6A,a

4.8.

48.

04 ±

0.4

32.

01 ±

0.1

3A,b

1.81

± 0

.22A

,b6.

72 ±

0.3

6A,a

2.01

± 0

.20A

,b2.

03 ±

0.1

0A,b

6.78

± 0

.31A

,a

Mic

roco

ccus

spp

.1.

2.3

8.04

± 0

.24

2.64

± 0

.16b

2.85

± 0

.22b

7.63

± 0

.16a

2.86

± 0

.15b

3.12

± 0

.16b

8.04

± 0

.24a

Aer

omon

as s

pp.

3.6.

27.

77 ±

0.3

71.

39 ±

0.0

8b1.

63 ±

0.1

4b6.

14 ±

0.3

6a1.

64 ±

0.1

9b1.

69 ±

0.2

7b6.

68 ±

0.2

5a

Pse

udom

onas

spp

.4.

8.6

7.63

± 0

.12

2.13

± 0

.13b

2.54

± 0

.26b

7.63

± 0

.12a

2.39

± 0

.22b

2.44

± 0

.17b

7.63

± 0

.12a

Stre

ptoc

occu

s sp

p.5.

10.1

7.64

± 0

.32

2.33

± 0

.28b

2.36

± 0

.29b

7.64

± 0

.32a

1.96

± 0

.18b

2.23

± 0

.06b

7.64

± 0

.32a

A–C

Mea

ns w

ithi

n a

colu

mn

for

indi

vidu

al m

icro

orga

nism

s no

t sh

arin

g co

mm

on s

uper

scri

pts

are

differ

ent

(P <

0.0

5).

a–c M

eans

withi

n a

row

not

sha

ring

com

mon

sup

ersc

ript

s ar

e di

ffer

ent

(P <

0.0

5).

1 Cle

an-in-

plac

e ch

emic

als

used

as

men

tion

ed in

Tab

le 2

.

6 ANAND AND SINGH


14 mo, and test them for their resistance. All isolates within a genus were also compared for their resistance against the cleaning steps. It was concluded that the re-sistance pattern for acid and sanitizer treatment steps, in general, was statistically similar for all isolates under a genus. The differences in effectiveness were, however, noticed for the other cleaning steps. Another important observation was the comparative lower logarithmic re-ductions noticed for some of the isolates obtained from the older (especially 10 and 12 mo) compared with the younger (especially 4 and 6 mo) biofilm consortia. These observations provide evidence of greater resis-tance of some isolates from older biofilms, and possibly reflect resistance development in these isolates during their prolonged exposure to cleaning chemicals. This is an important observation, as it provides direct evidence of the resistance development in biofilm-embedded cells during the long usage of membranes under industrial conditions. However, similar high resistances were not noticed in all the isolates. This may have the poten-tial for some selectively resistant organism to generate predominance and greater persistence in the biofilm matrix. Such organisms may have the ability to repeat-edly cross-contaminate the retentate, as also shown in one of our previous studies (Anand at al., 2012) Based on the individual cleaning steps, it was concluded that the sanitizer treatment was most effective against the planktonic cells, followed by the acid cleaning step. Despite this, resistant cells were observed in this study. These survivors have the potential to grow and form bio-films, as they would remain in contact with membrane surfaces for prolonged periods of time at a temperature of whey processing [at about 37.7°C (100°F)] conducive to their growth. The sanitizer’s broad antimicrobial ef-fect and the low pH of the acid treatment step are the main reasons for their observed effectiveness against planktonic cells.

Evaluation of Individual Cleaning Steps Against 12-h-Old Biofilms

Resistant planktonic microflora may attach to membrane surfaces, and gradually get converted into irreversible biofilms. In vitro biofilms were developed, under static conditions, to evaluate the effectiveness of different cleaning steps against biofilm-embedded cells, using individual isolates from different membrane consortia. During this segment of the study, 12-h-old biofilms with certain pretreatment counts were treated with CIP chemicals, as individual steps, and the cor-responding posttreatment reductions (log10 cfu/cm2) were calculated (Table 4). Approximately 8 log cfu/mL of planktonic cells were inoculated to the substratum at 37°C (98.6°F) for 12 h to develop in vitro biofilms.

Final counts of embedded cells in 12-h-old biofilms (termed pretreatment counts) were observed to be in the range of 3.26 to 5.28 log. A possible reason for the low pretreatment counts could be the short 12-h dura-tion for biofilm development under in vitro conditions. The most effective treatment was the acid treatment at pH 2.1. This was observed to result in 2.21 to 4.99 log reduction in biofilm-embedded cells for different isolates. The sanitizer treatment (step 6), on the other hand, showed only limited effectiveness against biofilm-embedded cells, as it reduced initial counts for different isolates by 0.64 to 1.52 log. No significant differences were observed within the treatments of alkali, surfac-tant 1, enzyme, and surfactant 2, which reduced the pretreatment counts to a different extent based on the isolate (Table 4).

More specifically, the acid treatment (step 3) was found to be effective against biofilms formed by isolat-ed species of Enterococcus, Staphylococcus, Klebsiella, Escherichia coli, Corynebacterium, Micrococcus, Pseu-domonas, and Streptococcus. The pretreatment counts were reduced by 4.26 to 4.99 log. Biofilms of Bacillus, on the other hand, showed a much lower reduction in the range of 2.21 to 2.76 log. This lower posttreatment reduction pattern indicates that biofilms developed by Bacillus were more resistant against the acid treatment (step 3) compared with other isolates. The sanitizer treatment (step 6) on biofilm-embedded cells resulted, in general, in much lower reductions (1.17 to 1.52 log). The lower effectiveness of the sanitizer treatment was more evident in Bacillus spp. biofilms, as this treat-ment (step 6) reduced pretreatment counts by only 0.64 to 0.87 log.

Based on this study, it may be concluded that sani-tizer treatment (step 6) was less effective in reducing biofilm-embedded cells compared with planktonic cells. One possible reason could be the sanitizer’s reduced penetration ability into the biofilm matrix. Some previ-ous reports also indicate greater tolerance of biofilms against sanitizers, such as chlorine, due to several reasons such as the lower penetration ability of anti-microbials (Marshall, 1992), the lower growth rate of biofilm cells, and the associated changes in cell physiol-ogy (Stewart, 2002; Shah et al., 2006).

Another important finding of this study is that the reduction pattern produced by the acid remained simi-lar for both planktonic cells and their biofilms (step 3). A possible reason for the greater effectiveness of the acid treatment compared with the sanitizer treatment may be due to OM present in biofilm matrices, which is more susceptible to acid treatment. Further studies are, however, necessary to study the exact reason for the greater effectiveness of acid treatment (step 3) for denaturation of the biofilm matrix.



Tab

le 4

. Pos

ttre

atm

ent

redu

ctio

ns o

f m

embr

ane

biof

ilm is

olat

es (

log 1

0 cf

u/cm

2 ; m

ean

± S

D o

f 2

repl

icat

es)

in a

n em

bedd

ed s

tate

(12

-h-o

ld in

divi

dual

isol

ate

biof

ilms)

by

differ

ent

clea

n-in

-pla

ce (

CIP

) ch

emic

als1

unde

r st

atic

con

dition

s

Mic

roor

gani

sms

Isol

ate

num

ber

Pre

trea

tmen

t

coun

t

CIP

cle

anin

g st

ep

Alk

ali

Surf

acta

nt 1

Aci

dE

nzym

eSu

rfac

tant

2Sa

nitize

r

Ent

eroc

occu

s sp

p.1.

2.1

5.16

± 0

.18

2.67

± 0

.06B

C,b

2.73

± 0

.04A

B,b

4.71

± 0

.06A

,a2.

75 ±

0.1

7AB

,b2.

59 ±

0.2

1A,b

1.36

± 0

.01A

,c

2.4.

14.

88 ±

0.1

02.

53 ±

0.0

1C,b

2.57

± 0

.19B

,b4.

38 ±

0.1

0A,a

2.41

± 0

.11B

,b2.

54 ±

0.1

8A,b

1.34

± 0

.08A

,c

3.6.

15.

25 ±

0.1

12.

76 ±

0.0

0AB

,b2.

75 ±

0.1

3AB

,b4.

70 ±

0.2

5A,a

2.67

± 0

.04A

B,b

2.65

± 0

.24A

,b1.

44 ±

0.0

6A,c

4.8.

15.

03 ±

0.1

62.

60 ±

0.0

8BC

,b2.

75 ±

0.0

3AB

,b4.

63 ±

0.1

6A,a

2.59

± 0

.04A

B,b

2.51

± 0

.08A

,b1.

33 ±

0.0

6A,c

7.14

.15.

21 ±

0.3

02.

96 ±

0.0

7A,b

3.02

± 0

.06A

,b4.

89 ±

0.4

7A,a

2.95

± 0

.15A

,b2.

78 ±

0.1

3A,b

1.52

± 0

.06A

,c

Stap

hylo

cocc

us s

pp.

1.2.

25.

23 ±

0.1

32.

84 ±

0.0

9A,b

2.88

± 0

.06A

,b4.

99 ±

0.1

9A,a

2.86

± 0

.12A

,b2.

87 ±

0.1

4A,b

1.45

± 0

.06A

,c

4.8.

24.

97 ±

0.1

82.

69 ±

0.0

5A,b

2.82

± 0

.11A

,b4.

49 ±

0.1

2A,a

2.76

± 0

.10A

,b2.

60 ±

0.0

6A,b

1.32

± 0

.08A

,c

5.10

.25.

12 ±

0.0

62.

64 ±

0.0

2A,b

2.75

± 0

.06A

,b4.

46 ±

0.0

9A,a

2.63

± 0

.22A

,b2.

59 ±

0.1

3A,b

1.27

± 0

.17A

,c

7.14

.24.

78 ±

0.1

82.

72 ±

0.2

8A,b

2.71

± 0

.16A

,b4.

47 ±

0.2

3A,a

2.68

± 0

.01A

,b2.

65 ±

0.1

5A,b

1.30

± 0

.02A

,c

Bac

illus

spp

.2.

4.3

3.50

± 0

.17

1.61

± 0

.04A

,b1.

62 ±

0.0

4B,b

2.57

± 0

.28A

,a1.

77 ±

0.0

6A,b

1.50

± 0

.10A

,b0.

85 ±

0.0

6AB

,c

3.6.

33.

59 ±

0.2

51.

64 ±

0.1

5A,b

c1.

64 ±

0.0

7B,b

2.56

± 0

.40A

,a1.

76 ±

0.1

4A,b

1.61

± 0

.11A

,bc

0.87

± 0

.08A

,c

4.8.

33.

64 ±

0.1

11.

69 ±

0.0

5A,b

1.75

± 0

.01A

B,b

2.71

± 0

.18A

,a1.

85 ±

0.0

4A,b

1.64

± 0

.05A

,b0.

84 ±

0.0

6AB

,c

5.10

.33.

26 ±

0.1

81.

43 ±

0.0

7A,b

1.45

± 0

.01C

,b2.

21 ±

0.2

7A,a

1.59

± 0

.05A

,b1.

37 ±

0.1

4A,b

0.64

± 0

.03B

,c

6.12

.33.

83 ±

0.2

11.

75 ±

0.2

0A,b

1.83

± 0

.01A

,b2.

76 ±

0.4

5A,a

1.92

± 0

.04A

,ab

1.74

± 0

.13A

,b0.

85 ±

0.0

2AB

,c

Kle

bsie

lla s

pp.

2.4.

25.

16 ±

0.1

12.

71 ±

0.1

1A,b

2.92

± 0

.05A

,b4.

57 ±

0.1

2A,a

2.72

± 0

.32A

,b2.

61 ±

0.1

3A,b

1.37

± 0

.09A

,c

6.12

.25.

01 ±

0.3

02.

60 ±

0.3

0A,b

2.74

± 0

.27A

,b4.

38 ±

0.2

0A,a

2.72

± 0

.20A

,b2.

68 ±

0.3

0A,b

1.38

± 0

.08A

,c

7.14

.45.

10 ±

0.0

52.

93 ±

0.0

8A,b

2.97

± 0

.06A

,b4.

63 ±

0.0

9A,a

3.06

± 0

.04A

,b2.

87 ±

0.0

8A,b

1.46

± 0

.07A

,c

Esc

heri

chia

col

i4.

8.5

4.75

± 0

.27

2.66

± 0

.29A

,b2.

85 ±

0.1

1A,b

4.44

± 0

.22A

,a2.

72 ±

0.1

6A,b

2.74

± 0

.06A

,b1.

32 ±

0.0

6A,c

6.12

.14.

94 ±

0.2

32.

56 ±

0.0

2A,b

2.72

± 0

.08A

,b4.

36 ±

0.2

4A,a

2.64

± 0

.13A

,b2.

74 ±

0.3

0A,b

1.26

± 0

.04A

,c

7.14

.34.

58 ±

0.2

62.

65 ±

0.1

3A,b

2.74

± 0

.14A

,b4.

26 ±

0.2

1A,a

2.66

± 0

.19A

,b2.

62 ±

0.2

6A,b

1.28

± 0

.16A

,c

Cor

yneb

acte

rium

spp

.2.

4.4

5.28

± 0

.18

2.49

± 0

.27A

,b2.

31 ±

0.2

8A,b

c4.

78 ±

0.1

0A,a

2.35

± 0

.22A

,bc

2.18

± 0

.30A

,bc

1.46

± 0

.13A

,c

4.8.

45.

21 ±

0.2

22.

33 ±

0.0

1A,b

2.39

± 0

.12A

,b4.

47 ±

0.2

0A,a

2.41

± 0

.05A

,b2.

30 ±

0.1

2A,b

1.41

± 0

.16A

,c

Mic

roco

ccus

spp

.1.

2.3

4.83

± 0

.11

2.50

± 0

.04b

2.57

± 0

.03b

4.28

± 0

.04a

2.48

± 0

.02b

2.52

± 0

.04b

1.49

± 0

.08c

Aer

omon

as s

pp.

3.6.

24.

60 ±

0.2

82.

17 ±

0.0

8b2.

07 ±

0.0

9b3.

80 ±

0.2

1a2.

12 ±

0.1

7b1.

98 ±

0.1

5b1.

28 ±

0.1

7c

Pse

udom

onas

spp

.4.

8.6

4.85

± 0

.25

2.79

± 0

.09b

2.61

± 0

.08b

4.49

± 0

.14a

2.67

± 0

.18b

2.50

± 0

.29b

1.17

± 0

.16c

Stre

ptoc

occu

s sp

p.5.

10.1

5.12

± 0

.21

2.50

± 0

.08bc

2.80

± 0

.13b

4.51

± 0

.06a

2.44

± 0

.18bc

2.30

± 0

.02c

1.24

± 0

.06d

A–C

Mea

ns w

ithi

n a

colu

mn

for

indi

vidu

al m

icro

orga

nism

s no

t sh

arin

g co

mm

on s

uper

scri

pts

are

differ

ent

(P <

0.0

5).

a–d M

eans

withi

n a

row

not

sha

ring

com

mon

sup

ersc

ript

s ar

e di

ffer

ent

(P <

0.0

5).

1 Cle

an-in-

plac

e ch

emic

als

used

as

men

tion

ed in

Tab

le 2

.

8 ANAND AND SINGH


Tab

le 5

. Pos

ttre

atm

ent

redu

ctio

ns o

f m

embr

ane

biof

ilm is

olat

es (

log 1

0 cf

u/cm

2 ; m

ean

± S

D o

f 2

repl

icat

es)

in a

n em

bedd

ed s

tate

(24

-h-o

ld in

divi

dual

isol

ate

biof

ilms)

by

differ

ent

clea

n-in

-pla

ce (

CIP

) ch

emic

als1

unde

r st

atic

con

dition

s

Mic

roor

gani

sms

Isol

ate

num

ber

Pre

trea

tmen

t

coun

t

CIP

cle

anin

g st

ep

Alk

ali

Surf

acta

nt 1

Aci

dE

nzym

eSu

rfac

tant

2Sa

nitize

r

Ent

eroc

occu

s sp

p.1.

2.1

5.49

± 0

.23

2.44

± 0

.08A

,b2.

55 ±

0.2

1A,b

4.77

± 0

.21A

,a2.

50 ±

0.2

3A,b

2.52

± 0

.28A

,b1.

33 ±

0.1

3A,c

2.4.

15.

33 ±

0.1

12.

39 ±

0.1

3AB

,b2.

53 ±

0.2

6A,b

4.63

± 0

.11A

,a2.

52 ±

0.2

8A,b

2.54

± 0

.35A

,b1.

30 ±

0.0

2A,c

3.6.

15.

17 ±

0.1

22.

15 ±

0.0

3AB

,b2.

30 ±

0.0

7A,b

4.35

± 0

.21A

,a2.

28 ±

0.1

3A,b

2.27

± 0

.13A

,b1.

03 ±

0.0

4A,c

4.8.

15.

20 ±

0.1

02.

11 ±

0.0

4B,b

2.28

± 0

.01A

,b4.

28 ±

0.0

6A,a

2.29

± 0

.04A

,b2.

26 ±

0.0

7A,b

1.04

± 0

.09A

,c

7.14

.15.

30 ±

0.1

02.

32 ±

0.0

6AB

,b2.

61 ±

0.1

0A,b

4.69

± 0

.07A

,a2.

54 ±

0.1

0A,b

2.38

± 0

.09A

,b1.

36 ±

0.2

0A,c

Stap

hylo

cocc

us s

pp.

1.2.

25.

57 ±

0.2

82.

57 ±

0.1

5A,b

2.68

± 0

.09B

,b5.

02 ±

0.2

1A,a

2.68

± 0

.01A

B,b

2.65

± 0

.07A

,b1.

50 ±

0.1

4A,c

4.8.

25.

45 ±

0.3

82.

48 ±

0.2

3A,b

2.54

± 0

.04B

,b4.

70 ±

0.2

1A,a

2.59

± 0

.09A

B,b

2.56

± 0

.11A

,b1.

38 ±

0.1

6A,c

5.10

.25.

41 ±

0.1

32.

46 ±

0.0

6A,b

2.45

± 0

.17B

,b4.

49 ±

0.0

4A,a

2.41

± 0

.17B

,b2.

38 ±

0.1

5A,b

1.24

± 0

.10A

,c

7.14

.25.

58 ±

0.0

12.

82 ±

0.0

6A,b

3.15

± 0

.00A

,b4.

95 ±

0.1

7A,a

2.94

± 0

.17A

,b2.

78 ±

0.1

3A,b

1.58

± 0

.06A

,c

Bac

illus

spp

.2.

4.3

3.76

± 0

.09

1.64

± 0

.04A

,b1.

64 ±

0.1

1A,b

2.55

± 0

.17A

,a1.

84 ±

0.0

6A,b

1.61

± 0

.09A

,b0.

69 ±

0.0

1A,c

3.6.

33.

72 ±

0.2

11.

60 ±

0.1

6A,b

1.51

± 0

.13A

,b2.

47 ±

0.0

4A,a

1.71

± 0

.09A

,b1.

55 ±

0.1

3A,b

0.67

± 0

.09A

,c

4.8.

33.

69 ±

0.1

01.

64 ±

0.0

6A,b

1.62

± 0

.01A

,b2.

42 ±

0.2

1A,a

1.75

± 0

.04A

,b1.

63 ±

0.0

6A,b

0.75

± 0

.04A

,c

5.10

.33.

51 ±

0.1

11.

51 ±

0.0

2A,b

1.50

± 0

.01A

,b2.

09 ±

0.2

1A,a

1.68

± 0

.11A

B,b

1.43

± 0

.06A

,b0.

59 ±

0.0

8A,c

6.12

.33.

84 ±

0.1

81.

70 ±

0.0

5A,b

1.66

± 0

.01A

,b2.

48 ±

0.0

6A,a

1.82

± 0

.10A

,b1.

70 ±

0.0

1A,b

0.70

± 0

.13A

,c

Kle

bsie

lla s

pp.

2.4.

25.

67 ±

0.0

72.

58 ±

0.0

1A,b

c2.

70 ±

0.0

4A,b

4.82

± 0

.02A

,a2.

54 ±

0.0

6A,c

2.53

± 0

.04A

,c1.

48 ±

0.0

4A,d

6.12

.25.

33 ±

0.0

42.

32 ±

0.0

3B,b

2.33

± 0

.06B

,b4.

51 ±

0.0

2B,a

2.28

± 0

.03B

,b2.

19 ±

0.0

1B,b

1.24

± 0

.13A

,c

14.4

5.36

± 0

.08

2.49

± 0

.04A

,b2.

63 ±

0.0

3A,b

4.75

± 0

.05A

,a2.

62 ±

0.0

7A,b

2.49

± 0

.08A

,b1.

48 ±

0.0

9A,c

Esc

heri

chia

col

i4.

8.5

5.42

± 0

.26

2.49

± 0

.03A

,b2.

67 ±

0.0

4A,b

4.78

± 0

.12A

,a2.

55 ±

0.0

7A,b

2.44

± 0

.08A

,b1.

39 ±

0.1

7A,c

6.12

.15.

40 ±

0.0

12.

36 ±

0.0

6A,b

2.43

± 0

.08A

,b4.

50 ±

0.0

9A,a

2.37

± 0

.11A

,b2.

39 ±

0.0

4A,b

1.26

± 0

.13A

,c

7.14

.35.

10 ±

0.2

12.

45 ±

0.0

3A,b

2.59

± 0

.07A

,b4.

53 ±

0.0

3A,a

2.41

± 0

.07A

,b2.

52 ±

0.1

3A,b

1.32

± 0

.07A

,c

Cor

yneb

acte

rium

spp

.2.

4.4

5.23

± 0

.11

2.29

± 0

.11A

,b2.

09 ±

0.1

2A,b

c4.

60 ±

0.0

1A,a

2.13

± 0

.06A

,bc

1.93

± 0

.10A

,c1.

24 ±

0.0

8A,d

4.8.

45.

31 ±

0.1

32.

30 ±

0.0

6A,b

2.26

± 0

.16A

,b4.

32 ±

0.0

8B,a

2.15

± 0

.04A

,b2.

15 ±

0.1

1A,b

1.28

± 0

.13A

,c

Mic

roco

ccus

spp

.1.

2.3

5.08

± 0

.09

2.35

± 0

.10b

2.24

± 0

.06b

4.17

± 0

.13a

2.40

± 0

.08b

2.16

± 0

.01b

1.21

± 0

.18c

Aer

omon

as s

pp.

3.6.

24.

90 ±

0.0

92.

02 ±

0.0

5b2.

09 ±

0.0

5b3.

84 ±

0.1

6a2.

05 ±

0.0

1b1.

92 ±

0.0

7bc1.

64 ±

0.0

1c

Pse

udom

onas

spp

.4.

8.6

5.26

± 0

.14

2.47

± 0

.02b

2.73

± 0

.08b

4.58

± 0

.16a

2.50

± 0

.13b

2.36

± 0

.21b

1.18

± 0

.04c

Stre

ptoc

occu

s sp

p.5.

10.1

5.50

± 0

.26

2.40

± 0

.29b

2.54

± 0

.41b

4.52

± 0

.42a

2.32

± 0

.37b

2.42

± 0

.42b

1.23

± 0

.25b

A,BM

eans

withi

n a

colu

mn

for

indi

vidu

al m

icro

orga

nism

s no

t sh

arin

g co

mm

on s

uper

scri

pts

are

differ

ent

(P <

0.0

5).

a–d M

eans

withi

n a

row

not

sha

ring

com

mon

sup

ersc

ript

s ar

e di

ffer

ent

(P <

0.0

5).

1 Cle

an-in-

plac

e ch

emic

als

used

as

men

tion

ed in

Tab

le 2

.



Evaluation of Individual Cleaning Steps Against 24-h-Old Biofilms

Standard CIP cycles performed in the industry were once every 24 h during the continuous whey RO process. In view of this, further studies were conducted to evalu-ate the effectiveness of CIP chemicals against 24-h-old in vitro biofilms developed under static conditions and treated with individual cleaning steps. Pretreatment counts and posttreatment reductions (log10 cfu/cm2) are presented in Table 5.

Pretreatment counts of 24-h-old biofilms for all iso-lates were slightly higher than pretreatment counts of 12-h-old biofilms. From these results, a reduction pat-tern is evident for all individual cleaning steps against 24-h-old biofilms similar to the 12-h-old biofilms. Once again, the acid treatment (step 3) was observed to be the most effective (2.09 to 5.02 log) in reducing pre-treatment counts of different biofilms. Some of the previous studies have recommended the enhancement of CIP by caustic and acid blends (Bremer et al., 2006). Similarly, Shaheen et al., (2010) showed inactivation of resistant spores by hot 0.9% nitric acid. Sanitizer treat-ment (step 6) remained less effective against embedded cells compared with their planktonic counterparts and reduced initial counts by only 0.59 to 1.58 log. Other CIP chemicals (alkali, surfactant 1, enzyme, and sur-factant 2) resulted in initial counts reductions of 1.43 to 3.15 log for the different constitutive microflora of in vitro biofilms. The finding related to the greater resis-tance of Bacillus spp. was further evident even in the 24-h-old biofilms.

The overall logarithmic reductions for 12- and 24-h-old biofilms indicated similar reduction patterns for all CIP chemicals treated under static conditions. A pos-sible reason for similar reduction patterns for 12- and 24-h-old biofilms could be the similar nature of biofilm matrices developed under in vitro conditions. Based on the results obtained from the sanitizer treatment (step 6) against 12- and 24-h-old biofilms, it can be conclud-ed that resistant cells attached to membrane surfaces remained protected against the sanitizer treatment. The reduction pattern of membrane isolates against the sanitizer treatment also revealed that embedded cells were more resistant than their planktonic counter-parts. Previous studies also showed greater resistance of embedded cells with respect to their planktonic state against different cleaning agents (Stewart and Coster-ton, 2001; Chmielewski and Frank, 2003; Shi and Zhu, 2009). In a previous study by Lindsay et al. (2002), the differential efficacy of chlorine dioxide-containing sanitizer was studied. The co-cultured bacteria in bio-films were reported to influence each other with respect to attachment capabilities and sanitizer resistance. In

view of this, some of the other studies being conducted in our laboratory are focused on cleaning process modi-fications for multispecies membrane biofilms.

CONCLUSIONS

Based on the present study, it can be concluded that the sanitizer treatment was the most effective treat-ment step against planktonic cells, followed by the acid step. On the other hand, the biofilm-embedded cells showed greater resistance to sanitizer treatment. This indicates the potential of resistant planktonic cells to grow and form biofilms during the long processing runs of over 20 h. As the embedded cells were more resis-tant to the sanitizer compared with their planktonic counterparts, it may be useful to modify the enzyme cleaning step for more effective breaking of the complex biofilm matrix. This may help in greater inactivation of biofilm microflora due to direct contact with the sani-tizer. The greater resistance of some of the isolates in older biofilms may also pose a unique challenge to the cleaning efficacy of older membranes. As the present study was conducted under in vitro conditions, further experiments are being conducted under a dynamic sys-tem using a Center for Disease Control (CDC) biofilm reactor, to be followed by the industrial trials.

ACKNOWLEDGMENTS

This research was supported by a Midwest Dairy Foods Research Center (Brookings, SD) grant, and a graduate research assistantship from the Agricultural Experiment Station of South Dakota State University (Brookings).

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