Controlling biofilm development on ultrafiltration and reverse osmosis membranes used in dairy plants : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Food Technology at Massey University, Manawatu, New Zealand
This study aimed to develop improved cleaning strategies for controlling biofilms on
the surfaces of membranes used in dairy ultrafiltration (UF) and reverse osmosis (RO)
Eleven UF / RO membrane modules from 7 different New Zealand dairy membrane
processing plants were received after typical cleaning-in-place (CIP) procedures.
Microorganisms were isolated from both the retentate and permeate sides of these
membrane surfaces and from the liquids collected from a UF membrane plant. Also
some foulants scraped from a RO membrane were tested. The routine CIP currently
used in the dairy plants was not adequate to completely remove organic material,
including microbial cells, proteins and carbohydrates from the membrane surfaces.
These residues may influence the surface characteristics and interactions between
microorganisms and membranes and thus affect biofilm formation. Thirteen isolates
including both bacteria and yeast were identified using biochemical techniques.
Klebsiella oxytoca were isolated from 3 different membrane plant sites. This is, so far as
we know, the first report of K. oxytoca being isolated from dairy membrane surfaces.
The ability of the 13 strains to attach to negatively charged polystyrene surfaces was
tested using a microtitre plate assay. Three K. oxytoca strains demonstrated higher
ability to adhere than the other strains, suggesting that these strains might play an
important role in developing biofilms on dairy membrane surfaces. Two K. oxytoca
strains (K. B006 from plant A, UF and K. TR002 from plant C, RO) that performed best
in the microtitre screening assay with respect to attachment capabilities were chosen for
the remainder of the study.
The cell surface hydrophobicity of all isolates was determined using the microbial
adhesion to hydrocarbon assay (MATH) and the cell surface charge was determined by
measuring the surface zeta potential. These two characteristics did not show a clear
relationship with the adherence of the isolated strains. However, it was found that
bacterial attachment was enhanced in the presence of whey or mixed strains.
A commercial biofilm reactor CBR 90 was modified for developing biofilms on
membranes and investigating strategies for biofilm removal. Biofilms of single and dual
K. oxytoca strains were developed under a continuous flow of whey. The saturated
biofilm was approximately 8 log10 CFU cm-2. The results of our study suggested that the
whey protein concentration, membrane type including membrane material
(polyethersulfone (PES) and polyvinylidene fluoride (PVDF)), membrane age (used and
new), bacterial strain and the interactions between different microorganisms are all
significant factors for biofilm development on membrane surfaces.
Three enzymatic cleaners and four sanitisers, including sodium hypochlorite (pH 6.5,
200 ppm free available chlorine (FAC)), Perform® (peracetic acid/hydrogen peroxide,
2% v/v), ozonated water (pH 7.0, 0.5 ppm free available ozone (FAO)) and anolyte of
MIOX® electrolysed water (EW) (pH 6.8, 120 ppm FAC) were tested for their efficacies
in killing culturable cells from biofilms formed by single or dual K. oxytoca strains on
used PES membrane surfaces. With no sanitation applied, two of three enzymatic
cleaners performed better than sodium hypochlorite (pH 10.8-11, 200 ppm FAC)
commonly used for CIP of UF membranes in the dairy industry. The four sanitisers
were used to treat the membranes after a CIP wash regime. The results indicated that if
a dairy processor were to use a standard CIP on membrane systems, then a further flush
with MIOX® EW anolyte would reduce residual attached microbial populations further.
In addition, using protease followed by a sanitation (sodium hypochlorite, Perform® or
anolyte of MIOX® EW) produced the best clean based on a greater than 2 log reduction
in residual cells and left no culturable and viable cells at a detection limit of 0.1 log10