Note: Descriptions are shown in the official language in which they were submitted.
53B
Case 4339
PPLIC~TION OF GLUC~N~SE TO CONTROL INDUSTRI~L SLIME
Backqround of the Invention
The present invention relates to glucanase enzyme systems
for treating microbially produced extracelLular polymers, present
or which build up on surfaces of cooling water towers and in
paper making broke water. Such extracellular polymers plus
microbial cells are also known as biofilm or microbial slime.
~ icrobially produced extracellular polymers can build up,
retard heat transfer and restrict water flow through cooling
water systems. Controlling slime-forming bacteria by applying
toxic chemicals is becoming increasingly unacceptable due to
environmental problems. In addition, the efficacy of the
toxicants is ~inimized by the slime itself, since the
extracellular polysaccharide enveloping microorganisms are
largely impenetrable.
Toxicants cannot adequately control large populations of
attached bacteria and they are effective mainly against floating
microorganisms. Qlthough surfactants and dispersants which
penetrate and help loosen slime can enhance the activity of
toxicants~ they are nonspecific and may have deleterious effects
on the industrial process.
This invention describes the use of glucanase which has the
advantage of being both specific and non-toxic. The approach is
designed to ~a) enchance the removal of slime where it has
formed, ~b) prevent the build-up of slime, and ~c) improve the
efficacy of biocides against sessile bacteria. The gluconase
specifically attacks the slime layer surrounding the bacteria.
Consequently, the microorganisms become planktonic--harmless in
terms of biofilm production--and are rendered susceptible to
biocides. The enzymes also act to maintain a clean surface (see
Figure h and remarks).
2~L5~
Examples of prior art single enzyme formulations are: those
found in 3,773,623, Hatcher, Economics Laboratories~ Inc., ~here
the slime formulation in industrial water such as white water
from pulp and paper mills is retarded by controlling amounts of
enzyme levan hydrolase.
Qlso, 4,055,~67, Christensen ~Nalco) describes a slime and
an industrial process whereby slime can be dispersed and
prevented by treating said slime with a few ppm of the en~yme,
Rhozyme HP-150, a pentosanase-hexosanase and 3,824,184, Hatcher
(Economics Laboratories, Inc.) describes a slime formation
contro11ed by intentionally adding to industrial water the
controlled amounts of enz~me levan hydrolase.
~ dditionally, 4,684,469, Pedersen et al. (Qccolab, Inc.)
discloses a method of a two-component biocidal composition
suitable for controlling slime. The preparation consists of a
biocide and a polysaccharide deqrading enzYme.
~ s to the biocides, generally methylene-bis-thiocyanate has
been preferred. Other operable biocides include chlorophenate
compounds, such as pentachlorophenates and trichlorophenates;
organomercurial compounds, such as phenylmercuric acid; carbamate
compounds, such as methyldithiocarbamates,
ethylenebisdithiocarbamates, and dimethyldithiocarbamates;
carbonate compounds such as cyanodithioimidocarbonates;
thiocyanates such as chloroethylene thiocyanate compounds; and
other biocides such as bromo-hydroxyacetophenone compounds,
benzothiazole compounds, ethylene diamine compounds,
nitrilopropionamides, bromopropionamides, bromo-acetoxybutenes.
bromopropanolaldehyde compounds, bis-trichloromethyl sulfones
bimethyl hydantoin compounds, and the like mixtures of biocides
can also be used.
The biocide methylene-bis-thiocyanate has proven to be
particularly effective in the context of this invention, as has
combination of dimethyldithiocarbamate and disodium
ethylenebisdithiocarbamate.
The advantages of the glucanase composition over the use Ot
biocides to control bacteria are that the biocides constitute
toxicants in the system and pollution problems are ever present.
The advantage of the present formulatirn over the formulation of
38
a single enzyme plus biocide i5 that the single enzyme attacks
only one narrow band of carbohydrate polymers whereas the present
invention improves the range of attack by combining activities of
a beta-glucanase and an alpha-amylase along with the basic
protease, broadly attacking the carbohydrate polymer and protein
surrounding the bacteria. ~ specific formulation embodying
ratios, far the present use of multiple enzyme preparations, is 2
parts beta-glucanase, I part alpha-amylase, and 1 part protease.
In this formulation, the alpha-amylase is at least 1 and can be
slightly over 1 part. The orotease which is set at 1 may
actually be .S to I part, and the beta-glucanase is set at Z
parts
~ preferred composition is 2 parts beta-glucanase, I part
alpha-amylase and 1 part protease. In the composition cerulase
may be substituted for beta-glucanase.
In general, glucanase is used in a dosage of 2 to 100 ppm
and may be from 2 to lO~parts per million. The glucanase can be
obtained from many chemical suppliers such as ~merican Cyanamid,
Betz, Beckman, Dearborn Chemical, Economics Laboratory, Inc.
Merck, Nalco, Vineland Chemical! and the like.
The concentration of glucanase required for effectiveness in
this invention varies greatly and can depend upon the conditions
such as temperature and pH of the water, the microbial count and
the type of industrial water being treated. The lower and upper
limits of the required concentrations will substantially depend
upon the specific enzyme or combination of enzymes used. For
example, a highly effective glucanase can require a concentration
of mainly about 1 or 2 parts glucanase to one million parts
industrial water in the context of this invention, or may require
a minimum concentration of 80 or 100 ppm.
In contrast to the prior art, this formulation is both more
specific and non-toxic. In view of this invention and in
comparison with the prior art, it can be said that the present
composition has the same over target polymers but digests them
more efficiently because of the enzyme activities of glucanase in
the mixture of [with?~ alpha-amylase, and the protease.
~oreover, the beta-glucanase is a unique enzyme component which
allows this efficiency to take place. The alpha-amylase and the
20~1~3~3
protease nick the microbial slime and allow the beta-glucanase
access to digest the slime exopolymer more effectively.
.:
It is noted as a matter of general mechanisms, that the
alpha-amylase alone does not give slime protection or remove
slime. It attacks the alpha-linkage between glucose molecules.
It nicks the outside of the slime molecule, so that the beta-
glucanase can enter and attack said carbohydrate molecule. The
protease attacks extracellular protein molecules.
~ ~j
Up to this time, enzvme treatment of industrial slime or
slime polvmer made by bacteria consisted of a single enzyme, for
:~
`. e~ample levanase. Levanase would break down a polymer of levan
into its subunits ~fructose). However, after the levanase would
be used on the slime levan. resistant bacteria would still remain
to proliferate. Further applications of levanase were
.
~i ineffective because the polymer it attacks was no longer present.
~` The levan polymer would be gone~ but other 51 ime polymers would
still be there and the bacteria would flourish. Although other
enzyme preparations have been used in the marketplace, for
'~''.'~ '
i: example ED~, a levan hvdrolyzer ~Sunoco), there has been no
, .:
-~ combination of enzymes that would actually attack polymer made b~
Pseudomonas bacteria and other bacteria ln the field, such as
~, Klebsiellal ~cinetobacter. Flavobacterium, Enterobacter, and
Aerobacter, which were rich in glucose, mannose and gulose sugars
:.:,
arranged in polymers.
Now, in a generalized process and in response to the prior
.,
'~ art above, the present invention has taken a clear culture of
: ~
Pseudomonas bacteria and made them produce a slime polymer in a
~` low substrate environment. Second, the invention has taken a
composite of microorganisms from the field blended with and grown
together both at the laboratory and under field conditions.
.~
simulated cooling tower water and utility water.
The results indicate that the maximum removal of
carbohydrate layers from pending bacteria has occurred. Thus
utili2ing glucanase has a superior result, especially if the
enzyme utilization was found to be useful in the very prevalent
Pseudomonas bacteria.
variety of enzymes were utilized in testing against
Pseudomonas bacteria. From 42 preparations of enzymes, three
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types of enzymes were found to be effective on slime produced by
Pseudomonas bacteria. First, alpha-amylase was found to attack
bacterial slime. Second, protease has been found also to have an
effect on bacterial slime. Then it was found that a combination
enzyme treatment with amylase. glucanase, and protease was
effective in removinq the biofilm.
Brief Descril~ion of the Drawi~s
Figure ~ is a plan view of the biofilm reactor sYstem.
Figure 2 is a graph indicating biofilm removal due to
biodispersant, cellulase. and a mixture of alpha-amylase, beta-
glucanase, protease in a 1:2:1 ratio.
Figure 3 is a plan view of the microbial fouling
reactor system.
Figure 4 i5 a graph showing biofilm mass versus time
for glucanase treatment and biodispersant.
Figure S is a graph showing pressure cdrop versus time
for glucanase treatment and biodispersant.
Figure ~ is a graph showing the results of a treatment
of enzyme versus biocide in a Microbial Fouling Reactor
experiment.
EX~MPLES
Example I
Preliminary activity screening of about forty enzyme
candidates was carried out using slimed microscope slides which
were treated with the enzyme candidates in small, stirred,
sterile beakers. The test slides were prepared in a slime
generation box using a colony isolate of Pseudomonas or a
composite of field microorganisms known to produce extracellula(-
polymers in industr-ial waters. 8acteria were propagated in
tryptic soy broth (TS8) and were enumerated on tryptone glucose
extract ~TGE). ~nhydrous dextrose ~D-glucose) was used to
supplement the TS8 nutrient.
5i3~
Enzyme digestion rates were determined at 1, 2 and 4-hour
intervals by assessing biofilm removal from the slides visually.
Enzyme candidates showing promising activity in this screening
test were explored more fully as below.
E _ ple II
The nine most promising carbohydratases and proteases from
the screening test were subjected to further examination using a
~iofilm Removal Reactor (~RR) which simulates water-tube fouling
in field applications. The reactor is shown schematically in
Figure 1. The reactor tubes were first slimed by exposure to
slime-forming bacteria in circulated minimal sùbstrate for a 72-
hour period.
Each of the candidate enzymes was tested in the reactor at a
level of 100 ppm for a 24-hour- period under the conditions shown
in Table I. The removal of biofilm in the 9RR was measured in
terms of the percent decrease in biomass resulting from enzYme
treatment of the fouled system. The results for the mixed
protease-carbohydratase are shown in Figure 2 and Table II. For
these tests, the reactor tubes were dried overnight at bO degrees
C and weighed; then cleaned, dried and reweighed to obtain the
recorded gravimetric data.
Further tests of these enzymes were conducted in a ~icrobial
Fouling Rea~tor (~FR)~ a similar apparatus which also provides
for a measure of pressure drop across the slimed reactor tubes as
a criterion of fouling. The apparatus is shown in Figure 3 and
the experimental conditions are listed in Table I. The
experimental procedure for the biomass measurements was generall~
similar to that used in ~RR, above. except that the biomass is
measured several times during the course of the experiment. In
addition, the effectiveness of the enzyme treatment is measured
by the decrease in pressure drop across the slimed tubes of the
reactor as well as by visual observation in the sight glass
section. Figures 4 and 5 show the results of tests of the mixed
enzyme compared to a polyol biodispersant.
538
Five of the enzyme preparations tested in the 8iofilm
Removal Reactor were effective in controlling slime. These are
tabulated in Table II with their relative effectiveness. Of
these, the qlucanase was clearly the best performer. This enzyme
composite is a combination of one protease and two
carbohydratases, namely alPha-amylase and beta-g1ucanase. It was
found to be effective In digesting slime la~ers produced by
cultures of pure and ml ecl strains of bacteria. ane commerciall
available mixed en~,me composition is shown in the table to give
37'~, biomass removal In ~he time period of the test.
The ~iofilm Rer~o al Reactor ~Figure 1) results are also
depicted in Figure 2. In the oiofilm remo~al experiments. the
enzyme cellulase remo~,ed r'3''. of the biomass ~b2 mn,XcmZ after
treatment as opposerl to 80 mg~cm2 before treatment) in 24 hour-,.
The l:2:1 combinatlon of alpha-amylase, beta-glucanase. and
protease enzymes remo,ed 3,'~/. of the biofilm in the same time
frame. The contl-ol (b!ank), which was untreated, continued to
increase in biomass ~5%. For comparison. a non-enzymatic
chemical biodispersant essentially checked overall develoDment of
biofilm but did not remove any biomass. Therefore, the multiple
enzyme approach was the best ~37'~.). -
The biomass removal results in the ~FR experiment agreed
essentially with 37% removal between 72.5 and 96.5 hr ~Figure 4).
The pressure drop data ~Figure 5) in the same ~MFR) experiment
support this finding.
EX~MPLE III
Focusing on the ml ed enzyme, further l~lrP .tudies were
conducted to deter~lne the effect of pH or 1--; effecti~eness ir
biofilm removal. r-- ~luc3nase was teste~1 i.lnq a pol~ol
biodispersant as 3 c~ ol ln single-c~cle ~,n~etic tap water
with DH maintainecl 3t '.-,, 8.5 or 9Ø rhe results are
summarized in Table III. The glucanase was effective up to pH ~.
The efficacy of the glucanase is also compared to that of the
dispersant at neutral and alkaline pH's in Table III.
38
Fxample IV
~ n experiment was run on the Microbial Fouling Reactor and
the results are shown in Figure 6. The experiment was designed
to test whether the enzyme product of this invention would keep a
surface clean. The conditions for the experiment differed in
substrate concentration and treatment dosage (Table IV). The
substrate concentration was low similar to substrate level in
cooling water. The dose was eit~,er 51ug of biocide or enzyme
product.
In Figure 6. the control or no treatment (-~-) curve
indicates what biofilm growth is possible in low substrate
conditions. The biocide curve indicates 100 ppm nonoxidizing
biocide slugged in the reactor at days 6, 9, 13, 16, 20, 23, 28
and 31 caused losses in biofilm, as measured by decreases in
pressure drop. The curve representing performance of the en7vme
combination also indicates biofilm loss after each treatment.
Qfter 31 days the difference between the biocide-treated line and
the untreated control was 2.4 inches (~ p = 2.4 in.); 2.7 inches
using the enzyme blend. Fiqure 6 indicates that after treatments
were stopped~ the biofilm in both lines grew.
The results were good. The experiment demonstrated that the
enzymes controlled the biofilm growth very well over one month.
The enzyme blend, which is nontoxic, performed at least as well
as the toxicant (nonoxidizing) biocides.
In the specification and claims glucanase is equivalent and
equal to beta-glucanase.
2(3~3~3
TA~L~ I
Biofilm Removal Test Conditions
Conditions Per APparatus
Parameter BRR MFR
pH 8.5 7.5, 8.5 or 9.0a
Temperature (C) 36+1b 33.0+1C
Make-up Water Synthetic Synthetic
Chicago Tapd Chicago Tapd
Substrate Concentration
TSB 50 ppm 50 ppm
D-glucose 50 ppm SO ppm
Inoculum Field Field
Composite Composite
Growth Period 72 Hr p < 10 ine
Treatment
Enzyme Concentration 100 ppm 100 ppm
Duration 24 Hr 24 Hr
a pH setting depended on experiment.
b BRR temperature is consisten~ly 36+1C resulting from
operation of recirculating pump.
c ~IFR tempera~ure is thermostatically controlled.
d Single cycle synthetic Chicago tap.
e MFR p of 10 inches occu~red at approximately 72 Hr.
~:0~38
TABLE II
Summary of BRR Studies at pH 8.5
Type of Enzyme ~ Remova
Neutral Protease -lO.0
Alkaline Protease~/~ -50.0
\ Alkaline Protease~Z? 18.0
Debranching Enzyme -l9.0
Alkaline alpha-amylase21.5
Beta-glucanase ~l~ 0
Beta-glucanase ~2) 14.0
Cellulasea 23.0
Alpha-amylase, Beta-b 37.0
glucanase + Protease
a Cellula~e attacks the beta-linkage between sugar
molecules.
b Alpha-amylas~, beta-glucanase and neutral protease
activities.
-- ~ 2~ LS~8
TABLE III
Effect of pH on Enzyme Treatment Performancea
% Removal of Biofilmb
pH Enzyme Biodispersant
7.5 48 3
8.5 35b lb
9.0 44b l1b
a Performance is evaluated at 100 ppm enzyme, 20 ppm
biodispersant concentration levels.
b Removal is an average of two experime~ts at 33 + 1C.
11
2~3L538
TABLE IV
Microbial Fouling Reactor Test Conditions
for Biofilm Control Experiment
Parameter Conditions
pH 8.5
Temperature 33.0+1.0C
Make-up Water Synthetic Tap Water
Substrate Concentration
TSB 10 ppm
D glucose 10 ppm
Inoculum Field Composite
Treatments
Enzyme Concentration 150 ppm
Duration Slug dose
Frequency Twice per week
Biocide Concentration 100 ppm
Duration Slug dose
Frequency . Twice per week
