BIOFILM REMOVAL
Biofilm is a substance that forms readily in water distribution lines, water storage tanks, and anyother aqueous environment. A biofilm forms when bacteria begin to excrete a slimy, sticky
substance that allows them to adhere to surfaces. The biofilm mass usually consists of many
species of bacteria, and can also include fungi, algae, and protozoa. Biofilm is resistant tochlorine and is difficult to remove once initial adhesion occurs. 1 The biofilm slime shelters
disease-causing microorganisms, protecting them from chlorine disinfection. In addition, biofilm
exerts an oxidant demand, consuming chlorine residuals in the distribution line and requiring
higher doses at the treatment station for residual maintenance at the end of the line.
A safe, user-friendly and viable method for controlling biofilms would have a significant impact
on any industry that utilizes treated purified water, including municipal drinking water, cooling
towers, and swimming pools. A mixed-oxidant solution generated on-site through the use of salt,
water, and an electrolytic cell has clearly exhibited the ability to remove biofilm, unlike traditional
chlorination technologies. The evidence includes third party research, visual documentationfrom municipal and industrial operators, and a number of improvements in water quality that areunderstood to result from removal of biofilms. MIOX Corporation, an equipment supplierproducing on-site mixed-oxidant generators, has proposed to work with the Montana State
University’s Center for Biofilm Engineering to conduct additional laboratory studies in the future.
Independent Research
Initial observations of biofilm removal were reported by Montana State University during a fieldstudy conducted at a KOA Kampground in Great Falls, Montana.2 The campsite has a small
potable water supply, as well as showers and a swimming pool. The site previously used
powdered sodium hypochlorite and experienced frequent positive coliform hits, even with freechlorine slug dosage levels as high as 1000 ppm. The positive hits in the presence of freechlorine were indicative of biofilm contamination, also evidenced by a black biofilm slime in theshowers. The distribution system contained accumulated biofouling that had to be flushed fromthe system whenever there was a power outage. The existing cartridge filters also had to becleaned every 2-3 days due to the heavy accumulation of biofilm.
After conversion to mixed oxidants, the KOA Kampground has not experienced a single noncompliance coliform event. The black slime in the showers disappeared soon after theconversion. Although biofilm initially sloughed visibly from the pipelines, the water eventually ranclear, and currently the filters only have to be cleaned every 3-4 weeks. Whenever power
outages occur now, no flushing is required and there is no discoloration of the water, indicatingthat the biofilm does not start to regrow, even when disinfection is temporarily interrupted.
The swimming pool was opened at the same time that the MIOX system was installed, so there
is no comparative data between operations with sodium hypochlorite versus mixed oxidants.

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However, there has been no growth of algae on pool surfaces, a common problem for outdoor
pools, and no algaecide is necessary, not even on the unpainted concrete areas. The pool pipes,connections, and filters remain visibly clean, and no slimy biofilm is apparent when touching thesurfaces. (In contrast, the potable water distribution system components rapidly coated with aslimy substance with use of sodium hypochlorite.)
The mixed-oxidant system was responsible for elimination of biofouling at the Great Falls KOA
Kampground, ultimately resulting in safer drinking water with no bacterial contamination, ease ofmaintenance with no flushing required, and greatly extended filter runs.
Hot Springs and Swimming Pool Facilities
Hot springs are very popular in Japan, with over 30,000 facilities across the country. The warm
aqueous environment provides an ideal breeding ground for bacteria, and a number of the sites
suffer from positive coliform hits and biofouling. Inono Hot Springs in Akita, Japan, was noexception. 3 Although dosing at 1.5 mg/L of free available chlorine (FAC) with sodium
hypochlorite, the site was often unable to maintain even a 0.2 mg/L residual, and experienced
positive coliform and Legionella hits. The decision was made to evaluate an on-site mixedoxidant generator to replace their existing sodium hypochlorite disinfection. Before removing
the sodium hypochlorite system, the MIOX distributor in Japan filmed the interior of the feedwater pipe and the filter recirculation pipe and recorded a heavy, filamentous mat of biogrowthin both locations. The interior of the pipes was subsequently videotaped at six and 22 days aftertreatment with mixed oxidants began.
Upon conversion to mixed oxidants, sloughing was immediately apparent as particles of biofilm
began to appear on the pool surface. Operators manually removed the biofilm and within one
week of operation, the pool water was clear.
INONO HOT SPRINGS

Biofilm removal started within hours
after MIOX system start-up

After 6 days, the pool was clean

Even more impressive was the videotape showing biofilm removal in the 4-inch pipelines. In thefeed water pipe, substantial removal was evident after 6 days, and during the last videotaping at2/Users/idokousei/Desktop/NSP・MIOX資料/NSP・MIOX関連資料/Biofilm Removal/Biofilm_Removal.doc /Printed: 02/23/2020/ Page of 9

22 days, removal of biofilms was total. The same phenomenon occurred in the filter
recirculation pipe with the entire filamentous mass completely eliminated.
FEED WATER PIPE

Feed water pipe withsodium hypochlorite

After 6 days with
mixed oxidants

After 22 days with
mixed oxidants

FILTER RECIRCULATION PIPE

With sodium hypochlorite

With mixed oxidants

After conversion to mixed oxidants and removal of biofilm, the chlorine dosage was reduced by
60% to only 0.6 mg/L, while the residual more than doubled to 0.4 mg/L. Positive bacterial hits
were eliminated, indicating a healthier recreational environment for the swimmer.

3/Users/idokousei/Desktop/NSP・MIOX資料/NSP・MIOX関連資料/Biofilm Removal/Biofilm_Removal.doc /Printed: 02/23/2020/ Page of 9Other hot springs in Japan using MIOX equipment have experienced similar improvements. For
example, the Hyuga Sun Park Hot Springs in Miyazaki, Japan, which utilized sodium
hypochlorite for disinfection, had such a severe Legionella outbreak during their first month ofoperation that 9 customers became infected and lost their lives. The facility was shut down forover a year to research alternatives after 1.5 million CFU/cm2 of Legionella was detected in thepipelines. In October of 2003, the hot springs opened again with newly installed MIOX
equipment. As part of the authorization to re-open the facility, daily Legionella counts arerequired. Since opening in October of 2003, there have been no positive counts for coliforms orLegionella. The absence of biofilm allows no protective covering for dangerous bacteria.
Installations at Shibasaki Gymnasium in Tachikawa City and Miwa Town in the Aichi Prefectureof Japan, both initially using sodium hypochlorite disinfection, reported drastically improved
clarity of the water after only 4-5 days of operation with MIOX mixed oxidants. Improved water
clarity indicates a removal of biofilm contamination and/or algae growth responsible for thecloudiness in the water.
MIWA TOWN

With hypochlorite

With mixed oxidants
TACHIKAWA CITYWith hypochlorite

With mixed oxidants

Another interesting incident validated the effectiveness of mixed oxidants at a hotel in Hawaii.
Mixed-oxidant generators at the Embassy Suites swimming pool in Honolulu were converted in
2002 to generation of sodium hypochlorite. Although the FAC residual in the treated water did
not change, black and brown algae deposits formed on the sides and bottom of the pool after

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only a few days. The operator converted one of his sodium hypochlorite generators back to its
original mixed-oxidant configuration, and the algae disappeared within a few days.
Another pool operator at the YMCA in Valdosta, Georgia reported similar results. Mixedoxidants eliminated a black algae deposit underneath a water slide that was not removed withhypochlorite disinfection.
Membrane Filtration
At the Orange County Water District in California, Dr. Don Phipps, Chemist and Microbiologist,
studied the effects of mixed oxidants and of sodium hypochlorite on the removal of
Psuedomonas putida biofilms on cellulose acetate RO membranes.4 He reported a higher initialoxidant demand of 17 mg/L with mixed oxidants versus only 9 mg/L with chlorine; however,mixed oxidants destroyed the polysaccharide biofilm substrate, while chlorine had no effect.
Many biofilms deposit an extra-cellular substrate upon attachment to a surface, which expedites
further attachments and protects the organisms from disinfectants. After removal of the oxidantdemanding substrate, FAC requirements with mixed oxidants would likely decrease, and thesurface would remain clean as long as mixed oxidants were periodically applied.
DAPI staining showed no intact DNA remaining on the mixed-oxidant samples. Phipps wasimpressed with the thoroughness of kill that occurred with mixed oxidants, the removal of thesubstrate itself, and the speed with which these events occurred once the proper dosage wasreached. Black and white pictures of the microscopic activity from his preliminary studies arereproduced below. The first view shows unchecked biofilm growth at 35 minutes. Removal ofbiofilms began suddenly at 4 hours and 40 minutes and was well underway at the 5-hour mark.
At 5 hours and 40 minutes, 1 hour after removal began, the biofilm was basically eliminated.

Biofilm Removal at 35 min

Biofilm removal at 5 hrs

Biofilm removal at 5 2/3 hrs

Cooling Towers
Rapid removal of biofilms and maintenance of a clean system has been observed in industrial
applications as well. Paul Petersen and Dr. Wes Bradford5 conducted a study of mixed oxidants
versus the organic biocide hydantoin on Gen-Probe cooling towers in California. Within a few
days of replacing the hydantoin biocide with MIOX disinfection, biofilm scales on the coolingsurfaces were eliminated. The scales did not reappear with continued use of mixed oxidants. Inaddition, the occurrence of detectable planktonic aerobic bacteria in the cooling water basin was

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below detection (< 1000/mL). Elimination of biofilms and aerobic bacteria to this extent incooling towers is considered unusual.
In an unrelated study, Charles Smith of Quality Water Systems installed a small MIOX systemon a bakery cooling tower for a pilot test. 6 Upon start of the test, the clear pipelines werecompletely obscured by a “leathery” black slime. Previous disinfection with hypochlorite had
minimal impact on the biofilm contamination, and the pipes required cleaning within 10 days
after disinfection was stopped. In contrast, mixed oxidants thoroughly broke down the biofilm.
When the MIOX pilot system was removed, the bakery owners reported that it took 3 weeks forthe biogrowth to reform, and even at that point, the “leathery” texture was not apparent, socleaning was unnecessary. Mixed oxidants removed even the tough substrate that allows more
rapid regrowth of biofilm.

Black biofilm coating before
application of mixed oxidants

Breakdown of biofilm begins
with mixed oxidants

Tests conducted at Cadbury Trebor Bassett in the United Kingdom7 showed significant activity
against biofilm bacteria with the start of MIOX equipment operations and removal of biofilms thatwere prevalent with earlier use of biocides. Controlling microbiological growth, including biofilms
on cooling surfaces, is one of the major challenges in cooling tower maintenance. Of particularconcern is Legionella, which tends to be present in biofilm masses, and can be difficult toeradicate by conventional means. In contrast to disinfection with biocides, no Legionellae
species were present on any coupon test throughout the 1-year mixed-oxidant trial, representing
a 99% reduction from previously observed biofilm formation. Mixed oxidants not only eliminated
all existing biofilm but also prevented the re-growth of new biofilm. Removal of biofilms not onlyis useful for control of Legionella, but also enables more efficient cooling tower operations andeliminates under deposit corrosion by iron- and sulphate-reducing bacteria.
Municipal Installations
Diana Water Supply in Texas has a number of well sites and has been gradually convertingfrom chlorine gas disinfection to MIOX mixed-oxidant generation, beginning in 1996. Severalyears ago, two line breaks occurred around the same time, one on a line treated with chlorine
gas and the other on a line treated with mixed oxidants. Both pipelines had been in use formore than ten years, and both were originally on chlorine gas. The conversion to mixed oxidants
on the second line had taken place about one year prior to the line break. The chlorine treated

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line was only 200 feet from the disinfection station, yet it was coated with a brown slimy
substance. In contrast, the mixed-oxidant treated line was half a mile from the disinfection
station, but the line was completely clean.
Visual removal of biomaterial has been
evident at a number of other municipal sites.
A pilot study of a CBI Claricone™ unit and a
MIOX SAL-80 system was conducted at a
midwestern water system. The MIOX solution
was applied in concentrations sufficient tomeet the chlorine demand of the raw water.
Resulting TTHMs were only 3 ppb and algae
growth in the clarifier was visibly impacted.
Sloughing occurred on tube settlers with 8hours, and within a week, solids on the tube
settlers turned brown and dropped off.8
The Village of Hazlet in Saskatchewan
converted from sodium hypochlorite bleachto mixed oxidants at their well site. Within
one month of operation, the black film on thecistern ladder and walls was virtually gone.9

Biofilm contamination on chlorine-treatedline (left) versus mixed-oxidant line (right)
at Diana Water Supply

Thibodaux, Louisiana converted from chlorine gas to mixed oxidants. With the use of chlorine
gas, they had to manually scrub their filter walls to remove a black deposit from iron andmanganese. 10 Even with vigorous effort, the deposits were very difficult to remove. After
conversion to mixed oxidants, the black film would rinse off with a hose with no scrubbing
required. It is likely that the black deposits were held in place by a biofilm substrate that wassubsequently destroyed with mixed oxidants, expediting the removal of the iron and manganesedeposition.
The Village of Swain’s Lake, New Hampshire utilized mixed oxidants for a pilot study during thepeak of a severe algae bloom.11 With use of chlorine gas, they had to backwash their filters
every 6 hours on average. With mixed oxidants, the filter backwash interval was extended to37.6, showing dramatic control of algae contamination.
Greenfield Municipal Utilities in Iowa monitored total trihalomethanes (TTHMs), by-products ofchlorine disinfection applied to water containing organic material.12 TTHMs in the distribution
system are typically higher than at the plant, due to organic material (biofilm) in the lines thatcontinues to react with chlorine in the water stream. While Greenfield’s TTHM levels both at theplant and in the distribution system were lowered with mixed-oxidant treatment, the results weremore drastic in the distribution system. At the beginning of the monitoring period in late July,
TTHMs were 36% higher in distribution than at the plant. At the end of the 3-month study,
TTHMs were only 14% higher in distribution than at the plant, indicating removal of organic
material from the pipelines.

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TOTAL TRIHALOMETHANES - GREENFIELD MUNICIPALUTILITIES
1009010/29/96 - MIOX disinfection inplace 3 months. TTHMs indistribution only 14% higherthan at plant.
Distribution

807060504030Plant

7/30/96 - 1st MIOX sample 7/30/96(1 week after start-up). TTHMs in
distribution 36% higher than at plant.

206/24/1996

7/14/1996

8/3/1996

8/23/1996

9/12/1996

10/2/1996

10/22/1996 11/11/1996

Conclusion
One of the common concerns of municipal customers removing biogrowth is the possibility ofexcessive flushing that may cause customer complaints or difficulties. Although this is apossibility, it is important to note that very few customers experience this dilemma. Shouldexcessive sloughing occur, the recommended remedy is to flush the water lines for the first
several weeks of operation. The distribution system typically stabilizes after a few weeks, andafter the biofilm is removed, water quality will improve, disinfection by-products will be lowered,and required dose rates will be reduced.
Although few peer-reviewed publications have been produced on removal of biofilms using
MIOX mixed oxidants, the anecdotal evidence is compelling. Biofilm experts in the industry,including Montana State University, continue to investigate the MIOX technology. Future studies
are planned with these organizations as the importance of removing biofilm becomes anincreasing priority in municipal, commercial, and industrial applications.

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1Montana State University, The Center for Biofilm Engineering web page. “What is biofilm?”
http://www.erc.montana.edu/CBEssentials-SW/bf-basics-99/bbasics-01.htm

2Crayton, Cyndi, et al. Montana Water Resources Center, Montana State University. Final Report on theValidation of Mixed Oxidants for the Disinfection and Removal of Biofilms from Distribution Systems.
October 1997.

3

NSP Co., Ltd. Okinawa, Japan. Videotaping, photography, and customer interviews.

4

Phipps, Don Jr. and Grisel Rodriguez, Water Resources and Technology Department, Orange County
Water District, California. Comparison of the Efficiency of Bacterial Removal and Reduction of Bacterial
Viability by Mixed-Oxidant Solution (MOS) and Chlorine (as NaOCl) on a Reverse Osmosis Membrane.
June 2001.

5Petersen, P. and W.L. Bradford, 2000, “Cooling Water: Potential Benefits from Mixed-Oxidant
Application in Cooling Tower Maintenance”, UltraPure Water, 17(5):53-61, May/June, 2000.

6Interview with Charles Smith, Quality Water Systems. April 2004.

7Lewis, J.A., et al. Waterman Environmental Services. An Evaluation of the Efficacy of the Mixed-Oxidant
Solutions Produced from “Activated Water” in Cooling Tower Biological Control. December 24, 2001.

8Gould, Brad, Lawrence A. Lipe & Associates. Midwestern Water System Claricone and MIOX Pilot
Study Results. Fall 2000.

9Interview with Larry Sletten, Operator for the Village of Hazlet, Saskatchewan, Canada. April 20, 2000.

10Conversation with Joe Van Marcke, former operator of the MIOX equipment. May 2002.

11Skip Wolfe, Kinetico Incorporated. Pressure Filtration System Pilot Plant Study Final Report. Swain’s
Lake Village Water District, Barrington, NH. October 31, 1995.

12Duben, Mark. Howard R. Green Company Consulting Engineers. Pilot Study Final Report: MixedOxidant Disinfection System at Greenfield, IA. December 5, 1996.

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