Non-biocidal treatment alternative in the paper machine short loop white water circuit works to prevent buildup and the resulting wet end deposits

Inhibitor Treatment Program Offers Option for Clearing Biofilm Buildup

A biofilm is a complex organic matrix formed by microorganisms growing on surfaces and is composed primarily of microbial cells, extracellular exo-polysaccharides (EPS), also referred to as glycocalyx or more commonly as “slime,” and water (Table 1).¹ The characteristics of discrete biofilms are exceptionally variable with this variability adding significantly to the difficulty of analysis. The organic matrix of a biofilm acts as a habitat for the microorganisms and serves several functions including holding the biofilm together and providing a favorable environment for growth and long-term survival of the resident cells.

This organic matrix also provides a local environment in which the cells are protected from extreme changes in physio-chemical parameters of the water (e.g., pH). Furthermore, the matrix is important in sequestering nutrients from the water column and protecting the cells from drying if the water level decreases.

In a paper system, the composition of biofilms is often more complex because of the presence of fillers, fibers, and organic and inorganic materials (Table 2).^2,3 Because of the nature of the process water in paper production, biofilms and subsequent wet end deposition can form rapidly. Particulate and dissolved organic and inorganic materials aggravate biofilm and deposit-related problems such as breaks, holes, and wash-ups.

FIGURE 1: Summary results from a North American alkaline fine paper mill.

BIOFILM FORMATION IN PAPER SYSTEMS. Biofilm formation is a dynamic process that begins with bacterial cells attaching to, or “colonizing,” a submerged surface and continues until a mature biofilm develops. The process of biofilm formation can be divided into five stages as indicated in Figure 1.

Stage 1: Conditioning Layer. Although a conditioning layer is not always necessary, in many models of wet end deposition, the process is initiated by organic and/or inorganic conditioning occurring on the machine surfaces. A surface immersed in water is quickly covered with organic and inorganic substances through reversible and irreversible adsorption processes. As the complexity of the composition of the water increases, so will that of the “conditioning layer” on a submerged surface. The conditioning layer reflects the composition of the process water.

The composition of the conditioning layer itself can vary temporally and spatially as a result of the adsorption processes. Materials in whitewater (e.g., polysaccharides and hydrocolloids) are especially effective in adsorbing to surfaces and causing changes in the properties of those surfaces. Thus, the conditioning layer will have a significant influence on the subsequent interactions between microorganisms and the boundary of the surface. Generally, the conditioning layer will make submerged surfaces more favorable for colonization by microorganisms.⁴

TABLE 1: Primary characteristics of biofilms
Characteristic	Range
Water Content	High: 70 - 95%
Organic Content	High: 70 - 95% (dry weight)
Bacterial Content	High: ( 106 cells/gm [wet wt.])
Biological Activity	High
Inorganic Content	Low

Stage 2: Bacterial Attachment (Colonization). For biofilm to develop on a surface, bacterial cells must first colonize that surface. Bacterial adsorption begins through the action of cell transport to the surface by either motility or fluid eddies. Studies have demonstrated that, near the surface, repulsive electrical double layer forces between the cell and the inert surface are opposed by both attractive van der waals forces and chemical bonding (covalent, hydrogen, and ionic bonds). The initial contact of bacterial cells to surfaces is considered reversible (i.e., the cell may remain there or leave).⁵

During the early stage of colonization, bacterial cells are loosely held in place by attractive forces, and the cells can be easily removed from the surface by the effect of slight shear forces, such as that provided by running water. If a bacterial cell remains on the surface, however, the association becomes more permanent as a boundary layer EPS is produced to secure the cell to the surface. In addition, the presence of cell adhesive organelles (i.e., fibriae) may enhance adsorptivity. The composition and structure of an EPS can vary depending on the bacterial species, prevailing nutrient conditions, and other physio-chemical parameters.^1,6

FIGURE 2: Effectiveness of a range of surfactants at preventing biofouling of stainless steel surfaces.

FIGURE 3: Effectiveness of a range of commercially available biodispersants and enzymes at preventing biofouling of stainless steel surfaces.

Stage 3: Biofilm Formation and EPS Production. After cells become firmly associated with a surface, cell growth and division tend to increase, forming microcolonies on the surface. Because paper process streams favor microbial growth, microcolonies can develop within a few hours. Microcolony development is important because this is when the actual formation of the biofilm begins.

As microcolonies form, the cells continue to synthesize EPS which, in turn, forms a thicker matrix around the cell population. The result is a localized habitat that favors continued, if not enhanced, growth of the resident cells. This matrix acts as a boundary to protect the resident cells from adverse environmental conditions (e.g., pH) and chemicals detrimental to cell growth (e.g., microbicides). For all practical purposes, this is the stage where a deposit on the machine can be detected by touch (the biofilm has a characteristic “slippery” feel) although it may not be possible to collect this deposit.

Stage 4: Biofilm Maturation. After a biofilm becomes established on a submerged surface, the cells in the biofilm continue to grow, producing EPS and developing the biofilm matrix, as long as favorable conditions persist. This continued growth leads to a thicker and more readily detectable biofilm. As the amount of EPS produced and the thickness of the biofilm increases, conditions become favorable for particles in the water, such as fillers, fiber, pitch, etc., to become trapped by the biofilm matrix, adding bulk to the deposit.

At this point, the deposition becomes massive enough that it is visible and easily collected. Thus, in paper process streams, biofilm formation is considered to be a two step process. “Primary” biofilm formation is accomplished mainly by motile single-celled bacteria, and “secondary” biofilm formation involves the incorporation of stalked and filamentous bacteria subsequent to the primary biofilm formation.⁷

Stage 5: Detachment. Once a biofilm reaches a certain thickness and age, single cells or large clumps, including the EPS matrix and associated particles, are routinely dislodged via shear forces. The thicker the biofilm, the greater the probability that large clumps will be dislodged. These actions have negative effects on paper production and quality and cause problems, such as breaks, spots, and holes, that lead to downtime for boilouts and wash-ups.

COMMERCIAL BIOFILM CONTROL TECHNOLOGIES. Several treatment strategies have been used to control deposition on paper machines, with varying degrees of success, including both oxidizing and non-oxidizing antimicrobial agents, ‘biodispersants,’ solvents, enzymes, various derivatives of these strategies, and different combinations of these approaches. Deposits at the wet end of a paper machine are typically composed of a varied mixture of microorganisms, organics, and inorganics.

The extent to which any of these deposit control strategies works is to some degree dependant upon the causative or binding portion of the deposit. In our experience, a deposit is considered microbiological in nature if it contains greater than 30% unicellular bacteria, greater than 15% fungi, or greater than 15% fresh water microorganisms. The microorganisms in the resultant biofilm are often responsible for serving as the binder for the deposit and for entrapping fillers, fibers, and naturally occurring inorganics or by simply adding bulk to the deposit. A unique approach to controlling biofilm has been developed that inhibits biofilm formation by preventing the attachment of exo-polysaccaride on the outside of the cell wall. In this capacity they function very differently from that of microbicides, dispersants, enzymes, or solvents.

(a) No Sulfosuccinate Present in Culture   (b) Sulfosuccinate Present in Culture

FIGURE 4: Electron photomicrographs of EPS formation around cells (a) in absence of sulfosuccinate and (b) in presence of sulfosuccinate.

Microbicides. Arguably, control of microbiological deposition has been fought the longest with the aid of antimicrobial agents. Microbicides such as DBNPA (2,2-dibromo-3-nitrilopropionamide), dithiol, oxidizers, quaternary ammonium chloride or “quat,” and isothiazolin are the most common products used for treating the short loop of a paper machine. These chemicals range in their modes of action from affecting cell membranes (e.g., quats) to inhibiting enzymes in various metabolic pathways (e.g., isothiazolin).⁸

Regardless of the mode of action, microbicides are non-selective and affect any susceptible bacterial cells. Thus, they tend to significantly reduce the number of free-floating or “planktonic” microorganisms in a paper process system. However, the effect of a microbicide on biofilm-associated cells may be significantly less, depending on the resident species and the age of the biofilm, because cells in a biofilm matrix are protected from the effects of microbicides.⁹

It has been shown that bacteria from biofilms show different physiological properties in their response to environmental influences compared with bacteria growing planktonically. Furthermore, recent studies have shown that the transport limitation of a microbicide into biofilms can negatively impact antimicrobial performance of both oxidizing and non-oxidizing agents.¹⁰ Even if a system is cleaned (e.g., boilout) before a microbicide program begins, this treatment approach can only slow, not eliminate, biofilm formation.

Due to the inherent nature of microbicides, they are scrutinized by regulatory agencies worldwide. Furthermore, there are hazards associated with microbicides if not handled properly, and if they are overfed, they have the potential to inhibit advantageous microorganisms in the effluent treatment plant. Although these concerns have prompted research into the discovery of environmentally friendly microbicide actives, such as dithiol, as well as the reformulation of proven actives into friendlier stabilization packages, much work has been done on the investigation into microbicide alternatives or “non-biocidal technologies.”¹¹ Three types of non-biocidal technologies are biodispersants, enzymes, and solvents.

Biodispersants. Anionic lignosulfonates, in various formulations and programs, have been utilized as biodispersants for several decades with limited success. Biodispersants are typically non-ionic or anionic surfactants (e.g., ethylene oxide/propylene oxide co-polymers) that stabilize or disperse particles to inhibit attraction and accretion of both microbiological and specific chemical or constituents of wet end deposition without inhibiting cell growth at use concentrations. This mode of action loosens the deposition, which promotes the penetration of microbicides into the deposit and may remove ‘dead’ or aged biofilm thereby preventing a rapid recovery.

Although the name “biodispersant” suggests the activity on a biological entity, laboratory testing has shown that many commercially available biodispersants, in the absence of a microbicide, have little or no ability to prevent or remove an existing biofilm.¹² Indeed, the majority of commercial biodispersant applications include the use of a microbicide. Properly applied biodispersant technology can increase the overall efficiency of a given microbicide program.

Enzymes. Enzymes have been evaluated in both the laboratory and in paper process streams for biofilm control, but are not widely utilized. The accepted theory behind biofilm control with enzymes is that the enzymes degrade extracellular polysaccharides by cleaving a specific bond in the EPS, thus “dissolving” the biofilm. For example, the gluconase enzyme can be used to hydrolyze bonds between glucose molecules that may be present in the EPS.

In biofilms, however, it is important to note that several different types of polysaccharides exist, depending on the species present in the biofilm. Moreover, changes in furnish and different seasons can dramatically change the type of microorganisms and their subsequent biofilm generation making a change of enzymes likely. Therefore, a mixture of specific enzymes is typically needed to effectively control biofilm formation in a paper system. As with dispersants, enzymes interfere with biofilm formation at a late stage of deposit formation.

Solvents. Solvents are proposed to work by enveloping bacteria cells and thereby preventing them from depositing on paper machine surfaces. Their function is random in nature and their mode of action is more of a cleaner or wetting agent. These organic solvents or wax dispersions may act to dissolve certain components of the deposition and in the case of pitch and stickies have been known to re-deposit further in the process, when the solvent concentration is decreased. Laboratory studies have shown that such products do not prevent biofilm formation but to a varying degree can remove existing deposits.

BIOFILM INHIBITORS. Various non-destructive techniques for biofilm measurement—including microscopic, spectrochemical, electrochemical, and piezoelectric analysis methods—have been reviewed in the literature. Of these techniques, biofilm determination based on measurements utilizing flow cells have been the most encouraging.

In continuing the search for effective alternatives to microbicides in the short loop of the paper system, a laboratory method was developed to compare the ability of non-biocidal compounds to prevent biofilm formation. This test protocol incorporates the use of a modified Robbins Device and subsequent epi-flourescent and biochemical testing. The modified Robbins device was used to compare hundreds of commercially and non-commercially available surfactants (Figure 2). A family of molecules discovered to be very efficacious at inhibiting deposit formation was the sulfosuccinates.^13,14 Furthermore, a unique, specially designed sulfosuccinate molecule was found to provide the best control in that family.¹²

Inhibition of biofilm formation by the substances tested was determined by measuring quantities of selected cellular components. Test compounds A - O represent a broad range of surfactants (e.g., EO/PO copolymers). All test compounds were applied at a concentration of 50 ppm of the active ingredient. Percent colonization means colonization relative to control (untreated) samples (i.e., 100% indicated no prevention of the biofilm, 0% indicated absence of biofilm).

Testing was also done to compare sulfosuccinate with commercially available biodispersants and enzymes (Figure 3). Again, compounds were tested at 50 ppm of active ingredient and percent colonization means colonization relative to control sample. Some compounds actually promoted the growth of selected cellular components. This may be caused by the bacteria using the test compound as a food source, thereby promoting their growth. Although the majority of biodispersants and enzymes had little to no effect on the formation of biofilm in this assay, these results should not be interpreted to mean that biodispersants and enzymes do not work in paper process streams; their activity is dependent on several parameters such as dosage, type of deposition, and how much biocide is present. These data only indicate that sulfosuccinates work to prevent biofilm development at an earlier stage than biodispersants and enzymes.

The effectiveness of sulfosuccinate in this assay has prompted further investigation into this compound’s mode of action. Figure 4 shows electron photomicrographs of bacteria (a) in the presence and (b) in the absence of sulfosuccinate. These two samples were treated to visualize any EPS on or near the surface of the cells. The abundant EPS associated with cells grown in the absence of sulfosuccinate is in marked contrast to similarly treated cells grown in the presence of sulfosuccinate.

These observations provide some insight into the possible mode of action of sulfosuccinate. Bacteria lacking EPS:

1. Have less capability to permanently attach to surfaces

    

2. Cannot easily protect themselves from the adverse effects of microbicides

    

3. Do not provide “glue” to hold wet end deposits together.

Thus, in the idealized model of biofilm formation (Figure 1), sulfosuccinate works in Stage 2, actually preventing the bacteria from forming an intact EPS matrix. The cells are then only loosely associated with the surface.

Due to the absence of the concentrated EPS layer around the bacterial cell, sulfosuccinates are described as biofilm inhibitors to distinguish them from microbicides, biodispersants, and enzymes (Table 3).

Figure 5: Photomicrographs of bacterial accumulation from coupon deposition on a European alkaline fine paper machine following a boilout (1000x).
	Control	Treated
Day 2
Day 4
Day 7

FIELD RESULTS. The first generation of biofilm inhibitors have been evaluated in a European alkaline fine paper mill, a Scandinavian board mill, and a European tissue mill, in addition to a North American tissue mill and two North American alkaline fine paper mills. Prior to running any of the field trials, lithium tracer studies were conducted from which the system cycle-up was determined and product application rates were accordingly determined.

The control data from a European trial location, shown in Figure 5, reflect deposition on a machine with a conventional proprietary microbicide (dodecylguanidine hydrochloride and methylene bis (thiocyanate)) and biodispersant in the short loop white water and a conventional proprietary microbicide (bronopol and quaternary ammonium chloride) in the saveall. The control consisted of the paper machine running with a commercial biocide program.

The treated system received a program consisting of the biofilm inhibitor. This conventional program was effective at keeping the machine clean and providing the mill with a desired four-week interval between boilouts. The microbicide and biodispersant feeding the short loop were totally replaced with a biofilm inhibitor program on an equal cost per ton basis.

A side stream monitoring device was used to measure visual appearance of the biofilm and the growth of certain biofilm constituents over a two-week period. Both measurements showed a marked decrease in biofilm growth with the biofilm inhibitor program. Over an 11-month period, testing on this European fine paper machine trial indicated that the biofilm inhibitor program reduced the number of breaks by 40%. This provided the mill with a 103% return on investment (benefits received minus program cost divided by the program cost). In addition to the favorable ROI, the mill totally eliminated microbicides from the short loop of the machine, thus providing a much safer program.

Results indicate that, for the specific paper machine in Figure 6, continuous feed is preferential over semi-continuous. The trial consisted of various production runs with boilouts in between runs. Of interest in this evaluation is the seemingly reproducibility of the results over many production runs and variations to the dosing strategy employed. This is in part due to the consistency of the production cycles for this paper machine. This trial location has requested to be a trial site for the second generation biofilm inhibitor validation testing. In addition, this location will be evaluating an online monitor utilizing infrared spectroscopy techniques.

Similar results to those achieved in Figure 6 were achieved in another North American alkaline fine paper machine evaluation where a short loop oxidizing biocide treatment, hypobromous acid, was replaced with a biofilm inhibitor. The previous microbiological control program on the machine was hypobromous acid fed to the silo and saveall clear water, controlled by oxidation/ reduction potential (ORP) measurements, and glutaraldehyde fed to the broke. The mill wanted to eliminate their hypobromous acid treatment at the silo because it interfered with their dyes when it was increased during times when additional control was necessary.

Furthermore, the biofilm inhibitor program simplified the entire control strategy (i.e., no ORP needed) and reduced safety and handling concerns of hypobromous acid at the mill (e.g., less equipment and pump corrosion). During the trial, the hypobromous acid treatment to the silo was replaced with a continuous treatment of the biofilm inhibitor. Cost restraints, imposed by the mill, precluded achieving theoretical dosage, as determined by pre-screening data. However, at costs equal to the previous program wash-ups, in between boilouts were reduced by 50%.

FIGURE 6: Summary results from a North American alkaline fine paper mill.

CONCLUSIONS. As indicated by the field results herein, biofilm inhibitors are an effective, environmentally friendly alternative treatment for short loops. A conventional microbicide program is still recommended for the incoming furnish to maintain a consistent microbiological population level (i.e., prevent uncontrolled growth) and as a treatment for the mill fresh water.

The mechanism of biofilm inhibitors seems to be different from that of biodispersants and enzymes in that biofilm inhibitors prevent bacterial attachment and biofilm formation by hindering the creation of a concentrated EPS layer around the bacterial cell. Therefore, biofilm inhibitors prevent biofilm formation at an earlier stage than biodispersants and enzymes. Furthermore, in all the technical validation trials that have been run to date, no adverse effects were observed on the sheet properties or operating parameters (e.g., size, strength, first pass retention, color/brightness).

Paramount to the success of non-biocidal technology is the development of noninvasive field monitoring techniques. Of the techniques worked with thus far, DNA measurement of biofilm on stainless steel surfaces in modified Robbins Devices, online bioactivity determination by extraction and analysis of bacterial adenosine triphosphate (ATP), and the use of field adapted infrared spectroscopy appear to be the most promising.

REFERENCES

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12. J.B. Wright, “Significantly Reduced Toxicity Approach to Paper-Machine Deposit Control,” Proceedings of the 1997 TAPPI Engineering & Papermakers Conference, TAPPI PRESS, Atlanta, pp. 1083-1088.
13. J.B. Wright and D.L. Michalopolous, U.S. Patent No. 5,512,186.
14. J.B. Wright and D.L. Michalopolous, U.S. Patent No. 5,593,599

WILLIAM J. BUNNAGE is business development manager-microbiological control and FRED L. SINGLETON is program manager-microbiological control, Hercules Pulp and Paper Div., Jacksonville, Fla. KEVIN CROSS is business manager-microbiological control, Hercules Pulp and Paper Div., Singapore.