Biofilm experiments

There are currently no recommendations or guidelines concerning the standardized cultivation of biofilms methods which give indications of practical efficacy are the most useful. Attempts have been made to unify procedures for determining the efficacy of biocides however, the findings of the available literature are difficult or impossible to compare. In principle, this can be described under several headings with respect to the duration of the investigations  

Herruzo-Cabrera, 2000 Bredholt et al., 2001 Spoering and Lewis, 2001). The obvious advantage is that the organisms are well defined the obvious disadvantage is their lack of representativity to environmental conditions. Defined mixed cultures are rarely employed (Alasri et al., 1992, Fatemi and Frank, 1999). The advantage of biocide studies with monocultures or defined mixed cultures lies in the better reproducibility of the experiment. In this way, variations in the experimental parameters can be chosen or kept constant. 

Only in few publications is the action of biocides described for aqueous systems with undefined biofilms with mixed composition (Exner et al., 1987 Mathieu et al., 1990 Goroncy-Bermes and Gerresheim, 1996 Morin, 2000 Holtmann and Sell, 2001 Walker et al., 2001). The advantage of efficacy testing on natural biofilms lies in the greater relevance to the practical situation. 

Vastly different methods are available for cultivation of biofilms. Basically, these can be described as either batch-mode or continuous mode methods. In batch studies, growth substrata in the form of coupons or glass slides are placed in e.g. Petri-dishes or other holders filled with medium. Under the action of undefined shear forces, investigations in so-called beaker reactors are conducted. A further test system which operates in batch mode and continues to become prominent is the miniaturized test system comprising microtitre plates, in which 96 wells enable the simulation of various experimental conditions simultaneously under static conditions (Geneveaux et al., 1996 O Toole et al., 1999). In general, biocide tests in batch systems are simpler to run, have shorter durations, and are very simple to carry out. Therefore, they are well-suited for initial comparisons or screenings of different biocides, or different concentrations and contact times of a particular biocide. 

After defined exposure times, the test substrata can be removed from the system and treated with biocide, or they remain in the system and are analysed at the end of the experiment to determine biocide efficacy. Other, new variations of the CSTR can be suitable as test systems. These include the Calgary Biofilm Device, which utilises shaken microtitre plates (Ceri et al., 1999 Ceri et al., 2001), the flow-cell (Stoodley et al., 2001), the artificial biofilm system (Harkonen et al., (1999), the colony biofihn system (Anderl et al., 2000) and the drip flow reactor (Xu et al., 1998). 

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Theis and Leder (1993) demonstrated in aerobic biofilm experiments with an oil field isolate of aerobic bacteria, containing predominantly Pseudomonas species, that OPA is more effective than formaldehyde (FA, 2.1a.) or glutaraldehyde (GA, 2.5.) in killing or inhibiting the growth of sessile microorganisms (see Table 16). OPA is effective without activation and is able to inactivate GA-restistant strains of Mycobacterium chelonae. On the other hand OPA unlike GA acts not sporicidal at its in-use concentration of 0.5% (w/v) and normal pH (6.5.). 

Table 6 Performance of equivalent bromine antimicrobial treatments against biofilms in well-controlled laboratory experiments to simulate industrial applications % Removal % Decrease in Fluid Biofilm Disinfection Oxidizing of Biomass Frictional Resistance (Log Reduction in...

The new stabilized bromine antimicrobial is an excellent antimicrobial having been proven superior in field and laboratory experiments compared to chlorine, stabilized chlorine, and equal to or better than solid hypobromite antimicrobials. The product is effective for the control of microbial biofilms and highly diverse microbial communities, including those that harbor Legionella. 

Investigations have been performed to exemplify the DO surface removal rates from biofilms grown on different types of wastewater (Bjerre et al., 1998b). Such investigations may indicate if Equation (5.3) can be considered an appropriate description of the aerobic activity. The wastewater for these studies originates from an open sewer system, the Emscher river, Germany. The results of the experiments are outlined in Table 5.2, and further details are shown in Figures 5.6 and 5.7. 

TABLE 5.2. DO Surface Removal Rates for Sewer Biofilms. The Biofilms were Grown by Continuous Supply of Wastewater from the Sewer, and the Experiments were Performed under Organic Substrate Nonlimiting Conditions (Bjerre etal., 1998b). 

The expressions shown in Table 6.1 all include constants that have been found good approximate values based on experiments. These values may of course be adjusted to account for specific cases. As an example, different flow conditions in continuously and intermittently pumped mains may affect the transfer of substances and products across the biofilm-water interface, and, thereby, the production of sulfide (Melbourne and Metropolitan Board of Works, 1989). 

The example of a biofilm reactor setup shown in Figure 7.1 demonstrates how an experiment can be performed under controlled conditions (Raunkjaer et al., 1997). The objective of the study is to determine substrate (acetate) and DO surface removal rates of biofilms that were grown on wastewater. Careful control is needed to do so during conditions where both the substrate and the DO should be studied as limiting factors for the removal rates. A great number of specific details that will not be dealt with here were considered for this experiment. 

A simplification of the aerobic heterotrophic in-sewer microbial processes is depicted in Table 5.3. By omitting the reaeration and the growth of the biofilm biomass in this description, the remaining processes proceed interactively in the water phase under the conditions established in the OUR experiments. Furthermore, the processes take place at a constant temperature and at DO nonlimiting growth conditions affecting the formulation of the relevant rate expressions (see Table 7.1). 

In addition to sampling in a sewer followed by analysis of specific components or use of a sample for further laboratory or pilot-scale experiments, a number of direct or indirect measurements must typically be performed in the sewer itself. Important measurements related to sewer process studies are DO, reaeration, biofilm characteristics and odor. 

Measurement of biofilm activity can be performed based on laboratory reactor experiments or with a technique combining biofilm growth taking place in a sewer followed by measurements in laboratory scale (Raunkjaer et al., 1997 Bjerre et al., 1998). Huisman et al. (1999) developed a sewer in situ biofilm respiration chamber. It includes a DO sensor and a chamber that can be pressed onto the sewer wall. It is designed to achieve an even and unidirectional flow distribution over the entire measurement area. Pure oxygen is injected for oxygenation. 

The production of Ss depends on the duration of the anaerobic period, whereby an average production rate can be estimated. The Ss produced is basically a net production however, the consumption of substrate for the production of biomass is considered relatively low under anaerobic conditions in this experiment. It has been confirmed that under the conditions performed, no biofilm is generated and no sulfide production takes place (cf. Section 6.3 and Figure 6.9). 

Zwiener C., S. Seeger, T. Glauner, and F.H. Frimmel (2002). Metabolites of the biodegradation of pharmaceutical residues of ibuprofen in biofilm reactors and batch experiments. Analytical and Bioanalytical Chemistry 372 569-575. 

Aside from adding defined compounds, experimental additions of natural DOM mixtures suspected to vary in lability have helped test ideas about the contribution of various DOM sources to aquatic ecosystems. In a nice example using manipulation of natural DOM sources, Battin et al. (1999) used flowthrough microcosms to measure the relative uptake rates of allochthonous and autochthonous DOM by stream sediments. They documented greater than fivefold differences or more in uptake and respiration, depending on whether the DOM was extracted from soil or periphyton. Moreover, they were able to show, via transplant experiments, several cases where prior exposure to a particular source of DOM increased the ability of that community to metabolize the DOM supplied. There appears to be some preadaptation of microbial catabolic capacity when these stream biofilms were re-exposed to a familiar type of DOM. Similarly, the response of heterotrophic bacteria to carbon or nutrient addition was greatest when the source community was particularly active (Foreman et al., 1998). Kaplan et al. (1996) showed that fixed film bioreactors, colonized on one water source, were unable to rapidly metabolize DOC in water from another source. 

Corn steep liquor (CSL), a byproduct of the com wet-milling process, was used in an immobilized cell continuous biofilm reactor to replace the expensive P2 medium ingredients. The use of CSL resulted in the production of 6.29 g/L of total acetone-butanol-ethanol (ABE) as compared with 6.86 g/L in a control experiment. These studies were performed at a dilution rate of 0.32 hr1. The productivities in the control and CSL experiment were 2.19 and 2.01 g/(Lh), respectively. Although the use of CSL resulted in a 10% decrease in productivity, it is viewed that its application would be economical compared to P2 medium. Hence, CSL may be used to replace the P2 medium. It was also demonstrated that inclusion of butyrate into the feed was beneficial to the butanol fermentation. A control experiment produced 4.77 g/L of total ABE, and the experiment with supplemented sodium butyrate produced 5.70 g/L of total ABE. The butanol concentration increased from 3.14 to 4.04 g/L. Inclusion of acetate in the feed medium of the immobilized cell biofilm reactor was not found to be beneficial for the ABE fermentation, as reported for the batch ABE fermentation. 

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