Biofilm Effect and Growth of Waste Frying Oil (WFO) on Bacteria

Chemicals

KCl, KH2PO4 was purchased from Carlo-Erba. NaCl, Na2HPO4, crystal violet were purchased from Merck. Yeast and peptone were purchased from Mast Diagnostics. Acetic acid was purchased from Acros. Ethanol was purchased from Riedel de Haen. All chemicals used were of analytical grade.

Bacterial strains

Bacterial strain Pseudomonas aeruginosa (ATCC 27853) was used in this study. Cells were maintained on agar plates at 4 ºC with transfers at monthly intervals.

Waste frying oil

Waste frying oil (WFO) was obtained and collected from the food Restaurant Malatya, Turkey. Waste frying oil was filtered for removing crude impurities and then, WFO autoclaved, and then used.

Growth conditions

Pseudomonas aeruginosa was firstly cultured in Luria- Bertani (LB) broth medium (g l-1); peptone (10), NaCl (10), and yeast extract (5). The final pH values of broth media was adjusted to 7.0. The same amounts of cells were grown at 37°C, 0 rpm on incubator for overnight (O/N). 100 μl of overnight cultures (OD600 nm ∼ 0,2-0,3) grown tube filled with 5 ml in 10 ml tubes was inoculated, and incubated for 24 h of time. Phosphate-buffered saline (PBS buffer) (gl-1; 8,0 NaCl,0,2 KCl, 1,44 Na2HPO4, 0,24 KH2PO4 and pH 7,4) and PBS+10% waste cooking oil (WFO), and cells were cultured at 37°C for 24 h; 0, 100 rpm or 200 rpm shaker.

Biofilm formation

After the incubation, the supernatant was removed. Biofilm tubes were washed four times with 1 x phosphate buffered saline (PBS) to eliminate any remaining cells. Cells attached to the tubes were then fixed with ethanol (%99) for 15 min room temperature and stained with 1% crystal violet. After staining, excess crystal violet was eliminated with water, and 33% acetic acid was used to dissolve the remaining dye. Biofilm mass was finally determined as a function of the concentration of this dye based on the absorbance at 570 nm [10, 11, 12, 13, 14].

Biofilm formation was standardized to growth with the biofilm index (BFI), which was calculated. The extent of biofilm formation was determined by applying this formula: “BFI = (AB - CW) ⁄ G in which BFI is the “Biofilm Formation Index was defined as the average percentage of bacteria grown as biofilms [15]”, AB is the OD570 nm of stained attached bacteria and CW is the OD570 nm of stained control tubes containing only bacteria-free medium, G is the OD600 nm of cells growth in suspended culture” [6, 7]. OD600 and OD570 were measured using a spectrophotometer directly from tubes. OD600 and OD570 versus time were plotted to obtain growth curves and biofilm formation, respectively. Each value is the average of three independent experiments.

Biofilm

The addition of WFO in all experimental conditions prevented biofilm formation and caused declines. This decline was seen in 14-fold to 100 rpm air agitation condition, with a minimum reduction of 2.7 folds to 200 rpm in air agitation condition. When we look at the difference; cell counts under non-shake conditions and 100 rpm air agitation condition, a 1.4-fold increase was observed at only 200 rpm air agitation condition. At 100 rpm shaking conditions a significant decline is evident (Figure 1).

BFI

An increase is observed at 200 rpm air agitation condition while a decrease is observed at 100 rpm air agitation condition at 0 rpm non-shake conditions. The

smallest value is 0.84, while the highest value is 0.98. The difference was only 1.2 times. This corresponds to only 16% difference (Figure 2).

cfu

When the PBS medium was not taken into account, the increase in cell count in the WFO-supplemented medium occurred at a maximum of 200 rpm in air agitation condition (4.90E + 07) while the lowest increase (1.29E + 07) in the non-shake environment occurred at 0 rpm. As

can be seen, as the ventilation conditions increase, the number of cells increases. When the 24th and the final time were compared, the highest difference was observed in 19 times and 100 rpm shaking conditions, while the lowest difference was observed in the non-shake conditions of 2.7 times and 0 rpm (Figure 3).

1,00E+08 Microbial counts [log (cfu ml-1)]

1,00E+07

1,00E+06

1,00E+05 PBS 0 rpm PBS +WFO 0

rpm When we look at the difference; a 6-fold reduction in cell count was achieved in non-shake conditions, a 3-fold increase at 100 rpm shaking conditions, and a 7-fold increase at 200 rpm shaking conditions. The biggest headlights were at 5735% at 100 rpm. This was followed by 754% and 200 rpm, respectively (Figure 4).

OD600

However, when we look at the OD600 values we call biomass; again the greatest increase occurred at 100 rpm air agitation (0.025) while the lowest rate and even decreased at 100 rpm air agitation condition. As a

different point of view; the highest rate was observed in the shake conditions at 1.4 times and 200 rpm, while the lowest rate was observed in the non-shake conditions at 0.7 times and 100 rpm (Figure 5). The reason for this is not fully understood.

PBS 200 rpm

PBS+WFO 100

PBS+WFO 200

rpm rpm Figure 5: Percentage of the difference between cell numbers at initial and after 24 hours.

When we look at the differences biomass (OD600); with is the biggest rate at 142% at 200 rpm and this was followed by non-shake conditions at 0 rpm with 117%, respectively (Figure 6).

The reason for the decrease in the OD600 value is not fully understood. The addition of WFO in all experimental conditions has prevented the formation of biofilm and blocking 2- fold more. Conversely, when WFO causes cell numbers to increase, no significant change in biomass has been observed. Under no circumstances OD600 = 0.05 was achieved.

The greatest increase in cell numbers (cfu/ml) occurred at 5735% at 100 rpm, while the greatest decrease (-76%) occurred in the same medium (OD600) in the same medium.