Besides improving the performance of microbiological systems the non-active biocatalysts like OrTec are used to control undesired biofilms. Therefore, a study was perform by Dong Kyu Kim and supervised by Dr. Bruce E. Ritmann to investigate how a non-active biocatalyst affects the accumulation and detachment of biofilm and the biodegradation of organic substrate.
Experimental Design
We performed the experiments with an acetate medium (3 mg/L) including inorganic nutrients (Table 1). We set up four porous-medium reactors similar to Stratton, Namkung, and Rittmann (1983). The reactors were 2.5-cm-diameter glass tubes 15 cm in length. Glass beads, 3 mm in diameter, were used as the biofilm supports. Sampling ports were located along the length of the reactor at 1-cm intervals. The feed solutions flowed from glass reservoirs through transparent vinyl plastic tubing and into the column. Table 2 provides the quantitative specifications for each biofilm reactor. The flow rate of the feed solution was 3 L/day, and the COD of the feed solution was 3.25 mgCOD /L.
Table 1
Inorganic nutrients in the feed solution
Nutrient Concentration-mg/L
Monobasic potassium phosphate 8.5
Dibasic potassium phosphate 33.4
Dibasic sodium phosphate 17.7
Ammonium chloride 1.7
Magnesium sulfate 11
Calcium chloride 27.5
Ferric chloride 0.15
Sodium bicarbonate 1.0
Note: The acetate concentration was 3 mg/L, which equals 3.25 mg/L as COD.
Table 2
Specifications of the biofilm reactors
Specification Value
Diameter (d) 2.5 cm
Height (h) 15 cm
Volume of reactor (V) 73.3 cm3
Porosity (ε) 0.40
Pore Volume (εV) 29.5 cm3
Flow rate (Q) 125 cm3/h
Hydraulic detention time (HDT = εV/Q) 14.1 min = 0.236 h
Superficial fluid velocity (4Q/d2π) 0.42 cm/min = 0.252 m/h
Specific surface area (a) 12.10 cm-1
Reynolds number (Re) 0.21
We initiated growth of the biofilm by seeding with diluted settled domestic sewage. Once biofilm formed, we began continuous feeding at a flow rate of 3 L/day. Once acetate removal reached steady state in all reactors, three reactors were dosed continuously with a non-active biocatalyst at
0.1, 0.5, or 1 ppm to investigate the effect of the non-active biocatalyst on the removal and the accumulation of biofilm, as well as acetate utilization. One reactor was a control and received no non-active biocatalyst.
The experiment took place over a 5-month period. We used approximately 1 month to set up the aerobic systems, grow biofilm in the porous reactor until reaching steady state, and make sure that all the analytical methods were reliable. The experiments with the three different mentioned concentrations (and a control) lasted about three months.
We also conducted a follow-up experiment during the last month in which the non-active biocatalyst at 0.1 ppm was added to the influent from the initiation of the biofilm.
We took samples from effluent every day and analyzed them for biomass and acetate concentrations. In addition, we removed glass beads periodically and stripped them of biofilm. Then, the removed biomass was assayed as filterable COD.
Analytical Techniques
1. To assay for biomass attached as a biofilm, we removed several glass beads from the column and sheared the biomass off the surface of beads using a sonicator or vortex mixer. We then measured the filterable COD of the sheared off biomass with a HACH COD digester and vials.
2. We measured the suspended biomass in the reactor effluent using a four-step procedure. The first step was to collect a volume of liquid in a vial and measure the mass of the vial. The second step was to filter the liquid to remove biomass onto the filter. The third step was to measure the mass of the empty vial. The difference in mass of the vial before and after the filtering gave the volume of liquid I filtered (assuming a density of water of 1 g/mL). The fourth step was to dry and weigh the filter. Prior to filtering, the clean filters also were dried and weighed. The mass difference between the filters before and after the filtering gave the mass of biomass in the liquid sample. Dividing the biomass by the volume gave the suspended solids in mg/L. The suspended solids were then converted to mgCOD/L by multiplying by 1.4 mgCOD/mgcells (Rittmann and McCarty, 2001).
3. We assayed acetate by the enzymatic technique of King (1991). The method couples the synthesis of acetyl CoA to the formation of AMP when acetate reaction with ATP and CoA.
acetyl CoA synthase
acetate + ATP + CoA ———————-> acetyl CoA + AMP + PPi
AMP, which is produced in proportion to the amount of acetate to react, is determined by HPLC. The detection limit for acetate is 100 nM, with a precision of 5%.
We added to 1 mL of effluent aliquots (20µL each) of (1) bovine serum albumin (BSA, 200 µg/mL), (2) disodium adenosine triphosphate (ATP) (10 mM), (3) coenzyme A (sodium salt, from yeast, 10mM), and (4) acetyl coenzyme A synthase (20 U/mL, 5mg protein) in a screw-cap 10-mL vials. We mixed the samples thoroughly and incubated them at 37’°C for 1 hr before termination the reaction by immersing the vials in a boiling water bath for 2 min. Once cool, all samples were measured by HPLC.
Results When Adding non-active biocatalyst to an Well-Established Biofilm
Figures 1, 2, and 3 summarize the results for effluent acetate (as COD), effluent suspended biomass, and biofilm biomass. By approximately one month of operation without non-active biocatalyst in any reactor influent, all three parameters had stabilized at the same values: effluent acetate = 0.2 mgCOD/L, effluent biomass = 8 mgCOD/L, and biofilm mass = 20 mgCOD/L/bead. These results document that the operating conditions of the biofilm reactors were excellent for growing and accumulating biofilm, which biodegraded most of the acetate when the non-active biocatalyst was absent.
On July 28, we added the no-active biocatalyst to the influent of three of the four reactors: Reactor A – 0.5 ppm, Reactor B – 1 ppm, Reactor C – 0.1 ppm. Reactor D was the control and didn’t receive non-active biocatalyst. All reactors continued to receive 3.25 mgCOD/L of acetate.
Figure 1 – Effluent acetate concentration before and after adding non-active biocatalyst on 7/28
Figure 2 – Effluent Suspended Solids before and after adding non-active biocatalyst on 7/28
Figure 3 – Biofilm mass before and after adding non-active biocatalyst on 7/28
Addition of non-active biocatalyst at 0.5 or 1 ppm dramatically and immediately affected biofilm accumulation, and the impacts increased for about two weeks. Biofilm accumulation on the glass beads decreased from 20-25 mgCOD/L/bead to about 10 mgCOD/L/bead (Fig. 3), a more than 50% decrease. At the same time, the suspended biomass increased from about 8 to about 40 mgCOD/L (Fig. 2) and acetate removal decreased sharply (Fig. 1): effluent acetate was 1.75 mgCOD/L, or less than 50% removal with 0.5- or 1-ppm of non-active biocatalyst. Thus, non-active biocatalyst applied at 0.5-ppm or more caused a very large loss of biofilm, and this results in a commensurate decrease in the biodegradation of acetate.
The trends for 0.1-ppm non-active biocatalyst were similar, but smaller: effluent biomass increased to around 15 mgCOD/L, effluent acetate increased to about 0.35 mgCOD/L, and biofilm mass was about 1 mgCOD/L/bead lower than the control.
8. Conclusions and Interpretations
The following conclusions and interpretations can be drawn from the laboratory experiments with fixed-bed biofilm reactors to which different concentrations of OrTec biocatalyst were applied.
• After OrTec was applied to a well-established biofilm, effluent suspended biomass and biofilm changed dramatically and in proportion to the OrTec concentration. Effluent suspended solids increased and biofilm mass declined due to increased detachment caused by OrTec.
• Due to the loss of biofilm, the effluent acetate concentration also rose when OrTec was applied. Thus, the OrTec reduced the amount of active biomass in the biofilm and may also have inhibited its ability to utilize acetate.
• The impact of OrTec was greatest for influent concentrations of 0.5 and 1 ppm, which gave similar long-term results.
• A 0.1-ppm influent concentration showed the same trends as 0.5- and 1-ppm concentrations, but the effects were smaller.