1. Up-flow Anaerobic Sludge Beds and
Chemical Oxygen Demand Analysis
Montana Weitzel
Lehigh in Ireland, Summer 2015
Biology Practicum
Microbiology lab of Dr. Gavin Collins
Graduate Assistant: Robert Dillon

2. Weitzel 2
Up-flow Anaerobic Sludge Beds and Chemical Oxygen Demand Analysis
Background:
Up-flow Anaerobic Sludge Beds, more commonly known as UASB reactors, are a form of
anaerobic digesters that utilize waste-conversion biofilms to treat toxic wastewater. As shown in
Figure 1, the influent wastewater is processed as it moves up through the reactor and is collected
as the treated effluent. The main component of such reactors is the sludge, which is comprised of
thousands of granules (see Figure 1). These granules are biofilms, in which microorganisms
adhere to one another when exposed to, or submerged in, aqueous solutions. The respective
microorganisms are joined together by the self-produced extracellular polymeric substance
(EPS). This matrix of polysaccharides and proteins is the major component in wastewater
biofilms.
Figure 1: Standard Up-flow Anaerobic Reactor
Diagram of an Up-flow Anaerobic Sludge Bed with each component.
The arrows represent the flow of effluent wastewater as it progresses through the system.

3. Weitzel 3
Microorganisms of the biofilm perform anaerobic digestion, in which compounds are
broken down in the absence of oxygen and produce a biogas of primarily methane and carbon
dioxide. As a result, the major by-product of UASB reactors is methane gas that can ultimately
be harvested as a source of energy.
To determine the quality of the treated wastewater, chemical oxygen demand (COD)
levels are taken. COD analysis measures the reduction of potassium dichromate under highly
acidic conditions and then compares the reaction to known standards of oxygen demand. The
biodegradation of oxygen demanding compounds correlates with aerobic respiration and the
resulting consumption of oxygen; COD levels are directly related to the amount of organic
compounds in water (LaPara, Allman, and Pope, 1999).
On average, UASB reactors reduce COD levels by 60-80% (Schmidt, 2000). For
wastewater to be released into an environment, the permissible levels of COD found in the
effluent range from 200-1,000 mg/L, or 0.2-1.0 COD/L. Influent wastewater has an average of 5
times the acceptable COD level range of about 5,000 mg/L. The success of UASB reactors is
determined by their COD percentage removal. The aims of this project are to assess the quality
of the effluent and investigate the microbial interactions of the biofilm.
Methods:
Three USAB reactors, R1, R2, and R3, were established with 16 portals to withdraw
samples from. The ports were positioned equally throughout the reactor to ensure an accurate
representation of wastewater undergoing treatment. Each reactor had a volume of 5.1 L with a
hydraulic retention time (HRT) of 20.6 hours. The reactors were fed with mock chemical waste
composed of volatile fatty acids (VFAs), powdered compounds, a sodium bicarbonate buffer and

4. Weitzel 4
a phosphate buffer. Sodium hydroxide was used as a strong base to pH the solution to that of
neutral. The VFAs used were acetate, propionic acid, n-butyric acid, and ethanol in a 1:1:1:1
ratio. The accompanying powders were yeast, peptone, cellulose, and lactose in a 1:1:1:1 ratio.
The ratio of VFAs to powders was 4:1; for every 1 g of VFAs in the feed, there was ¼ g of
powders. The influent feed had a concentration of 5,000 mg/L of COD, 5X the amount of COD
permitted in treated wastewater.
Samples were taken from the reactors three times a week, and the amount of biogas
produced by each respective reactor was measured once a week. Originally, 16 portals had been
established to withdraw samples from, but it became apparent that the cultures from ports 9-16
were mostly sludge and no COD analysis could be completed, as shown in Figure 2.
Figure 2: Standard Up-flow Anaerobic Reactor with Portals
A diagram of an Up-flow Anaerobic Sludge Bed.
Each portal is labeled numerically and is equidistant from one another.

5. Weitzel 5
Analysis of both “soluble” COD levels and “insoluble” COD levels were taken from each
culture to determine the COD differentiate between the COD levels caused by microorganisms
and organic compounds. To compare COD levels of the respective solubility, an analysis was run
of the original sample and then after the sample had been centrifuged. For each portal, two
identical samples were prepared to ensure quality data was collected; two soluble and two
insoluble.
The COD analysis method was the Closed Reflux, Colorimetric Method. This technique
uses a low range digestion solution and a sulfuric acid reagent (LaPara, Allman, and Pope,
1999). A 1/20 dilution of the influent with dH2O and a 1/10 dilution of the effluent with dH2O
cultures were performed in standard COD tubes. The total volume of each sample was 2.5 mL,
and two blanks of only dH2O were prepared as a control. Once the dilutions had been completed,
1.5 mL of the digestion solution was added using a hand pipette. 3.5 mL of the sulfuric acid
reagent was added by running the solution down the inside of the tube to create an acid layer
underneath the sample-digestion solution layer. The tubes were then inverted and placed in the
block digester at 150 °C for two hours.
The Closed Reflux, Colorimetric Method (COD) measures dichromate reduction by
absorption at the selected wavelength of 420 nm. The reagent water was used as a reference
solution, and all samples, blanks, and standards were measured against this solution (LaPara,
Allman, and Pope, 1999).
Results:
COD analysis data has been collected from the start of 2015. The ultimate goal of this
analysis is to determine the COD removal percentage and therefor the efficiency and success of

6. Weitzel 6
such USAB reactors. Because this project is ongoing, averages across the three reactors have yet
to be taken, a final calibration curve has yet to be established, and final calculations for
percentage removal have not been performed. The removal percentage calculations of COD are
as follows:
% REMOVAL = COD EFFLUENT÷ COD INFLUENT X 100
COD as MG O2/L = mg O2 IN FINAL VOLUME ÷ mL SAMPLE X 100
Table 1: Sample of Absorbance Readings
Absorbance readings of COD samples collected on 1/16/2015.
R_# is the respective reactor. P_# represents the corresponding portal number.
As shown above, a sample was not collected from R1P1.

7. Weitzel 7
The resulting absorbance readings from each respective portal are compared to one another.
Ideally, the difference between the two readings would be <0.005 (see Table 2).
Table 2: Sample of Absorbance Differences
A section of the data collected on 1/16/2015.
The difference between the R3P1 spectrometer readings is 0.001.
The smaller difference between sample values validates the accuracy of the analysis.
Over the course of the project, the samples and data obtained is unique to the specific collection
date. The resulting number of samples is not consistent and varies from three portal samples to
sixteen portal samples (see Table 1). As the experiment progressed, the number of cultures taken
fluctuated depending on the current status of the reactor, as leaks, tears, and breaks are common
occurrences and often times prevent a sample from one or many portals.
Discussion:
Due to the ongoing nature of this project, significant conclusions cannot be drawn
regarding the ultimate success of wastewater treatment by the three reactors. From the data
collected thus far, it is apparent that the reactors are capable of reducing COD levels. In addition
to COD analysis, other factors have been taken into consideration in hopes of understanding the

8. Weitzel 8
microbial processes of the biofilm. Currently, the size, density, and microorganism composition
of the biofilm granules at all depths of the reactor are undergoing assessment.
Until recently, it has been widely accepted that the microorganisms present in the reactors
are present throughout the entire system (Hulshoff, et. al, 1983). However, the research
conducted on this project suggests that the microbial composition differs depending on the stage
of the biofilm within the reactor. This breakthrough encourages eco-engineering to focus on the
assembly and activity of anaerobic waste conversion biofilms to optimize the effectiveness and
efficiency of wastewater treatment.
The demand for UASB reactors has significantly increased as an environmentally safe,
cost efficient, and consistent form of wastewater treatment. The research of this experiment will
continue to provide insight to the inner workings of biofilms and will further the advancement of
UASB reactors as the wastewater treatment system of the future.

9. Weitzel 9
Bibliography
Fang, H., & Chui, H. (1993). Maximum COD Loading Capacity in UASB Reactors at 37°C.
Journal of Environmental Engineering, 1991: 103-119.
Hulshoff, L. W., W. J. De Zeeuw, C. T. M. Velzeboer, and G. Lettinga. "Granulation in UASB-
Reactors." Water Science and Technology , 1983: 15.8-9: 291-304. Web.
LaPara, T., Allman, J., & Pope, P. (1999, September 21). Miniaturized closed reflux,
colorimetric method for the determination of chemical demand. Waste Management, 295-
298.
Schmidt, Jens E., "Granular Sludge Formation in Upflow Anaerobic Sludge Blanket (UASB)
Reactors." Biotechnology and Bioengineering 26 Mar. 2000: 229-46. Web.
Souza, M. (1986). Criteria for the Utilization, Design and Operation of UASB Reactors. Water
Science and Technology, 18(12), 55-69. Retrieved July 1, 2015.