CICEET Progress Report for the period 9/1/04 Through 3/15/05

Project Title: Wastewater Treatment to Minimize Nitrogen Delivery from Dairy Farms to Receiving Waters
Principal Investigator(s): Nancy G. Love
Additional Investigator(s): Katharine F. Knowlton, Barth F. Smets
Project Start Date: November, 1 2004 (Delayed due to date of funding arrival).

Figures

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Tables

Table 1

Table 2

Objective 1. Demonstrate the ability of the oxygen-limited autotrophic nitrification-denitrification (OLAND) process to remove nitrogen from anaerobically stabilized dairy wastewater.

Objective 2. Experimentally determine the operating parameters and treatment capacity for an OLAND-based biofilm treatment system.

Objective 3. Characterize the liquid, solid and gaseous effluent from the OLAND process.

Objective 4. Develop practical design equations and performance curves for the OLAND-based biofilm treatment system.

Objective 5. Ensure environmental compatibility by calculating a nitrogen balance for a model concentrated dairy operation located in the NERR region.

This report addresses activities associated with Objectives 1, 2 and 5. Objectives 1 and 2 will be reported simultaneously because the tasks associated with running the OLAND reactor system overlap significantly.

Tasks to meet objectives
Objectives 1 and 2:
Objectives and Tasks. To achieve objective 1, the following subtasks will be completed:
1. Construct two (2) OLAND biofilm reactors.
2. Construct a pilot plant-scale anaerobic storage tank using traditional design parameters so that a crust forms on the stored waste and holds the ammonia in.
3. Demonstrate the ability of the OLAND biofilm reactors to stabilized dairy waste by:
a. Establishing autotrophic nitrogen removal using synthetic wastewater, and
b. Demonstrating autotrophic nitrogen removal using anaerobically-stabilized dairy manure.

To achieve objective 2, the following subtasks will be completed:
1. Operate the constructed reactors while feeding anaerobically stabilized dairy waste and monitor performance.
2. Expose the reactor systems to increasingly higher nitrogen loading conditions and evaluate the performance.

Objective 5
Objectives and Tasks. To achieve objective 5, the following subtasks will be completed:
1. Complete a nitrogen balance using current operating protocols for a dairy farm in the Winyah Bay watershed in South Carolina.
2. Computationally assess the impact of implementing the OLAND technology on nitrogen cycling around the farm by recalculating the nitrogen balance, assuming OLAND is in place.
3. Revise the nitrogen balance calculations at the end of the project, once further information about OLAND performance with anaerobically stabilized dairy waste is completed.

Progress on Tasks
Progress on Objectives 1 and 2.
Significant work has been exerted during the current reporting period to demonstrate the ability of the OLAND process to achieve stable and efficient nitrogen removal using synthetic wastewater. Synthetic wastewater is used to achieve good operating performance of the OLAND process using methods previously established by others, and to ensure the presence of the appropriate anaerobic ammonia oxidizing (anammox) microorganisms. Since these microorganisms grow very slowly, this step is very important to ensure the presence of the correct community. Once we achieve efficient OLAND performance, anaerobically stabilized dairy waste will be applied to the system and the effectiveness of the OLAND system at removing nitrogen will be evaluated. Currently, an anammox culture is growing on synthetic wastewater, and anaerobically-stored (stabilized) solids separated dairy manure is being generated at the Virginia Tech dairy using a pilot -scale anaerobic storage unit.

A submerged fixed bed biofilm reactor (FBBR) was constructed by Mr. Paul Sweetman, graduate research assistant, as a first generation biofilm bioreactor (See Figure 1 and Figure 2). The reactor is constructed of acrylic pipe with an inner diameter of 10.1 cm. The total height of the reactor is 127 cm. The empty volume of the reactor is 10.3 L but is operated with a working (liquid) volume of 5 L and is filled with plastic carrier material (KONTAKT 565 from NSW Environmental Systems, Roanoke, Virginia, USA) ensuring a total surface area of 0.5 m² (See Figure 3). The media has a diameter of 2.2 cm, a thickness of 0.635 cm, a dry weight of 122 kg/m³, a surface area of 564 m²/m³ and a void space of 91 %. The reactor was seeded with 150 mL OLAND sludge obtained from Dr. Willie Verstraete’s laboratory in Belgium.

To inoculate the reactor, a batch phase was established in which 5 L of liquid media was poured into the column and the column was sealed. Over a period of 2 weeks, the reactor was maintained in a batch configuration with an internal recycle flow rate of 0.4 L/day (no influent or effluent flow). Once nitrogen removal was observed, feed was continuously administered at an initial flow of 0.4 L/day with an internal recycle flow of 1 L/day; this continued for approximately 50 days. Aeration was not provided to the reactor during operation, so the only oxygen provided was that which dissolved into the feed media due to open contact with the atmosphere. The reactor feed solution consisted of 100 mg-N/L (NH₄⁺/NO₂^- in a molar ratio of 1), 1g/L NaHCO₃, 30 mg/L KH₂PO₄ and 2mL/L trace element solution achieving a volumetric loading rate of 8 mg N/L-day (Kuai et al., 1998; Pynaert et al., 2004). During this operational period, it was discovered that ammonia volatilization in the feed solution (open to the atmosphere) contributed to a significant aspect of observed nitrogen removal. Such a finding is significant, especially when computing nitrogen balances for the OLAND system. On December 25th 2004, the reactor operation was turned over to Mr. Wendall Khunjar, graduate research assistant; in this report, the date Mr. Khunjar took over operation of the reactor is designated as day 0.

On day 5, the influent flow was increased to 1 L/day while the recycle flow was maintained at 1 L/day. During this period, the influent nitrogen concentration was increased to 125 mg N/L to achieve a volumetric loading of 25 mg N/L-day. Gas was collected in a 10-L gas bag connected to the headspace, which was monitored regularly for signs of gas production. This configuration was maintained for 44 days during which ammonia (NH₃), nitrate (NO₃^-), nitrite (NO₂^-), pH, dissolved oxygen (DO), total suspended solids (TSS) and volatile suspended solids (VSS) analyses were performed three times a week. Alkalinity analysis was performed weekly.

On day 44, the recycle flow was increased to 9 L/day in an effort to improve reactor performance. By increasing the shear force across the column, it was hoped that attachment of biomass would be enhanced. Additionally, the decision was made to make the reactor as anaerobic as possible. Thus the feed was deoxygenated with N₂ gas on a daily basis. The reactor has been operating under these adjusted conditions since day 44. Preliminary results detailing reactor performance up to the current period are listed in the Preliminary Data section of this report (See Figures 5.gif through Figure 9 and Table 1).

Construction of a second fixed film bioreactor is currently being delayed upon stabilization of performance of the first FBBR, which is currently operating. To augment the existing biomass present in the FBBR and maintain a source of biomass capable of anammox, a suspended growth continuous flow stirred tank membrane reactor (CSTMR) is currently being constructed (See Figure 4), and may ultimately replace the second fixed film reactor if its performance is strong. This reactor will initially be maintained under strictly anaerobic conditions to select for bacteria capable of the anammox process using the biomass in the current FBBR as a seed. The CSTMR will contain membranes so that conventional settling can be avoided, which has the potential to slow the accumulation of anammox bacteria further. Use of membranes for solid-liquid separation is ideal for enrichment of very slow growing bacteria, such as the anammox bacteria, because it retains them in the system and only allows dissolved constituents to leave the reactor in the liquid through the membranes. The CSTMR will have a working volume of 5 L, and effluent will be pulled out through the membranes constantly while influent is dripped into the top . Stirring can be provided by a stir bar, but it is anticipated that sufficient mixing will occur through bubble generation by the biomass as they generate N₂. The reactor will be kept anaerobic and pH balanced through deoxygenation with a blend of CO₂/N₂ gases. Online pH and ORP control will also be provided to maintain optimum growth conditions for anammox bacteria. Analyses of constituents similar to that performed for the FBBR will be carried out. Once the performance of the CSTMR is evaluated, it may continue to serve as the second bioreactor configuration. Because membranes are used to retain the bacteria, the biomass concentration can be very high, similar to what is achieved with a biofilm reactor system, and very competitive with the biofilm system. The rotating bioreactor configuration suggested in the proposal has been determined to be impractical due to scale-up problems with the design. Use of a high density suspended growth reactor (CSTMR) versus a high density biofilm reactor (FBBR) gives a broader range of comparison, and probably a more practical reactor design that will apply more readily to the field. The investigators believe that utilizing the membrane technology will be beneficial towards achieving more stable OLAND performance in light of current experience with the FBBR. Additionally, implementation of a CSTMR in the field will not warrant extensive cost increases as the technology is rapidly becoming more affordable.

Subtask 2, constructing a pilot plant-scale anaerobic storage tank using traditional design parameters has been accomplished. A 450 liter steel tank is being used, with a surface area to volume ratio typical of commercial anaerobic storage tanks. To mimic standard storage procedures, a starting volume of 150 liters of fresh manure slurry was used as a starting volume, and 1.5 liters of slurry (0.5% of total volume) is added once daily. Slurry is added through a submerged pipe (3 cm I.D.) to avoid disturbing the surface crust and ensure normal retention of ammonia in the tank. This storage tank will be operated for the standard 180 d retention time. Samples will be collected biweekly and at the end of the180 day retention time, and analyzed as outlined in the proposal.

Progress on Objective 5.
One of the PIs (Knowlton) has visited the Winyah Bay NERR site and met with both the reserve manager Wendy Allen and research coordinator Dr. Erik Smith. Application of the research results within the watershed was discussed; the NERR collaborators are excited about the project. Atmospheric deposition is a significant source of N contamination of the estuarine environment, so technologies to reduce ammonia emission will be of direct benefit.

Difficulties
Difficulties encountered with Objectives 1 and 2.
Initial efforts to achieve stable performance of the FBBR have been slow. The planctomycetes bacteria responsible for anammox are extremely slow growing with doubling times reported from 11 to 30 days (Van de Graf et al., 1996; Jetten et al., 2001). The process itself is also more than seven times slower than aerobic ammonia oxidation (Strous et al., 1998). Although slow growth is beneficial in a full-scale system, it is challenging when trying to start up a system. The anammox process is also inhibited by various chemicals, including nitrite, oxygen and phosphate (van de Graaf et al., 1996; Jetten et al., 2001). Oxygen concentration as low as 2 mM and nitrite concentrations between 5 and 10 mM inhibit the ANAMMOX activity completely but reversibly (Jetten et al., 2001). We are monitoring very carefully to ensure these inhibition thresholds are not exceeded.

The low concentration of seed biomass also contributed to slowed performance of the FBBR. Even after 60 days of continuous operation, the biomass concentration continues to be very low, which may contribute to the low removal rates observed. Compounding this is the fact that difficulties have been experienced in trying to maintain anaerobic conditions within the FBBR since the reactor itself is not fully gas tight. Modifications have been made in an effort to achieve anaerobic conditions but it is possible that oxygen may enter the reactor resulting in reduced removal rates as depicted in the preliminary data listed below. Initial data indicated that ammonia oxidizers were the primary active organisms. Whilst aerobic ammonia oxidation is essential for the OLAND system, it is also critical to establish sufficient biomass capable of anammox activity prior to allowing competition with ammonia oxidizers. Once a strong anommox community is established within the FBBR and CSTMR, we intend to reintroduce microaerobic amounts of air and aerobic ammonia oxidizing bacteria, available in our laboratory, to establish the blended OLAND community.

Difficulties Encountered with Objective 5.
Because of untimely animal health problems on the primary collaborator concentrated dairy farm, the planned initial visit to collect manure samples and begin collecting data to construct a whole farm N balance was delayed. The producer tightened biosecurity, and requested that we delay our visit. This is a temporary delay; we anticipate farm visits will have occurred by the next reporting period.

Project Objectives for Next Reporting Period

Objectives
All five objectives, listed previously, will be addressed to varying degrees during the next reporting phase.

Tasks to Meet Objectives
Specific tasks to be completed over the next reporting period are:

1. Complete construction and begin operation of CSTMR.
2. Operate the CSTMR and FBBR with synthetic feed.
   1. Determine the ease and speed of start-up of each reactor and also the control and operation of each reactor.
   2. Characterize the start-up of each reactor configuration and determine the most appropriate operational and control parameters for satisfactory operation by:
      1. Assessing stoichiometric validity of the OLAND process within reactor configurations.
      2. Determining degree of NH3 stripping that occurs within each reactor configuration
3. Expose the OLAND treatment systems to anaerobically stabilized dairy waste and evaluate performance.
   1. Automate and maintain stable operation over prolonged periods and achieve optimal biomass doubling time, as detailed in the literature.
   2. Assess stoichiometric validity of the aerobic ammonia oxidation and anammox processes using biomass from the OLAND reactors.
   3. Assess reactor performance by establishing volumetric removal rates of NH3 and NO₂^- after exposure to different oxygen and ammonia surface loadings.
4. Evaluate the degree of stabilization achieved by the pilot-scale anaerobic storage unit over time to assess its ability to achieve sufficient stabilization in support of the OLAND process.
5. Initiate development of loading curves to simplify design of the OLAND treatment systems for application to dairy waste management.
6. Complete the nitrogen balance on a dairy farm in the Winyah Bay watershed, and a “what if” evaluation on the impact of implementing OLAND on that dairy farm.

Work Plan for Next Reporting Period
To accomplish tasks as outlined previously, the diligent efforts of one full time graduate student and one part time technician in consultation with the Principal Investigators on the project will keep the project within deadlines.

Anticipated Success in Meeting Project Objectives
The slow growing nature of the planctomycete bacteria responsible for the anammox process within the OLAND system may prove to be a limiting factor. Obviously, the speed at which anammox biomass can be obtained through growth defines the progress of all elements of this report. Several minor modifications are currently being put into place to establish optimum growth conditions for the anammox biomass. We hold great hope for success in sustaining a dense anammox culture with the CSTMR, based on past experiences in Dr. Verstraete’s laboratory in Belgium. This culture will be used to reseed the FBBR to enhance its performance.

Preliminary Data
Preliminary results are given in Figure5.gif through Figure 9.gif and in Table 1. As shown in Figure 5, effluent nitrate concentrations increased over time before the reactor was adjusted to operate anoxically. This reflected a possible complete nitrification (nitrite formed by the aerobic ammonia oxidizing bacteria (AAOB) was subsequently oxidized to nitrate by nitrite oxidizing bacteria (NOB), which would outcompete the anammox bacteria and prevent growth of the latter). Poor ammonia removal was also observed during this period (Figure 6). Once the reactor was changed to anoxic conditions, effluent nitrate concentrations decreased, which is consistent with our suspicion that aerobic nitrite oxidizing bacteria were proliferating in the column. Although some nitrate is formed by the OLAND process, we were seeing too much relative to what we expected based on theoretical stoichiometry. Furthermore, by shifting the reactor to anoxic conditions, we undoubtedly reduced the degree to which AAOB proliferated. This was done on purpose to select for anammox metabolism under ideal growth conditions to enhance the amount of anammox biomass available.

As shown in Figure 7 and Figure 8, the percent total nitrogen removed has ranged from 20 to 50 percent so far. Furthermore, not all ammonia or nitrite is consumed. The reactor emulates a system that is either loaded to a higher degree than the biomass can withstand, or is chemically inhibited somehow. We have observed progressively greater amounts of biomass within the column, so we suspect inhibition. The pH of the system is running around 8.0 (Figure 9). This is the upper limit of optimal pH growth ranges recommended for anammox bacteria. Therefore, we assume some level of inhibition is occurring due to the slightly elevated pH. We are shifting our overhead gas to a blend of CO₂ and N₂ in order to buffer against loss of acid (as occurs over time with our highly alkaline feed, which causes the pH to increase more). The impact of this on performance with anaerobically stored, solids separated dairy manure will be determined once we change our feed to the field-generated waste.

Dissemination
Publications: none yet

Workshops: none

Conferences:
W. Khunjar, P. Sweetman, N. G. Love, K. F. Knowlton, B. F. Smets. Treatment of Anaerobically Stabilized Dairy Waste with an Oxygen Limited Autotrophic Nitrification plus Denitrification (OLAND) Fixed Film Reactor: Startup and Maintenance Issues. Submitted for consideration to be presented at the Virginia VWEA/AWWA Water Jam Conference, September 2005.

Manuals, Protocols none

Outreach Activities: none

Contact with End Users: The PIs have communicated with the primary end-user, Dale Gardner of the Virginia State Dairyman’s Association regarding our progress. His feedback is summarized below, and attached in a letter.

Patent, Copyright, Invention Disclosure Activity: none

Expenditures
The project is 25% complete based on time and only 12% complete based on expenditures. The low rate of expenditure reflects the fact that the major bioreactor construction costs have not hit the budget reports yet, and the primary student involved with this project is on a fellowship, so his stipend does not cost as much as budgeted. We anticipate adding an undergraduate research assistant to the project to assist this student, which will utilize the savings due to his fellowship.

End User Advisor Feedback
Click here to download pdf.

References
Jetten MSM, Wagner M, Fuerst J, van Loosdrecht M, Kuenen G, Strous M. Microbiology and application of the anaerobic ammonium oxidation (‘ANAMMOX’) process. Curr Opin Biotechnol 2001;12: 283­ 8.

Kuai L, Verstraete W. Ammonium removal by the oxygen-limited autotrophic nitrification­denitrification system. Appl Environ Microbiol 1998;64:4500­ 6.

Pynaert, K., Smets, B.F., Beheydt, D., Verstraete, W. Startup of Autotrophic Nitrogen Removal via sequential biocatalyst addition. Environ. Sci. Technol. 2004; 38: 1228-1235.

Strous M, Heijnen JJ, Kuenen JG, Jetten MSM. The sequencing batch reactor as a powerful tool for the study of slowly growing anaerobic ammonium-oxidizing microorganisms. Appl Microbiol Biotechnol 1998; 50: 589­96.

van de Graaf AA, de Bruijn P, Robertson LA, Jetten MSM, Kuenen JG. Autotrophic growth of anaerobic ammonium oxidizing microorganisms in a fluidized bed reactor. Microbiology 1996;142:2187 ­ 96.