CICEET Progress Report for the period 9/01/08 Through 2/15/09

Project Title: Phosphorus Removal in Retrofitted On-Site Wastewater (Septic) Systems by Stimulating Fe(III) Reduction: Insoluble Mineral Precipitation (Vivianite)
Principal Investigator(s): Kevin T. Finneran
Additional Investigator(s): Xiaoqi J. Zhang
Project Start Date: September 1, 2007

1. Continue all cell suspension experiments to quantify phosphate removal in the presence of various Fe(III) forms and concentrations;
2. Conduct experiments with Fe(III)-amended septic material experiments to asses both phosphate removal and total carbon removal
   1. These experiments utilized ¹⁴C-radiolabeled substrates and non-radiolabeled experiments (bulk COD);
3. Use MINEQL modeling to determine the equilibrium phase reactions that lead to vivianite versus alternate ferrous mineral phases;
4. X-ray diffraction (XRD) and Scanning electron microscopy-energy dispersive x-ray spectroscopy to characterize vivianite minerals;
5. Continue pure (chemical) phase experiments to determine the influence of pH and bicarbonate concentration on phosphorus removal in the presence of Fe(III);
6. Molecular microbial community analyses of native septic system wastewater and sludge versus Fe(III)-amended septic system wastewater and sludge;
7. Demonstrate continuous phosphorus removal using synthetic septic wastewater by iron reduction in the ASBR;
8. Characterize the precipitates accumulated in the ASBR to support the iron-reduction stimulated phosphorus removal; and,
9. Oversee and participate work done with co-PI work at University of Massachusetts at Lowell (Zhang);

Tasks to meet objectives

1. Objective a-a: cell suspension experiments with three different microbial genera amended with different forms of Fe(III) at circumneutral pH;
2. Objective a-b: batch incubations with septic material amended with different forms of Fe(III) including solid phase and soluble Fe(III); analyses include total phosphate and total chemical oxygen demand;
   1. Specific ¹⁴C-radiolabeled substrates tested included acetate, lactate, starch, glucose, butyrate, and propionate;
3. Objective a-c: batch studies with buffered solution at multiple pH values from 5.0-9.0 in the presence of different Fe(III) forms to quantify the effect of pH on iron-mediated phosphate removal (as Fe(III));
4. Objective b: batch studies with native septic material versus Fe(III) amended material to quantify the extent of mineralization of several key carbon compounds; Figures M1-M8 and Figure M10 are the experimental matrices for the carbon mineralization experiments:
   1. Carbon compounds:
      (1) Acetate
      (2) Lactate
      (3) Butyrate
      (4) Propionate
      (5) Glucose
      (6) Starch
   2. Fe(III) amendments
      (1) Ferrihydrite
      (2) Lepidocrocite
      (3) Fe(III)-NTA
      (4) Fe(III)-EDTA
      (5) Fe(III)-Phosphate (may stimulate greater phosphorus removal)
      (6) Fe(III)-Citrate;
5. Objective c: use MINEQL to develop equilibrium speciation models for vivianite versus siderite or ferrous iron based on iron, phosphate, or pH;
6. Objective d: ongoing analysis at the Seitz Materials Research Laboratory (MRL) at the University of Illinois;
7. Objective e: conduct experiments with three pure cultures for Fe(III) reduction mediated phosphorus removal; Figure M9 is the experimental matrix for the pure culture experiments:
   1. Geobacter metallireducens
   2. Shewanella oneidensis
   3. Anaeromyxobacter dehalogenans;
8. Objective f: DNA extraction and PCR amplification with universal Eubacterial primers is underway; ARDRA clone analysis will follow for native septic material versus Fe(III)-amended material;
9. Objective g-a: During the start up, ASBR will be inoculated with septic materials and fed with synthetic septic wastewater. FeOOH will be supplemented to the system at a Fe/P ratio of 1.5. Water samples and biogas produced from the reactor will be analyzed to monitor the performance of the reactor.
10. Objective g-b: The effect of iron dosage on continuous P removal will be studied after the reactor has reached a stable state. The Fe/P will be increased from 1.5 to 3.0; and,
11. Objective h: Solid samples from the reactor will be analyzed by TEM, SEM, EDS, and XRD to support phosphorus removal via vivianite precipitation. The tests will be conducted in the Center for Advanced Materials and Campus Materials Characterization Laboratory at UML and Center for Materials Science and Engineering at MIT.

Progress on Tasks
To date all tasks have been completed or are underway with multiple time points collected for ongoing experiments. Septic material was collected from a residential dwelling in early December 2007. Task b ¹⁴C-radiolabeled experiments are completed. Graduate students at both institutions are learning advanced mineralogy tools (XRD, TEM-EDX) for vivianite characterization. This is done outside of both laboratories using core facilities and therefore takes more time to complete than other tasks within the scope of work. Hossain Azam (Illinois graduate RA) is currently learning the necessary molecular biology tools for qualitative and quantitative nucleic acid (DNA) analyses of the septic materials. Molecular microbial ecology tasks are underway.

Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal? Please explain.
One additional project objective had been added to the research in the previous reporting period. This was to quantify the influence of Fe(III) amendment on total organic carbon (as bulk COD) degradation in septic material. While phosphorus removal is still the primary goal, during our characterization we measured very high rates of acetate mineralization in septic material. However, bulk COD removal rates in septic systems tend to be slow and the mineralization of lower molecular weight intermediates in septic systems is incomplete, based on known data from other sources. We postulated that adding Fe(III) will open up an untapped microbial niche ­ Fe(III) reduction ­ that would in turn increase the rate and extent of carbon removal. Previously un-oxidized organic carbon (i.e. COD that cannot be degraded in a typical septic system) would be degraded via Fe(III) reduction, which decreases the overall remaining BOD/COD load on the environment. As can be seen from the data below, our hypothesis was current and the rate and extent of carbon mineralization in the presence of Fe(III) is higher than native conditions. These data are critical because Fe(III) amendment may make septic system operation more effective in terms of its primary goal ­ carbon removal and sludge stabilization. Data for this portion of the project are presented below, and are currently part of a manuscript in progress for Water Research. Data are definitive with respect to Fe(III) amendment increasing carbon oxidation in septic system wastewater and sludge.

Dissemination activities during this reporting period (please include the number of participants where applicable).
Project related presentations/poster sessions at workshops/conference
Azam, H.M. 2009, Fe(III) Amendment Increases Carbon Mineralization in Septic System Material, University of Illinois Graduate Seminar Series

Azam, H.M., and K.T. Finneran. 2009 Fe(III) Amendment Increases Carbon Mineralization in Septic System Material, American Society for Microbiology General Meeting, May 17-21, 2009, Philadelphia, PA (Accepted)

Cheng, X, Zhang, X., Finneran, K, and Sun, D. (2009) Enhancement of phosphorus removal by iron amendments in a septic system. IWA 2nd Specialized Conference on Nutrient Management in Wastewater Treatment Processes. Krakow, Poland, Sept 6-9, 2009. (submitted)

Zhang, X. and Cheng, X. (2009) Phosphorus precipitation in septic systems induced by iron reduction: a novel process of phosphorus removal under anaerobic conditions. 2009 Wuhan International Conference on the Environment In conjunction with 2009 Annual Meeting of Chinese Society for Environmental Sciences. Wuhan, China, June 27-30, 2009 (invited speech)

Manuscripts published or submitted for publication
Xiang Cheng, Xiaoqi (Jackie) Zhang, Kevin T. Finneran, Dezhi Sun (2009) Phosphorus precipitation in septic systems induced by iron reduction: a novel process of phosphorus removal under anaerobic conditions. Wat. Res. (submitted)

Student activity (e.g. theses, dissertations, etc.) on the project (please identify students as graduate or undergraduate):
One graduate student is currently being trained at the University of Illinois; one student is being trained at the University of Massachusetts at Lowell; two undergraduate engineering students at Illinois participated during the summer semester. A visiting scholar from Harbin Institute of Technology has been working on this project at the University of Massachusetts at Lowell.

Difficulties
No difficulties have been encountered to date

Data Generated to date
See Figures attached.

Project Objectives for Next Reporting Period

Objectives
a. Determine the microbial community that develops during stimulated Fe(III) reduction in septic material;
b. Evaluate the potential for a field trial within the City of Bloomington IL;
c. Characterize the solid phase minerals that form in cell suspensions, pure chemical experiments, and septic material under stimulated Fe(III) reducing conditions;
d. Quantify the kinetics of phosphorus removal relative to the form and concentration of Fe(III) added;
e. Evaluate Fe(III) amendment strategies in the continuous reactor system;
f. Investigate and optimize the influencing parameters for phosphorus removal in the ASBR; and,
g. Analyze the population dynamics during the start-up and steady-state operation of the ASBR.

Work Plan to Meet Objectives
a. Objective a: ARDRA analysis of in situ microbial communities;
b. Presentation and meeting with Stakeholders in March 2009;
c. Perform XRD and SEM-EDX at the Materials Research Lab of the University of Illinois;
d. Conduct several rounds of experiments with different systems (pure culture, pure chemical phase-homogenous mixed, and septic material including newly collected septic material) to quantify the rate and extent of phosphorus removal;
e. Use the UML reactors to determine the best strategy for adding Fe(III) to an operating septic system;
f. Objective f: The effect of cycling time and temperature will be evaluated to optimize the operation process in order to achieve the most phosphorus removal; and,
g. Objective g: Biomass samples will be taken from different time during the start-up and steady-state operation and sent to our collaborators at UIUC. They will use amplified ribosomal DNA restriction analyses (ARDRA) to characterize the total microbial community composition. They will use quantitative polymerase chain reaction (Q-PCR) to specifically quantify the increase in Fe(III)-reducer biomass. According to the population dynamics, the relationship between dissimilatory Fe(III) reducing bacteria (DIRB), fermentative bacteria and methanogens will be established.

Dissemination Objectives for next reporting period
We anticipated the following communications within this reporting period, and the outcomes are in italics:
a. Two abstracts submitted to a national conference (ASM and AEHS annual conference): this objective was met
b. One manuscript in preparation related to carbon oxidation in septic material as a result of Fe(III) amendment: this objective was met; submission timeline is March 2009
c. Ongoing communications with Andrew Helminger of RTI: this objective is in progress
d. Data update to our NERRS partners: we have not completed this objective, but we have established a local relationship (see below) for the technology; we will update our NERRS partners once more data with respect to phosphorus are clearly defined

Overall Project Timeline Update
We do not anticipate changes to the project timeline at this stage.

Expenditures
There were two no deviations from the original expenditure forecast for this time period.

End User Advisor Feedback
Several potential end users have been identified with respect to this research:

Professor Finneran will be presenting this research to Rick Twait, who is the McLean County (IL), City of Bloomington superintendent of water for the city (about 60K population). He has coordinated a meeting with approximately five water-quality regulators interested in phosphorus removal from septic systems at several location in Illinois

• We have already identified a test location for launching a field-test of this technology at a multi-use facility in Bloomington
• Monitoring would be performed under the Bloomington TMDL program
• If successful, the water quality stakeholders are interested in discussing the future use of this research and potentially follow-on funding for the project
• The presentation will be held on March 12th at 2:30pm in Bloomington, IL

Pio Lombardo of Lombardo and Associates contacted the laboratory with respect to the research based on the CICEET web page description. Discussions began with respect to the work done to date.

We have opened a dialogue with Andrew Helminger of RTI, and he is helping use line up potential end users once data are more complete. NERRS partners have not yet requested a data update.

PI Response to End User Advisor Feedback
this will be updated in March after the City of Bloomington meeting.