Progress Report

CICEET Progress Report for the period 3/01/09 Through 8/31/09

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

Figures

Figures 1 - 19

Figures 20 - 27

A. Project Objectives for This Reporting Period

Begin field investigation with Fe(III) amendment for phosphorus removal at the “Davis Lodge Site” in Bloomington, IL;
Collect field septic samples from at least two additional sites for further lab experimentation;
Continue all cell suspension experiments to quantify phosphate removal in the presence of various Fe(III) forms and concentrations;
Continue experiments with Fe(III)-amended septic material experiments to asses both phosphate removal and total carbon removal
These experiments utilized ¹⁴C-radiolabeled substrates and non-radiolabeled experiments (bulk COD);
Use MINEQL modeling to determine the equilibrium phase reactions that lead to vivianite versus alternate ferrous mineral phases;
X-ray diffraction (XRD) and Scanning electron microscopy-energy dispersive x-ray (SEM-EDX) spectroscopy to characterize vivianite minerals in pure phase experiments and in incubations with septic material;
Continue pure (chemical) phase experiments to determine the influence of pH and bicarbonate concentration on phosphorus removal in the presence of Fe(III);
Begin molecular microbial community analyses of native septic system wastewater and sludge versus Fe(III)-amended septic system wastewater and sludge;
Investigate the factors affecting removal of phosphorus removal in the iron-amended ASBR;
Investigate the mechanisms of Fe(III) reduction and phosphorus removal by batch assays using the septic sludge from the ASBR;
Identify the precipitates formed in the Fe(III)-amended ASBR;
Oversee and participate work done with co-PI work at University of Massachusetts at Lowell (Zhang);

b) Tasks to meet objectives: The tasks to meet these objectives were:

Objective a: a field campaign began at the “Davis Lodge Site”, which is a user facility on Lake Bloomington in Bloomington IL. There are two septic tanks present on site and Fe(III) was amended into the upstream septic tank, where the bulk of waste is treated. Fe(III) was added as ferrihydrite (amorphous solid) and as Fe(III) citrate (soluble Fe(III)). The target concentrations in the tank were 2mmol/L for each Fe(III) amendment. Samples have been collected three times and a fourth is scheduled for this week. Samples takes at each time point include solids and aqueous for a variety of lab characterizations;
Objective b: samples were collected with the assistance of a pumper in Normal, IL and have been designated “Septic #2”. Bulk samples were also collected at the “Davis Lodge Site” and are a third batch for lab experiments;
Objective c: Cell suspensions currently focus on the pure culture Geobacter metallireducens to investigate the influence of Fe(III):phosphate ratio and pH on vivianite precipitation. In addition, these batch studies will provide minerals for ongoing mineralogy characterization since they are “cleaner” samples relative to the raw septic material;
Objective d-a: 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;
Objective d-b: 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));
Objective d-c: batch studies with native septic material versus Fe(III) amended material to quantify the extent of mineralization of several key carbon compounds; “Septic #2” samples have yielded different rates and extents of carbon mineralization but the general trend is the same – when Fe(III) is added mineralization to CO2 increases. One additional interesting finding is that methanogenesis is inhibited by Fe(III), as would be expected, but the extent of suppression is large and this will have an impact on methane flux from septic environments:
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;
Objective e: use MINEQL to develop equilibrium speciation models for vivianite versus siderite or ferrous iron based on iron, phosphate, or pH;
Objective f: ongoing analysis at the Seitz Materials Research Laboratory (MRL) at the University of Illinois. Data are presented in the figures to demonstrate the results of initial characterization;
Objective g: conduct experiments with three pure cultures for Fe(III) reduction mediated phosphorus removal; the cells included are:
1. Geobacter metallireducens
2. Shewanella oneidensis
3. Anaeromyxobacter dehalogenans;
Objective h: 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;
Objective i-a: Objective a-a: The effect of agitation on continuous P removal. It was studied after the reactor had reached a stable state. Three agitation rates were studied, 100, 50 and agitation-free.
Objective i-b: The effect of hydraulic retention time (HRT) on continuous P removal. It was studied after the reactor had reached a stable state. Four HRT were studied, 6, 4, 2 and 1 days.
Objective k-a: The mechanism of P removal. Several possible P removal mechanisms were examined. They included: P adsorption by the amorphous FeOOH, P adsorption by the biomass in the ASBR, and P precipitation by iron reduction.
Objective k-b: The precipitates collected in the iron-amended ASBR were analyzed by TEM, SEM and XRD.

c) Progress on Tasks.
Most tasks are progressing at the anticipated rate. The two tasks that are slightly behind schedule are: molecular microbial community analyses and mineral characterization. Both of these task timelines are related to the steep learning curve associated with the Illinois student learning to run the procedures on his own. The molecular analyses used are straightforward but require time to perfect for experimental tasks. The mineral characterization is not run by technicians, but rather the student has been trained and now runs/analyzes his own samples. While having him run the analysis increases the lag time of the task, we feel it is critical to his development as a scientist and researcher. The molecular biology task is underway and the student is now proficient in several PCR based assays needed for good DNA and RNA quantitation.

d) Have the results/data gathered during this reporting period changed the project objectives when compared to your original proposal? Please explain.
In mid 2008 we changed the project to include the carbon oxidation data (and now suppression of methanogenesis) to the project objectives. That has not changed. We now have added a field trial to the objectives. While this is earlier than I normally would like to run a field trial in a research effort, the opportunity was presented by an outside group and we took the chance because this is a low risk-high reward undertaking.

e) 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 (Presented)
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. (Presented)
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 (Presented)
Azam, H.M., and K.T. Finneran. 2009 Fe(III) Amendment Increases Carbon Mineralization in Septic System Material, Association for the Environmental Health Sciences Annual Conference, Amherst, MA, October 19-22, 2009 (Accepted)

Manuscripts published or submitted for publication

Azam, H.M. and K.T. Finneran, 2009, Ferric Iron Amendment Increases Carbon Oxidation and Suppresses Methanogenesis in Septic System Wastewater, submitted to Water Research
Xiang Cheng, Xiaoqi (Jackie) Zhang, Dezhi Sun. (2009) Phosphorus precipitation in septic systems induced by iron reduction. Journal of Environmental Engineering-ASCE. (submitted)
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. (in preparation)

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; four undergraduate engineering students at Illinois participated during the spring and summer semesters. A visiting scholar from Harbin Institute of Technology has been working on this project at the University of Massachusetts at Lowell.

f) Difficulties
No difficulties have been encountered to date

g) Data Generated to date:
The data are presented as figures and tables, which supplement this text. The figure and table legends below summarize the data and provide a brief discussion of the results. All tables and figures are provided as supplemental JPEG files and are numerically identified according to figure number.
a. Figures 1-7 refer to the material “Septic #2” (new material):
    Figure 1. Mineralization of [¹⁴C]-acetate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 2. Mineralization of [¹⁴C]-lactate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Mineralization of [¹⁴C]-propionate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 4. Mineralization of [¹⁴C]-butyrate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 5. Mineralization of [¹⁴C]-glucose in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 6. Mineralization of [¹⁴C]-glucose in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 7. Mineralization of [¹⁴C]-oleic acid (lipid) in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

b. Figures 8-14 refer to the second experimental series with “Septic #1”:
    Figure 8. Mineralization of [¹⁴C]-acetate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 9. Mineralization of [¹⁴C]-lactate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 10. Mineralization of [¹⁴C]-propionate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 11. Mineralization of [¹⁴C]-butyrate in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 12. Mineralization of [¹⁴C]-starch in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 13. Mineralization of [¹⁴C]-glucose in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

    Figure 14. Mineralization of [¹⁴C]-oleic acid (lipid) in incubations with native septic material versus septic material amended with different forms of Fe(III). The results are presented as short term and long term mineralization. Short term mineralization is critical as this is the fraction that will be degraded quickly, within 30 hours of being introduced into a septic system. The long term fraction is the material that will accumulate in the sludge layer and degrade slowly over time.

c. Figures 15-19 are images or scans from mineralogical characterization:
    Figure 15. TEM analysis showing vivianite precipitates in a pure phase suspension.

    Figure 16. TEM analysis showing vivianite precipitates in a pure phase suspension at lower magnification.

    Figure 17. TEM analysis showing vivianite precipitates in a pure phase suspension at lower magnification.

    Figure 18. TEM analysis showing vivianite precipitates in a pure phase suspension at lower magnification; sample is from buffered, pH controlled experimental series.

    Figure 19. XRD chromatogram indicating the Fe(II) peaks associated with vivianite; interferences were present in this sample.

d. Figures 20-27 are data from anaerobic sequencing batch reactors:
    Figure 20. The operational parameter table for the ASBR

    Figure 21. Figures 21, 22, and 23 show that the theoretical amount of Fe(III) was insufficient for practical applications. Agitation was necessary for Fe(III) reduction. Biological Fe(III) reduction by DIRB might need direct contact between cells and iron. HRT from 1 d to 6 d did not notably affect iron reduction and the consequent P removal. The slight increase in COD in the effluents under 1-d HRT indicates: (a) complete COD degradation needs longer HRT; (b) electrons from the degradation of this level of COD were adequate for iron reduction. Higher COD removal was obtained indicating that iron addition and reduction improved C mineralization.

    Figure 22. See above

    Figure 23. See above

    Figure 24. Figures 24 and 25 demonstrate the mechanisms of P removal. The potential mechanisms include ?-FeOOH adsorption, biomass adsorption and iron reduction.

    Figure 25. See above

    Figure 26. Figures 26 and 27 demonstrate that the precipitates formed in the Fe(III)-amended ASBR were indeed vivianite.

    Figure 27. See above

Project Objectives for Next Reporting Period:
a) Objectives: The objectives for the next period will be:

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

b) Work Plan to Meet Objectives: The tasks to meet these objectives will be:

Objective a: amplified ribosomal DNA restriction analyses (ARDRA) and quantitative polymerase chain reaction (Q-PCR) and Q-reverse transcriptase-PCR (Q-RT-PCR) analysis of in situ microbial communities;
Field trial is ongoing;
Perform XRD and SEM-EDX at the Materials Research Lab of the University of Illinois and assist with data analysis of UML mineralogy characterization;
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;
Use the UML reactors to determine the best strategy for adding Fe(III) to an operating septic system;
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,
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) and Q-PCR 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.

c) Dissemination Objectives for this reporting period: We anticipated the following communications within this reporting period, and the outcomes are underlined:

Two abstracts submitted/presented to a national conference (ASM and AEHS annual conference): this objective was met
One manuscript submitted related to carbon oxidation in septic material as a result of Fe(III) amendment: this objective was met
Ongoing communications with Andrew Helminger of RTI: this objective is in progress
Data update to our NERRS partners: we have not completed this objective, but we have established a local relationship for the technology; we will update our NERRS partners once more data with respect to phosphorus are clearly defined during the additional no-cost (to CICEET) year of research

d) Dissemination Objectives for next reporting period: We anticipate the following communications for the next reporting period:

Present one platform presentation at the AEHS annual conference
Submit at least two additional abstracts to national symposia
Submit two more manuscripts (one is in progress related to suppression of methanogenesis and microbial community and the other is related to phosphorus removal by Fe(III) reducing microorganisms)
Meeting with Bloomington IL working group

e) Overall Project Timeline Update:
We have requested and been granted a no-cost extension for one additional year of research to continue this project.

3. Expenditures:
There were two no deviations from the original expenditure forecast for this time period. We are currently operating in a no cost extension period.

4. End User/ Producer/ Adopter Advisor(s) 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 started research at a test location for launching a field-test of this technology at a multi-use facility in Bloomington
Monitoring is being 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 a date to be determined 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.

5. PI Response to End User Advisor Feedback:
This will be updated after the City of Bloomington meeting.

6. Additional Items not Described Above of Relevance to CICEET:
No additional items are anticipated for this reporting period.