CICEET Progress Report for the period 9/02/07 Through 3/01/08

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

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Tables

Table 1

Table 2

Table 3

Tasks to meet objectives
a. Objective a-a: cell suspension experiments with three different microbial genera amended with different forms of Fe(III) at circumneutral pH
b. Objective a-b: coordinate field sampling effort with IL department of public health officials and collect samples at a predetermined field location; this was followed by laboratory characterization including
i. Total anion concentration including phosphate
ii. Electron acceptor screening
iii. Bioavailable iron screening
iv. Dominant metabolic pathway screening using 2-[¹⁴C]-acetate
c. Objective a-c: 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
d. Objective a-d: 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))
e. Objective a-e: a conference call was held with all team members participating.

Progress on Tasks
to date all tasks have been completed or are underway with at least one time point collected for ongoing experiments. Septic material was collected from a residential dwelling in early December 2007.

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 has been added to the research. This is 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 postulate that adding Fe(III) will open up an untapped microbial niche ­ Fe(III) reduction ­ that will 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) may degrade via Fe(III) reduction, which decreases the overall remaining BOD/COD load on the environment. 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.

Dissemination activities during this reporting period (please include the number of participants where applicable).
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

Difficulties
No difficulties have been encountered to date

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.

a. Figure 1: Mineralization of 2-[¹⁴C]-acetate in septic material that was incubated unamended or amended with 200µM molybdate. The results are the individual replicates of a duplicate experiment. Molybdate is a specific inhibitor of sulfate reduction. These data demonstrate that acetate was rapidly mineralized in the septic material. It was not linked to sulfate reduction, as molybdate did not inhibit activity. All mineralization was to ¹⁴CO₂; ¹⁴CH₄ was not produced. The activity was likely linked to nitrite reduction or possibly to limited Fe(III) reduction.

b. Table 1: Initial characterization data for septic material collected in December 2007. Phosphate levels in this material are relatively low; therefore, phosphate will be added to experimental systems to increase the initial concentration.

c. Table 2: Phosphorus removal in septic material amended with different forms of Fe(III) relative to several amended or unamended controls. Data suggest phosphorus removal linked to Fe(III) amendment. Vivianite has not yet been identified in these samples. Although phosphorus adsorption to Fe(III) is possible, it is not likely in the Fe(III) citrate amended bottles ­ Fe(III) citrate is a soluble Fe(III) form. Therefore, at least some phosphorus removal may be due to ferrous iron mediated precipitation. All ND values are attributed to analytical error, and sampling will be continued.

d. Table 3: COD dynamics in Fe(III)-amended and unamended septic material incubations. Analytical issues in early samples preclude meaningful interpretation at this point. Trends up to day 12 suggest more COD removal in Fe(III)-amended bottles. Time 21 sampling will be redone to ascertain whether these are true values.

e. Figures 2-4: Phosphorus removal in abiotic, buffered suspensions (bicarbonate) at varied pH in the presence of 5mM (Figure 2), 1.5mM (Figure 3), and 0.5mM (Figure 4) Fe(II). The Fe(II) was maintained under anoxic conditions, and therefore no Fe(III) was present in these suspensions.

f. Figures 5-6: Phosphorus removal in cell suspensions of Shewanella oneidensis in bicarbonate buffer with varying concentrations of Fe(III) and phosphate. The data are incomplete because this specific culture did not apparently reduce the Fe(III) to Fe(II), which was likely a “learning mistake” on the part of the graduate student. However, the data are useful in that there was no apparent biosorption of phosphate to the cell mass, which is good for future experiments. Also, there was little if no abiotic loss of phosphate in the controls.

Project Objectives for Next Reporting Period

Objectives
The objectives for the next period will be:
a. Demonstrate that vivianite formation in cell suspensions will remove phosphate from solution;
b. Demonstrate that Fe(III) addition to septic material increases the rate and extent of mineralization of several low molecular weight carbon intermediates;
c. Demonstrate that Fe(III) addition to septic material promotes phosphate removal and vivianite formation;
d. Demonstrate that Fe(III) amendment to septic material stimulates an Fe(III)-reducing microbial community;
e. Operate and maintain the sequencing batch reactors (SBR) at UML and amend them with different forms of Fe(III) to quantify carbon removal kinetics and phosphate removal kinetics in a system that mimics an actual septic system; and,
f. Begin spectroscopic characterization of vivianite in our experimental systems.

Work Plan to Meet Objectives
a. Objective a-a: Cell suspensions will be continued with the three model cultures: Geobacter metallireducens, Shewanella oneidensis, and Anaeromyxobacter dehalogenans. The experimental methods will remain consistent with what has been described in detail within the proposal. If we identify a specific Fe(III) amendment or concentration with one series of cell suspensions we will focus on that single amendment for the remaining series.

b. Objective a-b: Batch experiments will continue with septic material collected in December 2007. In addition to monitoring total COD, we will also use radiolabeled substrates to quantify mineralization kinetics for some key carbon intermediates. The carbon substrates will include [¹⁴C]-radiolabeled acetate, lactate, butyrate, and propionate. [¹⁴C]-labeled starch and glucose can also be tested as moderate molecular weight intermediates, but their use is not definite at this time.

c. Objective a-c: Batch experiments will continue with septic material collected in December 2007. Phosphate will be monitored in solution to quantify loss of phosphate in the presence of the various Fe(III) amendments. This task will coordinate with the final task, which will include spectroscopic characterization of the samples we preserve to identify increases in the mineral vivianite.

d. Objective a-d: samples from both batch incubations described above and SBR experiments described above (UML tasks) will be analyzed using molecular analyses for specific Fe(III)-reducing microorganisms, as well as total microbial community analyses. The explicit methods are detailed in the proposal. Briefly, we will use amplified ribosomal DNA restriction analyses (ARDRA) to characterize the total microbial community composition. We will use quantitative polymerase chain reaction (Q-PCR) to specifically quantify the increase in Fe(III)-reducer biomass.

e. Objective a-e: The University of Massachusetts at Lowell startup has been limited by contract negotiations between CICEET and UIUC, and then UIUC and UML. Unlike UIUC, the UML group could not purchase major equipment until funding was in place. They have a reactor design quote ready for purchase, and the unit will be operational by early April to early May. Once it is operational it will be run continuously with influent septic material that UML personnel will collect at a location yet to be determined. The septic material collected for UML studies will be similar to the UIUC septic material in terms of sludge age. The SBR experiments will begin once the baseline phosphorus levels have been determined. Fe(III) amendments will be selected based on experimental data from UIUC batch studies.

f. Objective a-f: the graduate student research assistant from UIUC will being training at the UIUC materials research laboratory for transmission electron microscopy-energy dispersive x-ray spectroscopy (TEM-EDS) techniques that will be used to characterize the mineral products derived from the reduction of different Fe(III) species.

Dissemination Objectives for next reporting period
We anticipate the following communications within the next reporting period:
a. One abstract submitted to a national conference
b. One manuscript in preparation related to carbon and phosphorus removal in septic material as a result of Fe(III) amendment
c. Ongoing communications with Andrew Helminger of RTI
d. Data update to our NERRS partners
e. Presentation by PI Finneran to CICEET and UNH interested parties to be held in New Hampshire the week of June 1-5 (when PI Finneran is attending ASM Boston.)

Expenditures
There were two deviations from the original expenditure forecast; neither of which affects the overall budget amount. The first was a reallocation of funding originally earmarked for University of Massachusetts at Lowell to University of Illinois. The amount was $3,866 and it was personnel budget. The graduate student at University of Massachusetts at Lowell did not begin until January of 2008, and co-PI Zhang utilized undergraduate support for the fall 2007 semester. The funding difference calculated based on direct + indirect costs was $3,866, and that was retained by University of Illinois. PI Finneran allocated that to University of Illinois personnel.

The second expenditure deviation was in the sub-contract to University of Massachusetts at Lowell from University of Illinois. The year 1 budget was increased to $59,036, and the year 2 budget was decreased to $48,519. This did not alter the overall cost of the subcontract to University of Massachusetts at Lowell. The funding reallocation was made so UML could purchase large equipment in year 1, which will expedite experiments associated with the tasks to be performed at University of Massachusetts at Lowell.

End User Advisor Feedback
No end users have yet been identified with respect to this research. 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.