SUPPORTING INFORMATION. Influence of Feedstock and Pyrolysis Temperature of Biochar Amendments on

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1 SUPPORTING INFORMATION Influence of Feedstock and Pyrolysis Temperature of Biochar Amendments on Transport of Escherichia coli in Saturated and Unsaturated soil Sergio M. Abit, Carl H. Bolster,, Peng Cai,, and Sharon L. Walker U.S. Department of Agriculture Agricultural Research Service, 230 Bennett Ln., Bowling Green, Kentucky 42104, United States. Department of Chemical and Environmental Engineering, University of California, Riverside, California 92507, United States. State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan , China. Total of 9 pages, including 1 table and 2 figures. Corresponding Author: U.S. Department of Agriculture - Agricultural Research Service, 230 Bennett Ln., Bowling Green, KY. carl.bolster@ars.usda.gov; Phone: ; Fax:

2 Bacterial Surface Characterization. The electrophoretic mobility of the E. coli cells in leachate collected from representative columns was measured at 25 ºC using a ZetaPALS analyzer (Brookhaven Instruments Corporation, Holtsville, NY). The experimentally determined electrophoretic mobility values were converted to zeta potential values using the Smoluchowski equation. 1 The hydrophobicity of the cells in each of the various leachates was measured by the microbial adhesion to hydrocarbon (MATH) test 2 where the partitioning of cells between n-dodecane (Fisher Scientific, Pittsburgh, PA) and the leachate was determined spectroscopically. A 798 Titrino automatic titrator (Metrohm Ltd., Riverview, FL) was used to determine the amount of 0.3 N NaOH consumed by suspended cells ( cells ml -1 ) during titration over a ph range of 3.0 to Surface charge density was calculated from the resultant acidity values. 3 Column Preparation. Transport of the two E. coli isolates was evaluated under both water-saturated and unsaturated conditions using vertically oriented columns made of cylindrical transparent PVC pipe (inner diameter of 5.2 cm). Column lengths of 10.5 and 15 cm were used for saturated and unsaturated transport experiments, respectively, with glass frits that do not retain bacteria (10-15 µm pores; air-entry pressure > 60 cm H 2 O, Chemglass Life Sciences, Vineland, NJ) placed at the bottom. For both types of columns, the porous material was uniformly packed to a height of 10 cm by slowly adding porous material while the column was being vibrated. Twenty pore volumes of carbon dioxide (CO 2 ) were then passed through the columns followed by application of ~11 pore volumes of degassed 1 mm KCl electrolyte solution pumped in an upwards direction 2

3 through the column at a rate of 2.67 ml min -1 using a peristaltic pump (Masterflex L/S, Cole Palmer Industries, Vernon Hills, IL). Saturated Columns. Six tubes inserted through a rubber stopper that covered the top of the column were each attached to a three-way valve which connected to a syringe pump (Model 200 syringe pump, KD Scientific Inc., New Hope, PA) and a six-channel peristaltic pump. The drip point of the outlet tubing attached to the column base was then fixed above the saturated column and using the peristaltic pump, approximately four pore volumes of 1 mm KCl solution were applied through the top of the column at a rate of 2.67 ml min -1 (flow direction through column now downward). The last two pore volumes of effluent were collected for analysis as background solution. Unsaturated Columns. After application of 11 pore volumes of KCl, the water standing on the surface of the porous media was carefully removed. A layer of silica beads (3 mm diameter) was placed on the surface of the packed material and the column was mounted on an electronic top-loading balance (accurate to 0.1 g). A sprinkler made of six equally-spaced stainless steel hypodermic needles was positioned on top of the column. Each needle was connected by tubing to a three-way valve which connected to a syringe pump and a six-channel peristaltic pump. A digital manometer (Traceable TM, Fisher Scientific) was also attached to each tensiometer installed at 2.5 and 7.5 cm above the column base. A 1 mm KCl solution was then continuously sprinkled on the surface at a rate of 1.33 ml min -1 using the peristaltic pump. The drip point of the outlet tubing was dropped very slowly until the two tensiometers had consistent and similar readings (within 2 cm tension) and the top-loading balance indicated that enough water was removed to achieve ~ 50% saturation. Four pore volumes of KCl were sprinkled onto the 3

4 surface and the last two pore volumes of effluent were collected as background sample. Background samples were analyzed for ph (measured by Orion ph probe, Thermo Electron Corp., Beverly, MA), specific conductivity (SpC; measured using YSI 556 Multi-Probe, YSI Environmental, Yellow Springs, OH) and for the same parameters that the extracts of the porous materials were analyzed for. A set of five representative columns separately packed with the different porous materials was also leached with the same total amount of 1 mm KCl as the columns used in the bacterial transport experiments. Soils from these leached columns were air-dried and analyzed for soil ph using a 1:2.5 soil-to-kcl ratio and total organic carbon (TOC) by dry combustion with a Total N/C analyzer (Vario Max CN, Elementar Americas Inc). Leachate was also collected from all five columns for use in subsequent bacterial surface property analysis. Bacterial Batch Experiment. Two grams each of the porous materials were placed in designated pre-weighed centrifuge tubes. Twenty ml of bacterial suspension (~ cells ml -1 ) was then added to the tubes, re-weighed and then shaken in a reciprocating shaker at 100 oscillations min -1 for 1 hour to be consistent with the resident time of the bacteria in the column. The mixture was then centrifuged at 4 o C at 200 g for 5 min. One ml of the supernatant was then drawn and used to prepare pre-determined dilutions, plated on mfc agar plates and incubated overnight at 37 o C. Oven-dried mass of the soils were determined and single point sorption coefficients (k d ) were then computed. Sorption experiments were conducted in triplicate. 4

5 Bacterial Survival Evaluation Assessment of bacterial survival was conducted concurrent to each transport experiment. One ml of the bacterial suspension used in the transport experiment was added to a culture tube containing 9 ml of background effluent (collected prior to bacterial suspension application). The resulting 1:10 suspension was then homogenized in a vortex shaker from which 1 ml was immediately drawn, diluted and plated on mfc agar. A 1 ml-sample was also drawn from the same mixture at the end of a transport experiment. The sample was diluted and plated to quantify cells that remained culturable at the end of an experiment. 5

6 Table S1. Selected characteristics of the two E. coli isolates measured in effluent collected from each biochar treatment. Biochar Treatment Poultry Litter Pine Chips 350 o C 700 o C 350 o C 700 o C 0% 1% 2% 1% 2% 1% 2% 1% 2% SP2BO7 Surface charge density, µc cm a a --- b 667a a a a MATH (%) 5.01a a ab b ab Zeta Potential, mv -42.8b a a a a Sorption coefficient, ml g bc 2.36c 1.98c 3.56abc 3.09bc 4.1abc 3.62abc 6.07a 5.22a SP1HO1 Surface charge density, µc cm a a a a a MATH (%) c 23.8c b a c c Zeta Potential, mv -4.38a a a a a Sorption coefficient, ml g d 5.42d 4.69d 28.9cd 54.0cd 146b 115bc a a a Mean values in each row followed by the same lowercase letters are not significantly different using Tukey s HSD test at p < b Bacterial surface properties were not measured in effluent from columns with 1% biochar application rates. c MATH - microbial adhesion to hydrocarbon; a measure of hydrophobicity of bacterial surfaces 6

7 A C B D Figure S1. Scanning electron microscope images of pine chips biochar pyrolyzed at 350 o C (A) and 700 oc (B), and poultry litter biochars pyrolyzed at 350 oc (C) and 700 oc (D). Note: magnification and scale of A and B are similar to C and D. 7

8 Fractional Recovery (fr), % R 2 = 0.82 A R 2 = B Sorption Coefficient (k d ), ml g -1 Figure S2. Relationship between fractional recovery (fr) and sorption coefficient (k d ) obtained from single point isotherms for isolates (A) SP2BO7 and (B) SP1H01. 8

9 REFERENCES (1) Elimelech, M.; Gregory, J.; Jia, X.; Williams, R. A. Particle deposition and aggregation: Measurement, modeling and simulation; Butterworth-Heinemann: Woburn, MA, (2) Pembrey, R. S.; Marshall, K. C.; Schneider, R. P. Cell surface analysis techniques: What do cell preparation protocols do to cell surface properties? Appl. Environ. Microbiol. 1999, 65, (3) Walker, S. L.; Redman, J. A.; Elimelech, M. Influence of growth phase on bacterial deposition: Interaction mechanisms in packed-bed column and radial stagnation point flow systems. Environ. Sci. Technol. 2005, 39,