Influence of Feedstock and Pyrolysis Temperature of Biochar

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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 Lane, 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 430070, China ‡

S Supporting Information *

ABSTRACT: The effects of biochar feedstock, pyrolysis temperature, and application rate (1 and 2%) on the transport of two Escherichia coli isolates through a fine sand soil under water-saturated and partially saturated conditions were investigated in column experiments. Biochars from two feedstocks (poultry litter and pine chips) and pyrolyzed at two temperatures (350 and 700 °C) were evaluated. Both biochars pyrolyzed at 700 °C resulted in significant reductions in E. coli transport, with greater reductions observed with the pine chip biochars. For the low temperature biochars, increased transport was observed for the poultry litter biochar whereas reduced transport was observed for the pine chip biochar. In general, the effect of biochar application on E. coli transport was more pronounced in the unsaturated soils and for the 2% application rates. Large differences were also observed between the two isolates indicating that bacterial surface properties play a role in how biochar affects E. coli transport.



INTRODUCTION

One management practice which has the potential for reducing the leaching of pathogenic microorganisms is the incorporation of biochar into soils. While the term biochar is not clearly or universally defined, it generally refers to charred organic matteroftentimes derived from plant biomass or biowastegenerated for the purpose of carbon sequestration in soils.7,8 In addition to being used to sequester carbon, biochar is being viewed increasingly as a potential amendment for enhancing soil retention of agrochemicals and environmental contaminants such as nutrients,9 heavy metals,10 and pesticides.11 (The interested reader can consult recent reviews for more detailed information on biochar production and uses.7,8,12−14) When incorporated into soils, biochar can significantly alter soil properties known to influence microbial transport including soil structure and specific surface area, soil and solution pH, solution ionic strength and composition, and soil and solution organic matter content.9,15,16 Bolster and Abit16 recently tested the efficacy of poultry litter biochar pyrolyzed at two temperatures for reducing E. coli transport through a water-saturated sandy soil. They observed that the

The land application of animal and human fecal material poses a potential public health risk if humans become exposed to fecal-borne pathogenic microorganisms such as bacteria, viruses, and protozoa. One potential route of transmission for these pathogenic microorganisms is through drinking fecally contaminated groundwater. The land application of fecal material can lead to groundwater contamination when pathogenic microorganisms leach downward through the soil profile with irrigation or rainfall water.1−4 A significant number of waterborne disease outbreaks have been associated with drinking contaminated groundwater,4 and in many cases animal manure has been identified as the likely source of the outbreak.2 The transport of pathogenic microorganisms through soils and aquifer materials is controlled by soil physical and chemical properties including soil structure and texture, percent water saturation, pore-size distribution, soil solution ionic strength and composition, soil and solution pH, soil surface charge, and the concentration of organic carbon in solution and on the sediment phase.5,6 Implementing best management practices that alter one or more of these properties, therefore, has the potential for reducing groundwater contamination by pathogenic microorganisms following land application of manure or biosolids. © 2012 American Chemical Society

Received: Revised: Accepted: Published: 8097

February 27, 2012 June 4, 2012 June 28, 2012 June 28, 2012 dx.doi.org/10.1021/es300797z | Environ. Sci. Technol. 2012, 46, 8097−8105

Environmental Science & Technology



biochar increased, decreased, or had no effect on the transport behavior of three different E. coli isolates depending on biochar pyrolysis temperature and surface properties of the E. coli isolate. While this study demonstrated the potential of using biochar amendments as a method for reducing microbial movement through soils, if soil incorporation of biochar is to be seriously considered as a management practice for protecting shallow groundwater supplies from pathogenic microorganisms, further research is required to understand the factors controlling bacterial transport through biochar-amended soils and under what conditions biochar is most effective at limiting bacterial transport. The impact of biochar addition on soil and soil solution properties, which in turn is expected to affect microbial transport behavior, is largely dependent on the composition and the physical and chemical properties of the biochar. Biochar properties are in large part controlled by the biochar feedstock source and the temperature at which the biochar is produced.17 The physical properties of biochar, including poresize distribution and surface area, generally reflect the structure of the original feedstock.17 Moreover, the chemical composition of the feedstock will strongly influence the physical and chemical properties of the biochar.18 Pyrolysis temperature also determines the chemical and physical properties of the biochar including pH, surface area, and surface charge.10,19−21 Studies have shown that both biochar feedstock and biochar pyrolysis temperature can affect retention of contaminants such as pesticides and heavy metals in biochar-amended soils.10,22 While pyrolysis temperature has been shown to affect E. coli transport behavior,16 it is unknown whether biochar feedstock significantly affects bacterial transport through biocharamended soils. In the study of Bolster and Abit,16 the authors limited their investigation to water-saturated conditions. Because soil−water content plays an important role in bacterial transport through porous media,23 it will likely have a substantial role in bacterial transport through biochar-amended soils. For instance, retention of bacteria under water-saturated conditions results from mechanical filtration, attachment onto solid surfaces, and accumulation in a thin layer adjacent to the liquid−solid interface.6 For unsaturated soils, bacterial retention becomes more complicated because of additional contributing processes including partitioning to liquid−gas interfaces,24 capture in solid−liquid−gas interfaces,25 and storage in immobile zones.26 Thus, evaluating how biochar amendments affect bacterial transport under both saturated and unsaturated conditions is essential to improving our understanding of the complex interactions between bacteria and biochar in soils. In this study we compared how biochar produced from two of the most commonly used feedstocks (poultry litter and pine chips) and pyrolyzed at two different temperatures (350 and 700 °C) affects the transport of E. coli through a watersaturated and partially saturated (∼50% of saturation) fine sand when applied at rates of 1 and 2% (w/w). E. coli was chosen as the model bacteria because it is an indicator organism used by the U.S. Environmental Protection Agency for detecting fecal contamination of groundwater.4 Two E. coli isolates that have been shown to respond differently to biochar addition16 were utilized in our study. Our results provide important new insights on the factors controlling bacterial transport through biochar-amended soils.

Article

MATERIALS AND METHODS

Bacteria Preparation. The two strains of E. coli used in this study were isolated from a swine wastewater lagoon located at the Western Kentucky University farm. Isolates SP2BO7 and SP1HO1 differed in surface properties but are of similar sizes.27 The day prior to each column experiment, cultures were prepared by inoculating E. coli cells taken from Eosin Methyl Blue (EMB) Agar plates (BBL, Becton, Dickinson and Company, Sparks, MD) into 10 mL of Luria−Bertani (LB) broth (Fisher Scientific, Pittsburgh, PA) and grown overnight at 37 °C in an incubator. From the overnight culture, new LB broth cultures were grown to midexponential stage after which they were washed and diluted to a concentration of ∼1.0 × 107 colony forming units (CFUs) mL−1 as described in our previous study.16 Bacterial Surface Characterization. The electrophoretic mobility, hydrophobicity, and surface charge density of the E. coli cells suspended in column leachate was measured using previously published methods.28−30 See Supporting Information (SI) for greater details of methods employed. Porous Media Characterization. The soil utilized in this study was collected from the top 15 cm of a Crevasse soil (a mixed, thermic Typic Udipsamment). The soil material was airdried, thoroughly mixed and passed through a 2 mm sieve. The soil had a particle size distribution of 95.5% sand, 4.5% silt, and 0.5% clay as determined by the hydrometer method with median particle diameter of ∼311 μm. The biochars tested in this study were produced from two feedstockspine chip and poultry litterand were pyrolyzed at either 350 or 700 °C. Each biochar was mixed with the soil at rates of 0, 1, and 2% (w/w), placed in cylindrical glass jars and homogenized on a roller mixer for 3 days. The eight soil-biochar mixtures (1 and 2% biochar) along with the unamended soil are hereafter collectively called “porous materials”. Filtered extracts of porous materials using 1 mM KCl were analyzed for Al, Ca, Fe, Mg, and Na using inductively coupled plasma-optical emissions spectroscopy (Vista Pro, Varian Inc., Palo Alto, CA); SO4−S, PO4−P, and Cl− by ionic chromatography (ICS 3000, Dionex Corp., Sunnyvale, CA); and dissolved organic carbon (DOC) by loss on ignition (LiquiTOC, Elementar Americas Inc., Mt. Laurel, NJ). The electrophoretic mobility and surface charge density of the porous materials in 1 mM KCl was measured using same methods employed for bacterial surface characterizations. 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) packed to a depth of 10 cm. Prior to each experiment approximately 15 pore volumes of 1 mM KCl electrolyte solution was pumped through each column with the last two pore volumes of effluent being collected for analysis as background solution. 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 water quality parameters measured in the extracts of the porous materials. See SI for more detailed descriptions of column setup and operation. Bacterial Transport Experiments. The bacterial suspension containing ∼1 × 107 CFUs mL−1 of one of the E. coli isolates in 1 mM KCl solution was applied to the columns. For 8098

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Table 1. Selected Characteristics of Packed Fine Sand and Soil-biochar Mixtures Made by Mixing 1% or 2% of Poultry Litter or Pine Chip Biochars Produced at Two Temperatures (350 and 700 °C) and Background Effluent through Various Columns biochar treatment poultry litter

pine chip

350 °C 0%

700 °C

350 °C

700 °C

1%

2%

1%

2%

1%

2%

1%

2%

Porous Material Characteristics bulk density 1.59aa pH 6.94fg total organic carbon, % 0.02d acidityb, meq kg−1 16.2bc zeta potentialb, mV −51.3b Background Effluent Characteristics

1.58a 7.93 cd 0.16d −c −

1.57a 8.11c 0.49c 21.6b −44.0a

1.57a 9.04b 0.49c − −

1.58a 10.00a 1.03b 50.7a −44.5a

1.58a 6.94fg 0.57c − −

1.53b 6.60 g 1.43a 11.8c −43.0a

1.6a 7.28ef 0.53c − −

1.52b 7.58de 1.58a 13.8c −43.0a

pH SpC, mS cm−1 DOC, mg L−1 PO4−P mg L−1

6.74d 0.15c 3.05a ndd

7.42bc 0.19bc 6.98a 2.93abc

7.62b 0.26a 5.95a 4.44ab

8.63a 0.25a 3.39a 7.35a

6.84d 0.16c 4.54a 0.08b

6.78d 0.15c 3.73a nd

6.98 cd 0.16c 4.65a 0.08b

7.17bcd 0.16c 3.67a nd

pH SpC, mS cm−1 DOC, mg L−1 PO4−P mg L−1

6.95e 0.15c 4.34a nd

7.52bcd 0.22ab 9.86a 4.05c

7.55bc 0.26a 7.75a 6.86b

8.67a 0.26ab 5.20a 12.3a

6.92e 0.16c 6.95a nd

6.78e 0.16c 6.53a nd

7.01de 0.16c 9.92a nd

7.14cde 0.16c 8.37a nd

Saturated 8.23a 0.19b 5.14a 3.24ab Unsaturated 8.04b 0.22b 7.30a 4.37c

a

Mean values in each row followed by the same lowercase letters are not significantly different using Tukey’s Honestly Significant Difference test at p < 0.05. bAcidity and Zeta Potential of porous materials determined in KCl. cNot measured for 1% biochar application rates. dnd: non detectable.

measured parameters. Mean separations were performed using Tukey’s HSD test. All statistical analyses were performed using JPM ver 7.031 and differences were considered significant at p < 0.05.

the saturated columns, the bacterial suspension was applied at a rate of 2.67 mL min−1 for 30 min followed by bacteria-free KCl at the same rate for 105 min. To the unsaturated columns, the bacterial suspension and the 1 mM KCl leaching solution were applied at a rate of 1.33 mL min−1 for the same durations as the saturated experiments. Pore-water velocity for both saturated and unsaturated conditions was ∼0.31 cm min−1. Effluent was collected at 3.75-min intervals (roughly 10 and 5 mL effluent from the saturated and unsaturated columns, respectively) using a Spectra/Chrom CF-1 fraction collector (Spectrum Chromatography, Houston, TX). One mL was drawn from predetermined effluent samples to enumerate the concentration of E. coli in the effluent solution. Samples were diluted to 100 to 10−4and plated on mFC agar (Difco Laboratories Inc., Detroit MI) plates using the drop-plate technique (two replicates of four 10 μL drops per sample). Colony-forming-units were counted after the plates were incubated overnight at 37 °C. Bacterial concentrations in effluent were converted to normalized concentrations (C/C0), computed as the ratio of measured effluent concentration (C) and the measured influent concentration (C0). Separate columns were packed for each isolate, biochar, and water content combination with each treatment repeated on separate days to ensure true replication. Following completion of the transport experiments, the columns were dissected in 1 cm sections. Extraction and enumeration of bacteria recovered from each 1 cm section was performed as in our previous study.16 Concentrations of bacteria removed during the column dissections were normalized to the initial bacterial concentrations in the influent. Assessment of bacterial survival was conducted concurrent to each transport experiment as described in the SI. Bacterial batch sorption experiments were also conducted for each treatment and are also described in the SI. Data Analysis. One-way analysis of variance (ANOVA) was performed to identify statistically significant differences in



RESULTS AND DISCUSSION Soil, Effluent, and Bacterial Surface Characterization. Several of the measured soil properties were modified significantly by the biochar amendments (Table 1). With the exception of the low temperature (350 °C) pine chip (LTPC) biochar, addition of biochar increased the pH of the porous materials. Amendment with poultry litter biochars resulted in higher pH than did addition of pine chip biochars. In addition, soil amended with the high temperature biochars generally had greater pH than soil with low temperature biochars prepared from the same feed stock. Biochar additions led to at least an 8fold increase in total organic carbon (TOC). Addition of high temperature (700 °C) poultry litter (HTPL) and pine chip biochars led to higher increases in TOC than did low temperature poultry litter biochar (LTPL) amendments, with higher TOC observed for the 2% amendment rates (Table 1). Increases in soil pH and TOC following biochar application are commonly reported.9,16 Biochar addition had significant effects on the pH and composition of background effluent wherein observed trends were generally similar for both saturated and unsaturated conditions (Table 1). Trends in effluent pH agreed with trends in pH of the porous materials. Poultry litter biochar addition generally increased SpC of the effluent, with higher SpC for the 2% than the 1% treatments. Addition of pine chip biochars, on the other hand, did not result in significant changes in effluent SpC. No significant difference in DOC due to biochar amendment was observed. Incorporation of poultry litter biochar generally increased PO4−P in solution, whereas pine chip biochar had a very minimal or no discernible effect on 8099

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Figure 1. Normalized effluent concentrations for E. coli isolates SP2BO7 under saturated (A) and unsaturated (B) conditions and SP1H01 under saturated (C) and unsaturated (D) conditions. Columns were packed with fine sand soil and soil-biochar mixtures made by mixing 1% or 2% of poultry litter (open symbols) or pine chip (closed symbols) biochars produced at two temperatures (LT-350 °C and HT-700 °C).

PO4−P in solution. Changes in solution pH, DOC, and PO4−P concentrations have been reported elsewhere following biochar application to soils.16,32,33 The zeta potential for both isolates became less negative when suspended in solution collected from columns with biochar compared with the biochar-free soil column, though differences in zeta potential between the biochar-free soil and the biochar-added soils were only statistically significant for SP2B07 (SI Table S1). No statistically significant changes in surface charge density, as measured by NaOH titration, were observed for either isolate due to biochar amendments. No large differences in hydrophobicity among biochar treatments were observed for SP2B07. For SP1H01 a statistically significant increase in hydrophobicity was observed for cells suspended in the effluent from the poultry litter biochar amended soil whereas no significant difference in hydrophobicity was observed for cells suspended in effluent from the pine chip biochar-amended soils (SI Table S1). Relating Soil, Effluent, and Bacterial Surface Properties to Bacterial Transport. For the majority of treatments, the addition of biochar to the sandy soil resulted in significant changes in peak C/Co and fractional recovery (fr) values for both isolates, with the effects more pronounced in the unsaturated soil (Figure 1; Table 2). While applied biochars resulted in changes in soil, effluent, and bacterial surface properties, none of these observed changes can fully account for the observed trends in transport behavior of the two E. coli isolates. For instance, both poultry litter biochars raised

solution pH by 1−2 pH units, which is expected to increase the negative charge on the soil leading to enhanced bacterial transport due to greater electrostatic repulsion between the soil and bacteria.34 The 2% application of both poultry litter biochars also resulted in a near doubling of the solution ionic strength as measured by SpC, which in contrast, should lower the repulsive forces between the bacteria and negatively charged soil particles leading to reductions in bacterial transport.5,6 Addition of the pine chip biochars, on the other hand, had no effect on SpC and minimal effect on solution pH. However, measured zeta potentials for both the bacteria and the porous materials were similar for both biochar treatments. Biochar addition resulted in less negative zeta potentials for the bacteria (significant reductions only for SP2B07) and the 2% biochar-amended soils which would be expected to enhance bacterial retention due to reduced electrostatic repulsion between the soil and bacteria. While reductions in fr were observed for the 2% pine chip biochar and high temperature poultry litter treatments, decreases in fr were not observed with the low temperature poultry litter treatments. Moreover, the magnitude in changes in zeta potentials does not correlate with the magnitude in changes in fr. The lack of correlation between fr and zeta potential may be because zeta potential is a macroscopic parameter and is not sensitive to small-scale charge heterogeneities.30 Fractional recovery is also expected to increase with increasing concentration of PO4−P due to competition by phosphates for bacterial deposition sites.35 Addition of HTPL 8100

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Table 2. Percent of Total Bacteria Recovered in Effluent (fr), Percent of Total Cells Extracted (Ex) from Soil Following Column Dissections, and the Percent of Total Cells that were Unaccounted for and Assumed to be Unextractable (UEx) from Soil Following Column Dissections biochar treatment poultry litter 350 °C 0%

1%

pine chips 700 °C

2%

1%

350 °C 2%

1%

700 °C 2%

1%

2%

SP2BO7 Saturated fr Ex UEx Unsaturated fr Ex UEx Saturated fr Ex UEx Unsaturated fr Ex UEx

81aba 2c 18 cd

96a 1c 3c

88a 2bc 10bc

66bc 2bc 32ab

60c 5bc 35a

66bc 10abc 24abc

48 cd 21abc 31ab

33d 31a 36a

36d 25ab 39a

56b 10e 34ab

83a 8e 9c

46b 32bcd 22bc

47b 11de 41ab

11c 46abc 43ab SP1HO1

39b 26cde 35ab

10c 51ab 38ab

6.2c 57a 36ab

3.4c 50ab 47a

58c 16bc 26e

75ab 3c 22e

90a 5c 5f

61bc 5c 33de

23d 28ab 49c

18d 42a 40 cd

0.8e 12bc 87a

1.3e 26ab 72b

0.02e 16bc 83ab

16bc 48a 36d

31a 45ab 24e

21b 41abc 37d

9 cd 29bcd 62 cd

0.3de 32abcd 68bc

0.6de 40abc 60c

4 × 10−5e 14d 86a

2 × 10−4e 24 cd 76ab

5 × 10−5e 16d 84a

a Mean values in each row followed by the same lowercase letters are not significantly different using Tukey’s Honestly Significant Difference test at p < 0.05.

and LTPL biochars led to comparable increases in PO4−P (Table 1); however, while LTPL additions generally led to higher fr, HTPL addition generally reduced fr (Table 2). The most telling indication why solution properties by themselves cannot explain the observed trends in bacterial transport is the fact that while the pH, SpC, and PO4−P concentrations in effluent from pine chip biochar-amended treatments remained comparable to that from the unamended soil (Table 1), amendment with pine chip biochars resulted in decreases in fr ranging from ∼20% to ∼5.5 orders-of-magnitude (Table 2). A lack of correlation between effluent solution properties and E. coli transport through biochar-amended soil has been previously reported.16 In addition, changes in effluent solution properties resulting from biochar addition to fine sand, did not affect the survival of the bacteria for the duration of the study (data not shown) indicating that observed decreases in fr were not mainly due to adverse changes in culturability of the E. coli. While biochar addition did not significantly affect DOC concentrations in the column effluent, significant increases in soil TOC were observed for most treatments (Table 1). Increasing TOC, particularly in soils with low clay content has been shown to increase sorption of E. coli.36 This could explain why increasing biochar amendment from 1% to 2%, and thereby increasing TOC, generally lowered fr (Table 2). Furthermore, there was a strong negative correlation between TOC and fr (or in some cases log fr) for both isolates under both saturated and unsaturated conditions (r values ranging from −0.85 (p = 0.07) to −0.95 (p = 0.015)) suggesting that attachment of bacteria onto the added organic material (in biochar) indeed contributed to the observed trends in bacterial retention. Depending on treatment, the zeta potential of SP2BO7 ranged from 10- to 18-fold more negative than that of SP1HO1

(SI Table S1). This means a far greater tendency of SP2BO7 to be repelled by the negatively charged solid surface and be transported more effectively than SP1HO1, likely explaining the lower C/C0 and fr of SP1HO1 for nearly all treatments (Figure 1; Table 2). Another possible reason for the consistently lower fr of SP1HO1 compared with SP2BO7 may be the 5- to 10-fold higher hydrophobicity of SP1HO1 than of SP2BO7 (SI Table S1). As shown by a previous study involving 23 bacteria (including two strains of E. coli), the extent of retention of bacteria on a negatively charged surface increases with hydrophobicity of bacterial surfaces.37 For most treatments, biochar amendment to the fine sand enhanced bacterial retention resulting in a decrease in fr. The addition of LTPL, however, generally increased fr, results consistent with our previous study.16 It is not clear why this is the case, though it is possible that there exist some distinct qualities of the TOC or DOC from LTPL which may have resulted in unique modifications of sediment surfaces or changes in bacterial surface properties not measured in our study. Percent Water Saturation and Bacterial Transport. For all treatments, peak C/C0 and fr of both isolates in effluent from unsaturated (50% saturation) columns were noticeably lower than from the corresponding saturated columns, and trends in C/C0 and fr under unsaturated conditions resulting from incorporation of different biochars were generally the same as those under saturated conditions (Figure 1; Table 2). Reducing soil−water content promotes the formation of flow discontinuities or immobile zones where bacteria can be lodged making them inaccessible for further transport.26 Bacteria are also known to partition into liquid−gas interfaces24 and solid− liquid−gas interfaces25 which could further increase their retention. In unsaturated soils, water flow (and hence, also 8101

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whether biochar pore structure affects bacterial retention and transport through soils. Consistent with our earlier study,16 we observed some significant differences in E. coli transport behavior due to differences in biochar pyrolysis temperatures. When comparing the effect of biochars from the same feedstock, soil amendment with biochars pyrolyzed at 700 °C was generally more effective than biochars pyrolyzed 350 °C in reducing fr for the two isolates under both saturated and unsaturated conditions (Table 2). For instance, in pine chip biochar-amended treatments, reductions in fr for SP1H01 ranged from a factor of 3 for the 1% low temperature application compared with a factor of 45 for the 1% high temperature application under saturated conditions (Table 2). At 2% pine chip biochar addition, LTPC reduced fr by 1.8 orders-of-magnitude compared to a 3.4 orders-of-magnitude reduction in fr resulting from the HTPC amendment. Higher pyrolysis temperatures lead to increased microporosity of biochars41 and greater fraction of finer biochar particles17 which are both directly related to higher specific surface areas.17 Indeed, biochars produced at higher temperatures generally have greater specific surface areas.19,21,41 For example, specific surface areas of biochars produced from pine increased from ∼3 to 40 m2 g−1 when pyrolysis temperature was increased from 450 to 750 °C.42 In another study, specific surface area of poultry litter biochar (produced from the same batch of feedstock as the poultry litter biochar used in this experiment) increased from 4 to 51 m2 g−1 when pyrolysis temperature was raised from 350 to 700 °C.19 Given that E. coli has been shown to adhere to biochar surfaces,43,44 this may explain why for both isolates and both feedstocks, there was generally lower fr in soil amended with biochars pyrolyzed at 700 °C compared with those amended with biochars pyrolyzed at 350 °C. Batch Sorption Experiments. Sorption is a surface phenomenon and is generally directly related to specific surface area. Results from batch experiments show that sorption coefficients (kd) for each isolate were generally in qualitative agreement with results from the transport experiments (SI Table S1). That is, treatments with high kd values generally coincided with low fr and vice versa. Indeed, we observed strong and statistically significant (p < 0.001) correlations between kd and fr for SP2B07 and between log kd and fr for SP1H01 (SI Figure S2). Furthermore, kd values for the soils amended with the pine chip biochars were much greater than the soils amended with the poultry litter biochar (SI Table S1), results consistent with the transport experiments. This suggests that sorption-related mechanisms significantly contributed to differences in bacterial recoveries in biochar-amended soils. Column Dissections. Using measured values of total cells introduced into each column, total cells recovered in the effluent (fr), and total cells extracted from the soil during the dissections (Ex), we computed percentage of cells that were unrecovered during the column experiments. This unrecovered fraction represents the cells that were retained in the column but not extracted during the column dissections (UEx). For the majority of columns, the unrecovered fraction was considerably greater than the fraction of cells measured from the column dissections, with no discernible trends in the ratio of recovered to unrecovered cells among treatments (Table 2). Low recovery of the retained bacteria may be explained by one or more of the following: the culturability of the bacteria was adversely affected following attachment to the soil, a significant

bacterial transport) occurs in fewer connected water-filled pores that tend to be more tortuous and in interconnected water films surrounding particles.38 This may have increased the contact frequency between suspended bacteria and the pore wall/solid surfaces enhancing the effect of sorption-causing factors and leading to the more pronounced differences in fr between treatments under unsaturated conditions. Moreover, the fact that the general trends in fr as a result of the different biochar treatments were similar for saturated and unsaturated conditions suggest that the effects of sorption-influencing factors like TOC content and specific surface area of the porous material, and the effect of zeta potential and hydrophobicity of the bacteria are prevalent under both conditions. There were greater decreases in fr for SP1HO1 than SP2BO7 across treatments under unsaturated conditions. This could be at least partially attributed to greater hydrophobicity of SP1HO1 than of SP2BO7 and its effect on partitioning to gas interfaces. Partitioning to gas interfaces is facilitated by cell hydrophobicity. Previous studies involving multiple isolates have shown good correlation between hydrophobicity of bacteria and the accumulation at the air−water interface.39 Influence of Feedstock and Pyrolysis Temperature on Bacterial Transport. The type of feedstock used in biochar preparation significantly affected the observed trends in transport behavior. For both isolates, fr was consistently lower in pine chip biochar treatments than in corresponding poultry litter biochar treatments (Table 2). For instance, under saturated conditions, fr of SP2BO7 from columns with 2% high temperature (700 °C) pine chips biochar (HTPC) was 60% lower than columns with 2% HTPL. Also, fr of SP1HO1 from saturated columns with 2% HTPC was three orders-ofmagnitude lower than that of 2% HTPL. For the unsaturated columns, differences in reductions in fr between the pine chip and poultry litter biochars were even more pronounced. Notably, 2% addition of either HTPC or LTPC biochars proved to be most effective in reducing recovery of both isolates under unsaturated conditions. Biochars produced from different feedstocks have been shown to differ in their retention of dissolved contaminants in biochar-amended soils.22 One possible explanation for our observed differences in E. coli transport is the differences in pore size distribution between biochars produced from the two different feedstocks. Size distribution of internal pores of biochars is affected by the properties of the feedstock. For instance, wood-derived biochar retains the configuration of plant cells and its pores resemble interconnected chambers that usually have diameters of 5−10 μm17 which is the optimum pore size for the retention/ inhabitation of bacteria.17,40 In contrast, biochars from poultry manure are rather amorphous with relatively few larger pore sizes (up to 300 μm).17 In addition to having fewer pores where bacteria could be lodged, the larger internal pores in poultry litter biochar diminishes the likelihood that effective entrapment of the much smaller bacterial cell would occur.40 This could explain the consistently lower fr of E. coli in pine chip biochar-amended treatments than the soil amended with the same rate of the corresponding poultry litter biochar. Indeed, scanning electron microscope (SEM) images of our biochars show significant differences in pore structure between these two biochars (SI Figure S1). Given the strong correlation between the sorption coefficients from the batch experiments and fr values (see discussion below; SI Figure S2), the effect of pore structure, if any, is likely through sorption rather than physical straining mechanisms. Further research is needed to determine 8102

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Figure 2. Normalized concentrations of sediment-associated cells for E. coli isolates SP2BO7 under saturated (A) and unsaturated (B) conditions and SP1H01 under saturated (C) and unsaturated (D) conditions. Columns were packed with fine sand soil and soil-biochar mixtures made by mixing 1% or 2% of poultry litter (open symbols) or pine chip (closed symbols) biochars produced at two temperatures (LT- 350 °C and HT- 700 °C).

such as increasing soil retention of various nutrients and environmental contaminants, thereby potentially limiting their movement in the environment.7−11,16,22 Our results, and those of Bolster and Abit,16 suggest that biochar may also be effective for reduce leaching of bacteria through sandy soils, and that the efficacy of biochar amendments for this purpose will depend on the type of biochar used, moisture content of the soil, biochar application rates, and the surface properties of the bacteria. Because biochar addition to soils may result in increased soil concentrations of bacteria near the surface, the potential exists that biochar amendments may result in higher concentrations of bacteria in surface runoff and also potentially increase exposure to grazing animals. Continued research is needed to better understand the factors controlling microbial transport through biochar-amended soils as well as investigate whether biochar amendments affect runoff potential of microorganisms.

fraction of the retained bacteria was too strongly attached to the soil to become dislodged during the extraction process, or experimental error. The measured distributions of soil extractable E.coli with column length are presented in Figure 2. For saturated conditions, the concentrations of SP2B07 for the pine chip biochar treatments generally exceeded those for the biocharfree soil by a factor of 5 or greater throughout the column, whereas the concentrations for the poultry litter treatments were generally similar to the biochar-free soil (Figure 2A). For unsaturated conditions, soil concentrations of SP2B07 were also generally greater than the control or poultry litter treatments, though the differences were not as great as with the saturated soil (Figure 2B). For SP1H01, the treatments with the greatest overall soil retention (i.e., lowest effluent recoveries) did not have the highest concentrations of sorbed bacteria at depths greater than a few cm (Figure 2C and D). This is not unexpected given that high rates of bacterial attachment lead to reduced concentrations of bacteria in solution thereby leading to lower concentrations of sorbed bacteria downgradient from the column inlet. Environmental Implications. Given the high carbon content and recalcitrant nature of biochar, incorporation of biochar into soils has been viewed as a viable means for carbon sequestration to help address rising CO2 concentrations in the atmosphere.7,8,45 Recent studies have also demonstrated environmental benefits following biochar applications to soils



ASSOCIATED CONTENT

S Supporting Information *

Details of the procedures involved in the bacterial surface characterizations, column preparations, batch experiments, and the survival experiments. A table containing surface properties and sorption coefficients, and two figures in support of the discussion are also included. This material is available free of charge via the Internet at http://pubs.acs.org. 8103

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AUTHOR INFORMATION

Corresponding Author

*Phone: 270-781-2260; fax: 270-781-7994; e-mail: carl. [email protected]. Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS We thank Keri Cantrell for providing the biochar and Stacy Antle for assistance with the transport studies. This research was part of USDA-ARS National Program 206: Manure and Byproduct Utilization. The authors declare no competing financial interest. Mention of a trade name, proprietary product, or vendor is for information only and does not guarantee or warrant the product by the USDA and does not imply its approval to the exclusion of other products or vendors that may also be suitable.



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