Inactivation of E. coli O157:H7 in Cultivable Soil by Fast and Slow Pyrolysis-Generated Biochar

Abstract

An exploratory study was performed to determine the influence of fast pyrolysis (FP) and slow pyrolysis (SP) biochars on enterohemorrhagic Escherichia coli O157:H7 (EHEC) in soil. Soil + EHEC (inoculated at 7 log colony-forming units [CFU]/g of soil) + 1 of 12 types of biochar (10% total weight:weight in soil) was stored at 22°C and sampled for 8 weeks. FP switchgrass and FP horse litter biochars inactivated 2.8 and 2.1 log CFU/g more EHEC than no-biochar soils by day 14. EHEC was undetectable by surface plating at weeks 4 and 5 in standard FP switchgrass, FP oak, and FP switchgrass pellet biochars. Conversely, EHEC populations in no-biochar control samples remained as high as 5.8 and 4.0 log CFU/g at weeks 4 and 5, respectively. Additionally, three more SP hardwood pellet biochars (generated at 500°C for 1 h, or 2 h, or generated at 700°C for 30 min) inactivated greater numbers of EHEC than did the no-biochar control samples during weeks 4 and 5. These results suggest that biochar can inactivate E. coli O157:H7 in cultivable soil, which might mitigate risks associated with EHEC contamination on fresh produce.

Introduction

Fresh produce contamination has led to foodborne illness outbreaks; hence, there is need for effective mitigation strategies to minimize cross-contamination associated with soil (Gagliardi and Karns, 2002; Jiang et al., 2002; Solomon et al., 2002; Cooley et al., 2003; Islam et al., 2004a; Islam et al., 2004b; Sivapalasingam et al., 2004; Côté and Quessy, 2005; Islam et al., 2005; Johannessen et al., 2005; Jiang and Shepherd, 2009; Sharma et al., 2009; VanderZaag et al., 2010; Batz et al., 2011; Lynch et al., 2011; Monaghan and Hutchinson, 2012; Harris et al., 2013).

Various interventions, including antimicrobial application, methyl bromide fumigation, polyethylene tarp solarization, and field steaming have been investigated for eliminating pathogens in soil (Marshall et al., 2013). Drawbacks to these methods include equipment overhead, expenses related to antimicrobials and fumigants, maintenance and labor costs accompanying field tarping, harm to beneficial soil microorganisms, and large carbon footprints or environmental damage.

Another strategy to reduce pathogens in cultivable soil is application of biochar. Biochar is a form of black carbon, generated by pyrolysis of biomass (i.e., heating under anaerobic or low-oxygen conditions), resulting in incomplete combustion. Typical biochar is a very fine, highly porous material, 200–1000 μm in diameter. Benefits of biochar include (1) enhancing plant nutrient uptake and crop yield (Iswaran, 1980; Kishimoto and Sugiura, 1985; Glaser, 2002; Oguntunde et al., 2004; Tagoe et al., 2008; Noguera et al., 2010; Zhang et al., 2010; Haefele et al., 2011); (2) increasing water retention in sandy soils, reducing nutrient leaching and runoff leading to water hypoxia and eutrophication (Glaser et al., 2002); (3) reducing carbon emissions as a carbon sequestration sink (Lehmann et al., 2006; Lehmann, 2007a,b; Laird, 2008; Liang et al., 2008; Woolf et al., 2010); (4) producing bio-oils and syngas via fast pyrolysis (Demirbas et al., 2001; Boateng et al., 2007 and 2010; Granatstein et al., 2009); (5) improving soil microbiota (Perez-Piqueres et al., 2006; Warnock et al., 2007; O'Neill et al., 2009; Ball et al., 2010; Atkinson et al., 2010; Warnock et al., 2010; Anderson et al., 2011; Khodadad et al., 2011; Lehmann et al., 2011; Zimmerman et al., 2011); and (6) remediating contaminated soils by binding heavy metals (Toles et al., 1997; Toles and Marshall, 2002; Mohan et al., 2007; Lima et al., 2009; Lima et al., 2010; Beesley and Marmiroli, 2011; Novak and Busscher, 2011; Uchimiya et al., 2011; Ippolito et al., 2012).

Regarding carbon emissions, biochar binds biomass carbon into an environmentally stable form, as found in 2-millennia-old South American Amazonian Dark Earth (terra preta) resulting from slash-and-burn practices to enhance soil fertility (Glaser et al., 2001; Lehmannn et al., 2003; O'Neill et al., 2009). Arable land, in a world of over 7 billion people, is becoming less available; therefore, remediating poor soils via biochar may become an international priority, as has been suggested (Spokas et al., 2012a).

Kolton et al. (2011) reported that biochar enhanced numbers of chitin and cellulose-degrading bacteria, as well as beneficial root-associated bacteria in soil; however, the overall proportion of Proteobacteria decreased by 24%. Proteobacteria are a phylum containing many foodborne pathogenic microorganisms such as Salmonella, E. coli O157:H7, Shigella, and Vibrio. Others have noted a decrease in Proteobacteria in biochar-amended soils (Anderson et al., 2011), or have investigated effects of biochar on E. coli transport through soils (Abit et al., 2012; Bolster et al., 2012). Further work is needed to determine effects of biochar on individual pathogens, such as E. coli O157:H7. The objective of this study, therefore, was to determine the influence of 12 types of fast-pyrolysis (FP) or slow-pyrolysis (SP) biochars on the survival of enterohemorrhagic E. coli O157:H7 in autoclaved soil at 22°C over an 8-week period. The hypothesis was that biochar-amended soil would result in more rapid pathogen inactivation than would control soils.

Materials and Methods

Biochar preparation

Twelve types of biochar (Tables 1 and 2) were produced as follows: FP biochars were generated in a bench-scale FP reactor through a nitrogen-flushed fluidized silica bed at 500°C, as described previously (Boateng et al., 2006, 2007, 2010). Residence time of biomass feedstock (ground to ≤2 mm in diameter) was <1 s and biochar was separated from accompanying syngas via cyclone and charcoal catch. SP biochars were generated under conditions listed in Tables 1 and 2 in a standard biochar reactor, along with ambient air, as previously described (Boateng, 2006).

Table 1.

Survival of Escherichia coli O157:H7 (Log Colony-Forming Units/g) Stored in 10% Biochar-Amended Soil at 22°C as Recovered on Selective Modified EMB Agar ^a

	Storage time (weeks)
Biochar soil amendment	0	1	2	4	5	6	7	8
Switchgrass pellets: negative control	—^b	—	—	—	—	—	—	—
Soil negative control	—	—	—	—	—	—	—	—
Soil positive control	^cA 6.34±0.05 A^d	A 6.58±0.08 ABC	A 6.38±0.18 A	B 4.78±0.58 A	C 3.48±0.04A	D 0.00 A++	D 0.00++	B 0.00−−
Fast pyrolysis: Horse litter	A 5.47±0.02 A	A 5.34±0.38 CD	B 4.15±1.01 C	C 0.89±0.89 D	C 0.00 D++	C 0.00 A−+	C 0.00−−	C 0.00−−
Fast pyrolysis: Oak	AB 6.42±0.37 A	A 6.95±0.11 AB	B 5.51±0.47 AB	C 2.42±0.22 C	D 0.00 D++	D 0.00 A++	D 0.00+−	D 0.00−−
Fast pyrolysis: Switchgrass	A 6.15±0.09 A	B 4.88±0.58 D	C 3.23±0.65 C	D 0.00 D++^e	D 0.00 D++	D 0.00 A+−	D 0.00−−	D 0.00−−
Fast pyrolysis: Switchgrass pellets	A 5.82±0.28 A	A 5.88±0.62 ABCD	B 4.46±0.62 BC	C 0.00 D++	C 0.00 D++	C 0.00 A++	C 0.00−+	C 0.00−−
Switchgrass 700°C–30 min	A 6.12±0.23 A	A 6.51±1.10 ABC	A 6.15±0.52 A	B 3.56±0.48 ABC	C 1.64±1.64 BC	D 0.00 A++	D 0.00++	D 0.00++
Mixed hardwood pellets 700°C–30 min	A 6.14±0.23 A	A 6.44±1.06 ABC	A 6.55±0.44 A	B 4.12±0.58 AB	C 2.61±1.01 AB	D 0.00 A++	D 0.00−+	D 0.00−−
Mixed hardwood pellets 500°C–1 h	A 6.03±0.23 A	A 5.65±0.57 BCD	A 5.61±0.03 AB	B 3.55±0.00 ABC	C 1.15±1.15 CD	D 0.00 A++	CD 0.00++	CD 0.00−+
Mixed hardwood pellets 500°C–2 h	A 6.33±0.11 A	A 6.78±0.80 AB	A 6.50±0.73 A	B 3.28±0.37 BC	B 3.29±0.06 A	C 0.80±0.80 A	C 0.00−+	C 0.00−+
Mixed hardwood pellets 600°C–2 h	A 6.30±0.07 A	A 6.59±0.77 ABC	A 6.34±0.69 A	B 4.43±0.58 AB	C 2.97±0.07 AB	D 0.65±0.65 A	D 0.00++	D 0.00++
Switchgrass 500°C–1 h	A 6.16±0.67 A	A 6.96±0.42 AB	A 6.58±0.48 A	B 3.59±0.02 ABC	C 2.32±0.42 ABC	D 0.00 A++	D 0.00++	D 0.00−+
Switchgrass 500°C–2 h	A 6.40±0.28 A	A 7.03±0.39 A	A 6.61±0.41 A	B 4.42±0.49 AB	C 2.56±0.30 AB	D 0.80±0.80 A	D 0.00++	D 0.00−+
Switchgrass 600°C–1 h	A 6.29±0.01 A	A 6.63±1.29 ABC	A 6.58±0.54 A	B 3.96±0.10 AB	C 2.55±0.47 AB	D 0.00 A++	D 0.00++	D 0.00++

Data points are means of observations±standard error of the mean.

Indicates no samples tested positive by either surface plating or by selective enrichment.

Comparison of the effect of time: within the same biochar soil amendment type, values in the same row that are not preceded by the same upper case letter are significantly different (p≤0.05).

Comparison of the effect of biochar soil amendment type: within the same sampling time, values in the same column that are not followed by the same upper case letter are significantly different (p≤0.05).

Values of 0.00 are followed by+or – indicating positive or negative results of selective enrichments for replicate experimental trials.

Table 2.

Survival of Escherichia coli O157:H7 (Log Colony-Forming Units/g) in 10% Biochar-Amended Soil at 22°C, Recovered on Tryptic Soy Agar+0.1% Sodium Pyruvate ^a

	Storage time (weeks)
Biochar soil amendment	0	1	2	4	5	6	7	8
Switchgrass pellets: negative control	—^b	—	—	—	—	—	—	—
Soil negative control	—	—	—	—	—	—	—	—
Soil positive control	^cA 6.84±0.08 A^d	A 6.99±0.16 ABC	A 6.79±0.30 A	A 5.83±0.38 A	B 4.01±0.40 A	C 1.23±1.23 AB	C 0.00++	C 0.00−−
Fast pyrolysis: Horse litter	A 6.04±0.07 A	A 5.70±0.47 BC	A 4.72±0.82 BC	B 1.41±1.41 CDE	B 0.65±0.65 EF	B 0.00 B−+	B 0.00−−	B 0.00−−
Fast pyrolysis: Oak	A 6.87±0.27 AB	A 7.33±0.02 AB	A 6.02±0.31 AB	B 2.69±0.35 BC	C 0.00 F++	C 0.00 B++	C 0.00+−	C 0.00−−
Fast pyrolysis: Switchgrass	A 6.44±0.03 A	A 5.47±0.62 C	B 4.02±1.06 C	C 0.00 E++ ^e	C 0.89±0.89 DEF	C 0.00 B+−	C 0.00−−	C 0.00−−
Fast pyrolysis: Switchgrass pellets	A 6.49±0.25 A	A 6.69±0.24 ABC	A 5.59±0.09 ABC	B 0.80±0.80 DE	B 0.00 F++	B 0.00 B++	B 0.00−+	B 0.00−−
Switchgrass 700°C–30 min	A 6.74±0.18 A	A 7.45±0.28 AB	A 6.90±0.21 A	B 4.75±0.49 A	C 2.64±1.04 ABCD	D 0.00 B++	D 0.00++	D 0.00++
Mixed hardwood pellets 700°C–30 min	A 6.56±0.04 A	A 7.50±0.17 AB	A 6.77±0.35 A	B 4.36±0.80 AB	C 1.97±1.97 BCDE	D 0.00 B++	D 0.00−+	D 0.00−−
Mixed hardwood pellets 500°C–1 h	A 6.57±0.10 A	A 6.32±0.02 ABC	A 6.18±0.04 AB	B 2.16±2.16 CD	BC 1.04±1.04 CDEF	C 0.00 B++	C 0.00++	C 0.00−+
Mixed hardwood pellets 500°C–2 h	A 6.68±0.23 A	A 7.24±0.48 ABC	A 6.92±0.55 A	BC 2.86±2.86 BC	B 3.60±0.36 AB	CD 1.26±1.26 AB	D 0.00−+	D 0.00−+
Mixed hardwood pellets 600°C–2 h	AB 6.60±0.04 A	A 7.33±0.27 AB	AB 6.82±0.27 A	B 5.53±0.27 A	C 3.62±0.15 AB	D 1.60±0.32 AB	E 0.00++	E 0.00++
Switchgrass 500°C–1 h	A 6.74±0.38 A	A 7.35±0.40 AB	A 7.03±0.35 A	B 4.31±0.85 AB	C 2.81±0.91 ABC	C 1.95±0.65 A	D 0.00++	D 0.00−+
Switchgrass 500°C–2 h	AB 6.76±0.19 A	A 7.71±0.21 A	A 7.09±0.33 A	B 5.35±0.03 A	C 3.45±0.47 AB	D 1.78±0.00 AB	E 0.00++	E 0.00−+
Switchgrass 600°C–1 h	AB 6.73±0.02 A	A 7.96±0.12 A	A 7.33±0.14 A	B 5.13±0.02 A	C 3.33±0.17 AB	F 1.99±0.09 A	D 0.00++	D 0.00++

Data points are means of observations±standard error of the mean.

Indicates no samples tested positive by either surface plating or by selective enrichment.

Comparison of the effect of time: Within the same biochar soil amendment type, values in the same row that are not preceded by the same upper case letter are significantly different (p≤0.05).

Comparison of the effect of biochar soil amendment type: Within the same sampling time, values in the same column that are not followed by the same upper case letter are significantly different (p≤0.05).

Values of 0.00 are followed by+or – indicating positive or negative results of selective enrichments for replicate experimental trials.

Determination of total volatile content (VOC) of biochars

VOC was measured following the ASTM D1762-84 method (ASTM International, 2007). Briefly, oven-dried samples (0.5 g) were weighed into a crucible, lidded, and preheated with the furnace door open at 300°C for 2 min, then heated at 500°C for 3 min. Samples were then moved to the rear of the furnace and heated at 950°C for 6 min. Crucibles were cooled to room temperature in desiccators and weighed again. VOC was then calculated as: \documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document} \begin{align*}& \hbox{volatile content} (\%) = \\ & \quad \frac {\rm weight \ loss \ of \ biochar \ after \ heating \ ( g ) } {\rm dry \ mass \ of \ biochar ( g ) } \times 100 \end{align*} \end{document}

Bacterial strain preparation

Two strains of E. coli O157:H7 (ATCC #43894, and isolate #C9490) were inoculated into 50-mL tryptic soy broth+100 ppm nalidixic acid (TSBN) for 24 h at 37°C. The two 50-mL bacterial suspensions were centrifuged for 10 min at 1800×g, and each resuspended with 5 mL of 0.1% peptone water, increasing population densities 10-fold. Both suspensions were then composited.

Soil preparation

Soil was prepared as described previously (Douds et al., 2005) by combining a 0.75:1:0:1:0:0.75 (vol/vol) mixture of autoclave-sterilized soil, sand, vermiculite, and turface (calcined clay, Applied Industrial Materials, Corp., Deerfield, IL) (SSVT) for a final carbon content of 0.6%. SSVT is a material composite representing soils amenable for frequent watering and excellent drainage. SSVT was added to each of 14 hermetically sealed containers (90 g of SSVT in each) along with 10 g (10% total mass) of 1 of 12 SP or FP-generated biochars (Table 1). One container with 90 g of SSVT+10 g of SP switchgrass pellet biochar served as an uninoculated control and one container with 100 g SSVT and no biochar served as a no-biochar control.

Inoculation of soil

The SSVT+biochar treatments in 12 containers, in addition to the negative control, were each inoculated with 1 mL of the composited E. coli O157:H7 suspension. One milliliter of sterile deionized water was added to the uninoculated control sample. All biochar+SSVT treatments, as well as positive and negative controls, were adjusted with sterile deionized water to a final moisture content of 17.6% (66% of total water-holding capacity). Samples were mixed vigorously for 2 min to distribute bacterial inocula into soils. Samples were stored in a microprocessor-controlled incubator for 8 weeks at 22±1°C.

Soil sampling for presence of E. coli O157:H7 by surface plating

Soil was tested for presence and populations of E. coli O157:H7 on day 1 (week 0) as well as at the following storage times: weeks 1, 2, 4, 5, 6, 7, and 8 by cultural sampling methods, as modified from previous studies (Abit et al., 2012; Bolster et al., 2012; Gurtler et al., 2013). At each sampling time, containers were vigorously mixed for 60 s, 1 g was removed from the container, added to 9 mL TSBN, and vortexed thoroughly. One gram was determined to be an adequate sampling size, based on 60 s of mixing dry inoculated soil, thus eliminating problems related to soil/inocula heterogeneity. Serial dilutions in 0.1% peptone were plated onto slightly selective modified eosin methylene blue agar (MEMB) as well as onto tryptic soy agar+100 ppm nalidixic acid+0.1% sodium pyruvate (TSAPN) to assist recovery of injured cells (Gurtler and Beuchat, 2005; Gurtler and Kornacki, 2009). MEMB agar was composed of the following ingredients (per liter of water) added prior to autoclaving: 37.4 g BBL^® eosin methylene blue agar (Difco & BBL Manual, 2009; BD-Difco, Sparks, MD)+0.2 mg crystal violet+0.2 mg brilliant green+2 g ox bile (Difco) +1 g sodium pyruvate. Following autoclaving and cooling to ≤45°C, the following antibiotics were added and mixed thoroughly: 0.2 mg novobiocin and 0.1 g nalidixic acid. Petri plates of MEMB and TSAPN were incubated at 37°C for 24 h prior to counting. The remainder of the diluted soil sample was then incubated overnight at 37°C, as a pre-enrichment.

Selective enrichment for presence of E. coli O157:H7

Soil samples in TSBN testing negative by surface plating were incubated overnight at 37°C as a pre-enrichment, vortexed thoroughly, and 1 mL added into 9 mL of the selective enrichment medium, TSB-select (described in Gurtler et al., 2013). Selective enrichments were incubated for 24 h at 37°C, vortexed thoroughly, and 10 μL was streaked onto selective tc-SMAC-PN agar (i.e., tellurite, cefixime, sodium pyrurate, nalidixic acid–sorbitol MacConkey agar) for presumptive-positive confirmation. Tc-SMAC-PN agar was composed of the following ingredients (per liter of water) added prior to autoclaving: 50 g of sorbitol MacConkey II agar (BD Difco)+1 g (0.1%) sodium pyruvate. Following autoclaving and cooling to ≤45°C, the following antibiotics were added and mixed thoroughly: 0.1 g (100 ppm) nalidixic acid+rehydrated Difco cefixime and tellurite supplement (tc-SMAC supplement, BD Difco), composed of 0.05 mg (0.00005%) cefixime and 2.5 mg (0.00025%) potassium tellurite. Tc-SMAC-PN plates were incubated at 37°C for 24 h, and typical colonies were confirmed by serological agglutination (Remel RIM™ E. coli O157:H7 latex test).

Statistical analysis

Raw data were log₁₀-transformed prior to statistical analysis, performed with the MIXED procedure of the SAS Software package, Release 9.2 (Statistical Analysis System, SAS Institute). Data from a completely randomized design of two independent research trials were analyzed using a repeated-measures analysis to examine significance of main effects and interaction of treatment and time. Significant differences (p<0.05) among interaction means were determined by Fisher's protected least significant difference (LSD) technique and the slice option of the MIXED procedure.

Results

Recovery comparison of E. coli O157:H7 on selective MEMB agar versus TSAPN

All samples were inoculated with approximately 7 log colony-forming units (CFU)/g of soil on day 0. E. coli O157:H7 cells, recovered from soil/biochar treatments, were assessed for recovery on selective MEMB and TSAPN (Tables 1 and 2). Statistical analysis for main effects revealed that more viable cells were recovered on TSAPN than MEMB (p<0.0001). The mean difference between the two media was 0.40 log CFU/g and injury generally ranged from 0.3 to 1.3 log CFU/g, as determined by differences between populations recovered on TSAPN and MEMB. MEMB was included as a slightly selective medium in the event that TSAPN was overgrown with contaminants. Nevertheless, all random presumptive-positive E. coli O157:H7 colonies were confirmed on tc-SMAC-PN and via serological agglutination. Because greater numbers of E. coli O157:H7 colonies were recovered on TSAPN than on MEMB, only the TSAPN data will be described below.

Survival of E. coli O157:H7 (EHEC) over time as determined by surface plating on TSAPN

Populations of EHEC in no-biochar, positive-control soil remained statistically unchanged during the first 4 weeks of sampling and declined significantly (p<0.05) at week 5 (Table 2). By contrast, EHEC populations declined significantly (viz., 2.4 log CFU/g) by week 2 in soil amended with FP switchgrass biochar. EHEC populations in all other 11 biochar treatments decreased statistically, relative to controls, by week 4. Inactivation of EHEC to levels undetectable by surface plating occurred by week 4 in FP switchgrass biochar and by week 5 in FP switchgrass pellets and in FP oak treatments (Table 2). Conversely, average populations in positive-control no-biochar soil samples remained as high as 5.8 and 4.0 log CFU/g by weeks 4 and 5, respectively. The no-biochar control samples contained mean EHEC populations of 1.23 log CFU/g by week 6. Additionally, three more SP hardwood pellet biochars (generated at 500°C for 1 h, or 2 h, or generated at 700°C for 30 min) inactivated greater numbers of EHEC than did no-biochar control samples during week 4 and/or at week 5 (Table 2).

Survival of E. coli O157:H7 over time, as determined by selective enrichment

Samples testing negative for EHEC by surface plating were selectively enriched to amplify populations. Selective enrichments were required for all week-7 and week-8 treatments. Results indicated that all selective enrichments from FP horse and FP switchgrass samples were negative by week 7, while all positive-control no-biochar soil samples at week 7 tested positive for the presence of EHEC (Tables 1 and 2). By week 8, all positive-control soil samples, one slow pyrolysis treatment (i.e., hardwood, 700°C for 30 min), and all FP treatments were negative for EHEC by enrichment. In contrast, viable EHEC was detected in seven other slow pyrolysis biochars through week 8.

Comparison of the effects of biochars within a given sampling time on survival of E. coli O157:H7

Survival of EHEC within a given sampling time was affected by treatments as early as week 2, when significantly lower numbers (2.8 and 2.1 log reduction, respectively) were recovered from FP switchgrass and FP horse biochar than were recovered from positive-control, no-biochar soil. SP mixed hardwood (600°C for 2 h), as well three slow pyrolysis switchgrass treatments (500°C for 1 h, 500°C for 2 h, and 600°C for 1 h), never inactivated statistically greater numbers of EHEC (p>0.05) at any sampling time (weeks 1–8) than did positive-control, no-biochar soils. Furthermore, EHEC was detected by enrichment in all four of these SP treatments at week 8, while all positive-control, no-biochar treatments tested negative. The same conclusions that were just stated can also be drawn for the mixed hardwood 500°C for 2-h treatment, with one exception (week 4), where fewer cells were recovered than from the no-biochar positive-control soil. Overall, most statistical differences in populations of EHEC among varying biochar treatments were attributable to the FP biochars (Table 2).

Analysis of total VOC of biochars

The mean percentage of total VOCs for FP biochar samples was 27.10%, in comparison to 19.57% in SP biochar samples (Table 3). A direct and proportional correlation between VOC content and bacterial inactivation, however, cannot be drawn. For example, mixed hardwood pellets (500°C for 1 and 2 h), with higher VOC than FP horse litter, was, nevertheless, less efficient at reducing populations of EHEC (p<0.05), relative to FP horse litter at weeks 4 and 5. Furthermore, at week 8, no EHEC was detected by selective enrichment of FP horse-litter samples, although EHEC was still detected at week 8 in mixed hardwood pellets (500°C for 1 and 2 h).

Table 3.

Percent Mass of Total Volatile Content of Biochar

Biochar soil amendment	(% Mass) ^a
Fast pyrolysis: Horse litter	20.03±0.09
Fast pyrolysis: Oak	30.86±2.77
Fast pyrolysis: Switchgrass	34.05±0.28
Fast pyrolysis: Switchgrass pellets	23.45±1.33
Switchgrass 700°C–30 min	16.19±0.45
Mixed hardwood pellets 700°C–30 min	20.49±0.30
Mixed hardwood pellets 500°C–1 h	25.55±1.04
Mixed hardwood pellets 500°C–2 h	26.38±0.07
Mixed hardwood pellets 600°C–2 h	14.18±0.35
Switchgrass 500°C–1 h	21.55±0.89
Switchgrass 500°C–2 h	15.39±1.36
Switchgrass 600°C–1 h	16.82±2.19

Data points are means of observations.

Discussion and Conclusions

Our use of autoclaved soil was recently published in a leek internalization study (Gurtler, 2013). Spokas et al. (2012a) reviewed 46 studies in which investigators tested laboratory, greenhouse, or field application of biochar for response by vegetable crops at biochar concentrations ranging from 0.5% to 30%, by weight. Commensurately, use of 10% no-bichar control in soil in the present study was chosen to determine antimicrobial efficacy at high concentrations.

Our results reflect that soils with FP biochars, and some SP biochars (viz., SP hardwood pellets and SP switchgrass, both generated at 700°C for 30 min, as well as SP hardwood pellets, 500°C for 1 h) are capable of increasing inactivation of E. coli O157:H7, when compared with unamended soil at 22°C, for up to 7 weeks. A general trend was that greater inactivation of EHEC occurred when biochar was generated by shorter reactor residence times, as well as at lower temperatures. For example, the order of decreasing antimicrobial efficacy for all FP and three SP biochars was as follows: all FP biochars (reactor times of <1 s)>SP hardwood (500°C for 1 h)>SP switchgrass (700°C for 30 min)>SP switchgrass (600°C for 2 h).

Our results demonstrate that within 2 weeks, soil supplemented with FP switchgrass biochar underwent a 2.4 log CFU/g reduction of EHEC, which was 2.8 CFU log lower than EHEC populations in no-biochar soil at the same sampling time. Populations for FP switchgrass were also undetectable by surface plating at week 4, while 50% of selective enrichments were negative for the pathogen by week 6. The application of biochar could serve as a sustainable means to prepare field soil for crop production and mitigate contamination of fresh produce. Further work could address effects of FP biochar on survival of plant pathogens (molds and bacteria) as well as Salmonella and Listeria.

The fact that four SP biochar treatments sustained EHEC numbers 0.34–0.76 log CFU/g higher than populations in control soil (1.23 log) at week 6 may suggest a neutral effect on pathogen longevity. Treatments with neutral effects on pathogen survival were SP mixed hardwood (600°C for 2 h) and three SP switchgrass treatments (500°C for 1 h, 500°C for 2 h, and 600°C for 1 h). While populations within these treatments did not statistically differ from numbers surviving within control soil, all four of these biochars tested positive for EHEC by enrichment at week 8, while all control no-biochar soil samples tested negative at week 8. Further investigations may examine factors contributing to bacterial survival, such as biochar byproducts utilized as metabolic substrates or compatible solutes that could protect the cell membrane, or even biochar surface area and porosity, which could provide protection for entrapped cells.

Many volatile compounds are known to be strong antimicrobials (Elgaali et al., 2002; Tzortzakis, 2007; Singh, 2011; Ćavar et al., 2013; Jesionek et al., 2013; Zheng et al., 2013). The fact that a combination of shorter biochar reactor residence times+lower temperatures resulted in a general trend of greater EHEC inactivation could possibly relate to biochar volatiles. It is well established that lengthened reactor times and higher temperatures decrease the concentration and types of volatile compounds within char (Apayadin-Varol et al., 2007; Pütün et al., 2007; Boateng, 2010; Spokas et al., 2012a, 2012b). For this reason, chars generated via FP are typically higher in many volatiles than are SP chars, produced from the same feedstock. While it was beyond the scope of the present experiment, further research should correlate pathogen reduction with concentrations of identified volatile species in the biochar. Other variables that should be addressed include minimum biochar amendment levels necessary to inactivate pathogens; effects of varying moisture content, storage temperature, soil composition, and pH on pathogen inactivation; length of time biochar retains its antimicrobial properties following application to soil under natural field conditions; effects of various types of biochar feedstocks (e.g., peanut hulls, rice hulls, grass clippings, leaves, corn stover, pine wood or bark, paper sludge, sugarcane bagasse, straw, nut shells, hog manure, poultry litter, etc.) on pathogen inactivation; and response of various foodborne pathogens to biochar, including Salmonella, Shigella, Listeria, foodborne viruses, etc.

This is the first study to report inactivation of E. coli O157:H7 in soil. This work may be useful for designing future soil amendments to reduce the risk of cross-contamination events associated with higher-risk fruits and vegetables, such as leafy greens, melons, green onions, and tomatoes.

Footnotes

Acknowledgments

The authors thank John Phillips for help with statistical analysis, Rebecca Bailey for laboratory assistance, and Modesto Olanya and Nasir Malik for internal reviews.

Disclosure Statement

No competing financial interests exist.

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