Quantitative Effects of Biochar Oxidation and Pyrolysis Temperature

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Quantitative Effects of Biochar Oxidation and Pyrolysis Temperature on the Transport of Pathogenic and Nonpathogenic E. coli in Biochar-Amended Sand Columns Waled Omar Suliman, James B. Harsh, Ann-Marie Fortuna, Manuel Garcia-Perez, and Nehal I Abu-Lail Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b04535 • Publication Date (Web): 30 Mar 2017 Downloaded from http://pubs.acs.org on March 31, 2017

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Environmental Science & Technology

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Quantitative Effects of Biochar Oxidation and Pyrolysis

2

Temperature on the Transport of Pathogenic and Nonpathogenic E.

3

coli in Biochar-Amended Sand Columns

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Waled Suliman1,2, James B. Harsh2, Ann-Marie Fortuna4, Manuel Garcia-Pérez3, and Nehal I.

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Abu-Lail5,* 1

7 8

2

Department of Crop and Soil Sciences, Washington State University, Pullman, WA 99164, USA 3

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Department of Microbiology, Benghazi University, Benghazi, LYB2186, Libya

Biological Systems Engineering Department, Washington State University, Pullman, WA, 99164, USA

10 4

11 12

5

Soil Science Department, North Dakota State University, Fargo, ND, 58108, USA

The Gene and Linda Voiland School of Chemical Engineering and Bioengineering, Washington State University, Pullman, WA, 99164, USA

13 14 15 16 17

Corresponding author

18

*

19

1505 Stadium Way

20

313 Wegner Hall

21

Gene and Linda Voiland School of Chemical Engineering and Bioengineering

22

Washington State University

23

Pullman, WA 99164-6515

24

Tel: 509-335-4961

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[email protected]

Nehal I. Abu-Lail

1 ACS Paragon Plus Environment


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Abstract

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The present study quantifies the transport of Escherichia coli pathogenic O157:H7

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and nonpathogenic K12 strains in water-saturated Quincy sand (QS) columns amended with

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oxidized (OX) or unoxidized (UO) pine wood (PW) or pine bark (PB) biochar produced at either

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350oC or 600oC. Our results showed that: (1) the addition of oxidized biochar into QS columns

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enhanced the transport of E. coli O157:H7 by 3.1 fold compared to the unoxidized counterparts

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likely due to an increase in the repulsive forces due to their higher negative charge densities;

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(2) the retention of E. coli O157:H7 was 3.3 fold higher than that of E. coli K12 in all biochar-

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amended sand columns; and (3) increased application rates of unoxidized PW600 biochar from 0

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to 20wt.% led to a reduction in the transport of E. coli O157:H7 and K12 from 98 to 10% and

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from 95 to 70%, respectively. Our data showed that mixing sand with PW350-UO at a 20 wt.%

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application rate almost completely retained the pathogenic E. coli in the subsurface; suggesting

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that utilizing sand mixed with biochar can act as a promising biofilter capable of protecting

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natural aquafers from pathogens.

40 41 42 43 44 45 46 47 48


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1. Introduction

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The transport of pathogenic bacteria through soil represents a scientific and public concern due

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to their potential to cause groundwater contamination and outbreaks of waterborne diseases1. E.

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coli O157:H7, a Gram-negative, facultative anaerobic, enteric bacterium normally excreted in

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human and animal feces2,3, is a recognized pathogen capable of causing a wide range of diseases

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in humans3,4. In the US annually, E. coli O157:H7 causes 75,000 infections and 61 deaths5,6. In

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addition, E. coli O157:H7 is responsible for massive disease outbreaks such as the tragedy that

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occurred in Walkerton, Canada and that resulted in infections to nearly half of the 5,000 people

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living there due to contamination of drinking groundwater with E. coli O157:H77. Due to their

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abundance, exploring means to reduce the transport of E. coli O157:H7 in the subsurface is

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needed.

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The modification of the natural filtration capacity of sand is one of the means by which

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the risk of groundwater contamination by pathogenic bacteria can be reduced8. This can be

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achieved by the incorporation of organic amendments such as compost and/or biochar, a carbon-

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rich porous material, into sand. Similarly, but relying on the indigenous nonpathogenic bacterial

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strains in soil, researchers have suggested that biofilms of these strains can clog pores in sand

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and potentially be used to reduce the hydraulic conductivity in soil9. Although this is an

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environmental friendly approach, keeping bacterial viability under subsurface soil stresses such

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as reduced oxygen can be challenging10. Finally, planting and vegetative soil covering have been

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widely used as means to modify the filtration capacity of soil11. This option is viable only in

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locations where plants can grow12. In comparison, in addition to its potential role as a pathogen

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biofilter, biochar can enhance soil fertility and sequester carbon13. The ability of biochar to

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attenuate microbes depends largely on its physiochemical properties including surface area, pore



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volume, pH, volatile matter, oxygen content, and hydrophobic properties14. Biochar

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physiochemical properties are dependent on biochar production conditions as well as on the

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feedstock used to produce the biochar. Specifically, biochar charge and hydrophobicity can be

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largely controlled by manipulating biochar’s production temperature15 as well as post-oxidation

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processes16. In comparison, biochar’s ash content, total carbon, fixed carbon, and mineral

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concentrations are mainly affected by the feedstock properties17,18.

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Recent laboratory investigations of E. coli transport in biochar-amended soils

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documented positive effects of biochar’s ability to increase the attenuation of E. coli through

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modifying soil surface area, hydrophobicity, charge, organic carbon content, porosity, or solution

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chemistry14,19,20. However, most of biochar studies on bacterial transport were conducted with

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raw-heterogeneous biochar grains disregarding the role of biochar surface functionality on

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transport. Knowledge acquired with only bulk properties of fresh raw-biochar cannot be

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transferred easily to natural soil environments, where biochar gets exposed to biotic and abiotic

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oxidation processes. Better fundamental understanding of how biochar physiochemical

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properties affect bacterial transport in biochar-amended sand will enable better design of

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production processes to yield biochar with desired properties for enhanced bacterial attenuation.

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Here, we compared and quantified the transport of a pathogenic and a nonpathogenic E.

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coli strain in sand amended with oxidized and unoxidized biochars produced at two pyrolysis

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temperatures (350 oC and 600 oC) from two commonly used feedstock sources (pine wood and

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pine bark).

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characterized air-oxidized biochar simulating subsurface natural conditions was also

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investigated.

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2. Materials and Methods

The role of biochar’s surface functionality on E. coli transport using well-


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2.1 Bacterial Cultures

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E. coli strains investigated were obtained from Douglas Call, a professor of the Department of

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Veterinary Microbiology and Pathology at Washington State University (WSU). The strains

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were activated by growing for twelve hours at 37˚C on a shaker rotating at 150 rpm in Luria

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Bertani (LB) medium adjusted to a pH value of 7.0 using 2N HCl. Following incubation, 1%

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volume of the activated culture was transferred into 20 ml of LB medium and grown at the

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conditions above until the late stationary phase of growth. Cells were harvested when the

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absorbance at 600 nm (read using a Lambda 25 Perkin Elmer spectrophotometer) reached a value

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of 0.56 ± 0.02. The specific growth constants were found to be 0.018 and 0.014 h-1 for the

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pathogenic and nonpathogenic strains, respectively. After growth, bacterial suspensions were

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centrifuged at 5,100g for 10 minutes. Cell pellets were collected and washed three times with

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deionized water (DIW). Bacterial pellets were then resuspended in DIW to a concentration of

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0.05 optical density at 600 nm (equivalent to 1.2 x 108 CFU/mL). Ionic strengths of solutions in

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environmental systems can vary from < 0.01 M in groundwater (such as that in DIW), to about

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0.1 M (can vary between 0.03 and 0.3 M) in brackish water, to 0.57 M in seawater21. All studies

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done here were performed in DIW as the extreme low end of ionic strengths present in soil.

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2.2 Electrophoretic Mobility Measurements

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Bacterial cells grown as described above were washed twice by centrifugation at 5,100g for 10

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min each round. Collected bacterial pellets were then diluted with 0.2 µm filtered DIW adjusted

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to the pH of interest using either 0.1 M HCl or NaOH until an optical density of ~ 0.05 was

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obtained at λ = 600 nm. Electrophoretic mobilities of E. coli cells were then measured in pH

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values of 1.8, 2.8, 4, 7.7, and 8.5 for E. coli K12 and in pH values of 1.7, 2.1, 3.6, 5.1, 7 and 8

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for E. coli O157:H7. To measure the mobilities, 2 ml of the bacterial cell suspension was



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injected using a syringe into a nano-Zetasizer 3000 HSA (Malvern Instruments Ltd., Malvern,

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UK). All electrophoretic mobility measurements were performed five times at room temperature.

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The five times’ replicates were taken from different cultures grown on the same day.

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2.3 Biochar Preparation and Characterization

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Pine Wood and Pine Bark biochars were produced and characterized at the biomass

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thermochemical conversion laboratory at WSU according to details reported elsewhere15,22 and

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briefly summarized in Table (1). Here, biochars are denoted as PW for the pine wood feedstock

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and PW-350, and PW-600 for the resulting unoxidized (UO) biochars created at 350 oC and 600

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o

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was applied for the pine bark (PB) feedstock batches. For brief descriptions of how biochar was

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prepared and characterized, please refer to the supporting information sections 1 and 2.

C, respectively. The oxidized samples are labeled with an OX. Similar abbreviation procedure

129 130 131 132 133 134 135 136 137 138 139 140


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Table 1. General physicochemical properties of biochars used in this study15,22 PW350

PW600

PB350

PB600

Property UO

OX

UO

OX

UO

OX

UO

OX

Biochar yield (wt. %)

36.0

37.22

16.5

14.96

46.7

41.85

30.9

26.16

Carbon content (wt. %)

70.50

65.37

87.80

85.08

66.07

63.85

78.11

78.07

Oxygen content (wt. %)

23.15

29.17

7.06

10.27

23.67

27.11

8.98

10.21

Nitrogen content (wt. %)

0.26

0.26

0.38

0.32

0.67

0.60

0.75

0.73

Hydrogen content (wt. %)

5.50

4.79

3.63

3.44

4.88

4.49

3.32

3.29

Fixed carbon content (wt. %)

49.58

48.33

83.15

79.27

51.50

48.71

73.89

70.14

Volatile matter content (wt. %)

49.82

51.24

15.72

20.84

42.15

47.33

17.81

21.56

Ash content (wt. %)

0.58

0.42

1.13

0.89

4.72

3.95

8.85

7.70

Mg (mg kg )

0.11

0.17

0.21

0.20

0.16

0.19

0.76

0.19

K (mg kg-1)

0.41

0.40

0.72

0.74

0.61

0.71

2.01

2.20

-1

0.65

0.77

1.18

1.24

1.15

1.44

4.36

4.33

0.16

0.28

0.12

0.23

0.14

0.11

0.17

0.16

Bulk density (g cm )

0.14

0.14

0.24

0.24

0.61

0.61

0.21

0.21

Particle density (g cm-3)

0.56

0.56

0.7

0.7

0.45

0.45

0.48

0.48

71

71

78

78

64

64

68

68

Moisture content (%)

1.69

2.5

1.71

2.6

3.13

3.4

2.22

2.12

pH H2O

8.20

7.8

8.79

8.10

7.88

7.5

10.19

9.00

0.01

0.02

0.07

0.05

0.07

0.06

0.19

0.08

53.96

66.59

52.20

66.90

36.28

56.06

30.37

30.83

3.0

1.2

5.6

3.2

1.6

1.3

4.4

3.7

Total OFGs (atom %)

45.59

49.95

16.79

29.64

57.31

75.54

9.22

23.46

Total AFGs (mmol g-1)

0.15

0.61

0.03

0.13

0.16

0.34

0.02

0.12

0.01

0.00

0.03

0.02

0.04

0.03

0.11

0.11

146

190

500

570

172

187

424

550

0.14

0.23

0.57

0.75

0.20

0.24

0.43

0.81

-1

Ca (mg kg ) -1

Na (mg kg ) -3

Porosity (%)

EC H2O (ds m-1) -1

CEC (C-molc kg ) pHpzc

-1

Total BFGs (mmol g ) 2

-1

SACO2 (m g ) 3

-1

Total PVCO2 (cm g )

143 144 145

PW and PB: pine wood and pine park, 350 oC and 600 oC: pyrolysis temperatures, UO: unoxidized, OX: oxidized, EC: electric conductivity, CEC: cation exchange capacity, OFG: oxygen-containing functional groups, AFG: acidic functional groups, BFG: basic functional groups, SA: surface area and PV: pore volume.

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2.4 Quincy Sand

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Quincy sand (QS) was used in this study because it is present on nearly 700,000 acres in

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Washington, Oregon, and Idaho, and is an agriculturally important sand in the Pacific Northwest 7 ACS Paragon Plus Environment


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region of the US (NRCS, 2013). The sand was collected, air-dried, and sieved through a 2 mm

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mesh23. Some selected physical and chemical characteristics of the sand are shown in Table S1 in

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the supporting information. To measure the zeta potential of sand, a 5 g of sand was added to a

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100 ml of deionized water and agitated on an orbital shaker for 6 hours at 25oC. The aliquot of

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the supernatant was then decanted from the container and analyzed by a Nano-Zetasizer 3000

154

(Malvern Instruments Ltd., Malvern, UK).

155

2.5 Bacterial Transport in Porous Media

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72 experiments were conducted to evaluate the transport of pathogenic and nonpathogenic E. coli

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in biochar-amended sand columns. Three types of porous media were used. These were Quincy

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sand (QS) and mixtures of QS and 8 types of either unoxidized or oxidized biochars. Prior to

159

measurements, the QS was cleaned and washed with DIW five times in order to remove any

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residual fine particles that could interfere with measuring the bacterial concentration or clog the

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column tubing. The sand and biochar samples were sieved to a uniform size with an average

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diameter of 500 µm. Prior to each experiment, 20 wt.% of a biochar was mixed thoroughly with

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the sand and packed dry in columns. Packed columns were acclimated to DIW for 10 pore

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volumes (1 PV = 11 mL) using a peristaltic pump (Vera Manostat/Barnant). The absorbance of

165

the effluent was measured after equilibration and values of zero further ensured that residuals of

166

fine particles were removed. The pH of the effluent was continuously monitored using Hanna HI

167

9813-6 pH/EC/TDS meters (HANNA instruments Inc., US) to ensure a constant pH throughout

168

experimentation. After that, a bacterial suspension was pumped upward at 16 ml/min through

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sand-biochar columns. Upward flow was used in order to eliminate the gravity force’ effects on

170

transport and to displace air from most pores between column filling material.


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The bacterial suspension was allowed to flow through the column for at least 7 PV

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followed by 5 PV of bacteria-free solution to elute all cells that may not have been retained on

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the porous media. Samples of the column effluent were collected in 3.25-mL fractions using an

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automated fraction collector (Model CF1, Spectrum Chromatography). Subsequently, the

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bacterial concentration in the effluent was quantified using a spectrophotometer at 600 nm.

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Bacterial breakthrough curves (BTCs) were presented in a dimensionless form by dividing the

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outlet concentration (C) by the inlet concentration (C0) and the C/C0 fractions were plotted

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against the PV values. Each column experiment was repeated at least three times on separate

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days using different cultures taken from different inoculations to ensure a true replication.

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2.6 Mathematical Modeling of the Filtration Efficiency

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In porous media, the rate at which bacteria attach to sand can follow different forms of kinetics.

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However, a second order blocking kinetics’ model is commonly used to account for the

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reduction in the bacterial attachment rate as a function of time due to the saturation of the

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favorable sand available sites for bacterial attachment24-26. The lack of a steady state bacterial

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concentration region in the majority of our collected breakthrough curves suggests that blocking

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is occurring in the sand column in the presence and absence of biochar. To check if blocking

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indeed is affecting bacterial retention in our porous media (sand and biochar), an advection-

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dispersion model with a second order bacterial attachment kinetics was employed to the data24–28.

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According to Tan et al. 1994 and Saiers et al. 1994, the continuity equation for the one-

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dimensional transport of bacteria in the water filling the pores of the sand column can be written

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as 24,25:

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∂C ρ b ∂S q ∂ 2C q ∂C + = α1 − ∂t θ ∂t θ ∂x 2 θ ∂x 9 ACS Paragon Plus Environment

(1)


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Where C is the bacterial concentration in the pore water (mg dry cell weight/ cm3 of pore water),

194

t is time, ρb is the fluid bulk density (mg dry sand/ cm3 of bed), θ is bed porosity (cm3 pore

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volume/cm3 of bed), S is the concentration of bacteria attached to the sand (mg dry cell weight/

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mg dry sand weight), x is the dimension coordinate of flow (in the length direction of the

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column), q is Darcy’s velocity (cm/s), and α1 is the longitudinal dispersivity (cm). Equation 1

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assumes no growth of bacteria occurs in the pore water during experimentation time. Because the

199

columns we used were relatively short, bacteria were assumed to be well dispersed in the

200

longitudinal direction of the column and as such, the concentration of bacteria was assumed to be

201

constant as a function of column depth. As such, the right hand side of the equation was set to

202

zero.

203

When blocking is a part of the retention process, then a second order kinetics’ model is used to

204

describe the attachment rate and a first order kinetics’ model is used for detachment rate. In

205

equation 1, the attachment rate now depends both on the bacterial concentration in the pore water

206

(C) as well as the concentration of bacteria attached to the sand (S) and is described by:

ρ b ∂S ρ = k cψC − b k y S θ ∂t θ

207

208

(2)

ψ =

209

S max − S S max

210

(3)

211

Where kc and ky are the attachment and detachment coefficients (1/s), Smax represents the

212

maximum mass of bacterial cells attached to the sand divided by the dry weight of sand and can

213

be obtained experimentally through performing a mass balance on the bacterial cells in the 10 ACS Paragon Plus Environment

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column. ψ describes a fraction of the maximum bacterial attachment capacity as shown by

215

equation 3.

216

In equations 1-3, θ is measured experimentally and kc and ky are considered as fitting

217

parameters that can be tailored to allow the solutions of equations 1-3 to fit the experimental

218

data. Equations 1-3 were solved simultaneously using the initial conditions (at t=0, C=0, S=0).

219

The equations were solved numerically. The solutions of the equations were fit to the

220

experimental data of C/C0 as a function of time. The quality of the fit was judged by maximizing

221

the value of the coefficient of determination (R2) which is used to indicate the model’s ability to

222

fit the experimental data. Values closer to 1 indicate a better regression model. On average, R2

223

values were 0.94±0.03 (n=9). We choose to fit only the pseudo-steady state portion of the data

224

because we are mainly interested in comparing the strengths of the attachment for the different

225

biochar treatments investigated. Figure 1 shows a representative example of how the second

226

order kinetics’ model fit our data. For all the conversions and the parameters used in our

227

calculations, please refer to supporting materials section 4. For modeling results, please refer to

228

section 5 and Tables S3 and S4.



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1.0

C/C0

0.8 0.6 0.4 0.2 0.0 0

2

4

6

8

10

12

14

Pore Volume

229 230 231 232 233 234

Figure 1. A representative example of the ability of the second order kinetics’ model of bacterial attachment (equations 1-3) to fit the experimental data (kc=2.88x10-4 sec-1, ky =1.8x10-3 sec-1, and R2= 0.9315). Black circles show breakthrough data obtained for the transfer of E. coli O157:H7 in sand mixed with 5 wt.% PW-600 biochar. The red solid line is the blocking model fit to the data.

235

The values of kc obtained from fitting equations 1-3 to the experimental data can be

236

related to collision efficiency values (α) via the following equation:

kc =

237

3 q 1−θ ηα 2 θ dP

238

(4)

239

Where dP is the particle diameter and η is collector efficiency, taken as the sum of the physical

240

forces affecting collisions. These forces include diffusion, effects of neighboring particles,

241

London-van der Waals forces, interception, and gravitational settling. Using the model of


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242

Rajagopalan and Tien, (1976), as modified by Logan et al., (1995), the single collector efficiency

243

was calculated as: −2 / 3 1/8 /8 η = 4 As1 / 3 N Pe + As N Lo N 15 + 0 .0338 As N G1.2 N R−0.4 R

244 245

(5)

246

where As is dimensionless Happel correction factor, Npe is dimensionless Peclet number, NLo is

247

dimensionless number that incorporates London-van der Waals attractive forces, NR is

248

dimensionless interception number, and NG is dimensionless gravitation number. Hereafter, the

249

single soil/biochar grain is termed as the collector while a bacterial cell is called particle. Table

250

S3 in supporting information summarizes the values of the parameters used in the calculations of

251

η and α.

252

For comparison purposes between treatments, the average value of C/C0 in the pseudo-

253

steady state region (approximately present between pore volumes 2 and 10 for most treatments)

254

was used to calculate the retention rates. Table S4 in the supporting information provides all the

255

pseudo-steady-state C/C0-ss values and the calculated collision efficiencies using the blocking

256

model for the pathogenic E. coli O157:H7 strain and the nonpathogenic E. coli K12 strain.

257

3. Results

258

3.1 Zeta Potentials of Bacteria, Sand, and Biochars

259

The ζ-potentials of the bacteria, sand, and biochars under different pH conditions are shown in

260

Figure 2. Our data show that the ζ-potentials for all the biochars investigated decreased linearly

261

with increase in environmental pH with the oxidized biochars being more negatively charged

262

than the un-oxidized ones (Figures 2A-2D). The ζ-potential of PB-600 whether oxidized or not

263

(Fig. 2D) decreased linearly with increase in pH. Similarly, the ζ-potential for QS decreased 13 ACS Paragon Plus Environment


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264

linearly with increasing the pH (Fig. 2E). In the range of pH between 5.0 and 8.0, ζ-potentials of

265

both bacterial strains were negative and generally became more negative as the pH increased

266

(Fig. 2F). In a dramatic contrast, the negative ζ-potential of E. coli K12 increased sharply in the

267

pH range investigated while the negative ζ-potentials E. coli O157:H7 increased slightly over the

268

same range; indicating that the ζ-potentials of the nonpathogenic E. coli were more sensitive to

269

pH changes compared to those measured for the pathogenic strain. Our results also indicate that

270

the pathogenic E. coli was less negatively charged than the nonpathogenic E. coli. This finding

271

suggests that the repulsive electrostatic forces between the bacterium and sand or sand/biochar

272

mixture will be weaker for the pathogenic E. coli compared to the nonpathogenic E. coli.

273 274 275 276 277 278 279

40

A)

Zeta Potential (mV)

Zeta Potential (mV)

280

PW-350

20

UO

OX

0 -20 -40 -60 0

2

4

6

8

40

PW-600

B)

20

UO

OX

6

8

0 -20 -40 -60

10

0

pH


2

4

pH

10


40

PB-350

C)

UO

20

Zeta Potential (mV)

Zeta Potential (mV)

Page 15 of 36

OX

0 -20 -40 -60

D)

40

PB-600 UO

20 0 -20 -40 -60

0

2

4

6

8

10

0

2

4

6

E)

QS sand

Zeta Potential (mV)

40

8

10

pH

pH

Zeta Potential (mV)

OX

20 0 -20 -40 -60

40

F)

E. coli

20 0 -20 -40

K12

O157:H7

-60 0

2

4

6

8

10

0

pH

2

4

6

8

10

pH

281 282 283 284 285

Figure 2. Average values of zeta potentials for unoxidized and oxidized biochars [(A) PW-350, (B) PW-600, (C) PB-350, and (D) PB-600), (E) sand, and (F) pathogenic and nonpathogenic bacteria (n=3). Error bars represent the standard deviation and are included for all samples but sometimes are too small to be seen on the graph.

286

Figure 3 shows BTCs collected for E. coli O157:H7 and K12 transport using PW-600-UO at

287

concentrations (0, 1, 5, 10, and 20 wt.%). As shown in Figure 3, all BTCs displayed a relatively

288

sharp breakthrough front and a high degree tailing, except in the case of E. coli O157:H7 with 20

289

wt.% biochar, where the BTC was rising slowly. The pathogenic strain was more susceptible to

290

biochar addition compared to the nonpathogenic strain (Figure 3). At 20% biochar application,

291

~89% and 26% of the pathogenic and nonpathogenic bacteria, respectively were retained in the

3.2 Effect of Biochar Concentration on E. coli Transport



292

column. This is an indication of possible stronger interactions present between the surface of the

293

biochar/sand mixture and that of the pathogenic bacteria compared to the nonpathogenic one.

Page 16 of 36

294

For both bacterial strains and under all conditions investigated, bacterial retention in the

295

column was dependent on the biochar concentration. For example, at pore volumes between 4

296

and 9, the addition of only 1wt.% of biochar to sand influenced bacterial transport in the column

297

for both strains (Figures 3A-3C). The transport rates of both E. coli O157:H7 and E. coli K12

298

were reduced by 2 fold when 5 wt.% biochar was added to sand compared to the control (0%

299

biochar). Moreover, and as can be seen from Figure 3 A-C, the BTCs obtained for the two

300

bacterial strains were similar when no biochar was applied. The same holds true when only 1%

301

biochar was applied. However, with increase in biochar concentration from 5 to 20 wt.%, BTCs

302

obtained for both strains were quite different (Figures 3A & 3B). For E. coli O157:H7, pseudo-

303

steady state plateaus were significantly different among the BTCs displaying the different

304

biochar concentrations with most reduction in transport observed when the 20 wt.% biochar was

305

used. On the contrary, E. coli K12 pseudo-steady states obtained for 5, 10 and 20% biochar

306

concentrations were statistically similar (p > 0.05). Throughout the washing phase, C/C0 fell

307

sharply to near zero values in all experiments; indicating an irreversible attachment of both

308

strains to QS or QS/biochar mixtures. The pseudo-steady state values of C/C0 decreased linearly

309

with increased biochar concentrations for E. coli O157:H7 (Figure 3C). However, the pseudo-

310

steady state values reached a plateau after 5 wt.% biochar for the E. coli K12 strain. Because

311

using 20 wt.% biochar resulted in almost complete attenuation of pathogenic E. coli in the sand

312

column, only 20 wt.% biochar concentration was investigated in further experiments.


Page 17 of 36


1.2

1.2 1.0

C/C0

0.8 0.6

B ) K1 2

0% 1% 5% 10% 20%

0% 1% 5% 10% 20%

1.0 0.8 C/C0

A) O 157 :H 7

0.6

0.4

0.4

0.2

0.2 0.0

0.0 0

2

4

6

0

8 10 12 14 16 18 20 PV

2

4

6

8 10 12 14 16 18 20 PV

Steady State C/C0

1.2

C)

1 0.8 0.6 0.4 0.2 0 0

313 314 315 316 317 318 319 320 321 322 323

10 20 Biochar concentration (wt.%)

30

Figure 3. Bacterial breakthrough curves (BTCs) obtained for E. coli (A) O157:H7 and (B) K12 in saturated sandy columns amended with PW600-UO biochar at different application rates. Error bars indicate the standard deviation obtained from triplicate experiments. C) A scatter graph that shows the relationship between the biochar concentration (% wt.) and the steady state normalized effluent bacterial concentration (C/C0) for E. coli O157:H7 (black-filled circles) and K12 (yellow-filled circles). The solid line is described by: Y=-0.0395X+0.9276, r2=0.9931. Note that Figures 3A and 3B display some widening in our BTCs, especially for the 20% biochar application treatment. The widening observed was the result of running the experiment for longer times prior to the washing phase to capture more data that can be used to explain the blocking effects observed.



Page 18 of 36

324

3.3 The Effects of Low-Temperature Biochars on Bacterial Transport

325

Figure 4 shows the BTCs of E. coli O157:H7 and K12 transport through QS mixed with either

326

low-temperature (350 oC) PW or PB derived biochars at 20 wt.% application rate. The shapes of

327

the BTCs varied as a function of biochar used as well as microbe tested. For example, Figures

328

4C and 4D showed symmetrical BTCs with relatively sharp breakthrough front and sharp tailing.

329

This shape was the most dominant BTC shape for E. coli O157:H7 transfer in QS alone or in

330

QS-PW350-OX as well as for all cases of E. coli K12 transport. Such BTCs indicate that the

331

rates of adsorption and desorption of bacteria from biochar-sand mixture are similar. In

332

comparison, Figure 4B which represents the transport kinetics of E. coli O157:H7 in PB with or

333

without biochar, was asymmetrical exhibiting a degree of tailing; indicating that the adsorption

334

rates of bacteria are smaller than their desorption rates. Figure 4A showed a horizontal-like shape

335

BTC; indicating strong and irreversible attachment of E. coli O157:H7 bacteria to UO-PW350

336

sand mixture.

337

Our results revealed that feedstock source and oxidation of low-temperature biochar had

338

a relatively minor influence on the transport of K12 with almost no bacterial retention in the sand

339

columns (Figure 4C & 4D). In a dramatic contrast, both the feedstock source and the oxidation

340

status of the biochar significantly altered the transport of E. coli O157:H7. The PW-350-UO had

341

the highest performance with 98.5% of cells retained. Oxidation of PW and PB reduced the

342

retention of E. coli O157:H7 by 9- and 7.5- fold, respectively.


Page 19 of 36


1.2

1.2 QS/Control

A) O157:H7

B) O157:H7

QS+PW-350-UO

1

QS/Control

1

QS+PB-350-UO

QS+PW-350-OX

0.8

QS+PB-350-OX

C/Co

C/Co

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0

0 0

2

4

6

8 PV

10

12

14

16

0

2

4

6

1.2

1.2 1

12

14

16

QS+PB-350-UO

1

QS+PW-350-UO

10

QS/Control

D) K12

QS/Control

C) K12

8 PV

QS+PB-350-OX

QS+PW-350-OX

0.8

C/Co

C/Co

0.8 0.6

0.6

0.4

0.4

0.2

0.2 0

0 0

2

4

6

8 PV

10

12

14

16

0

2

4

6

8 10 PV

12

14

16

343 344 345 346

Figure 4. Bacterial breakthrough curves (BTCs) for column experiments run with: E. coli O157:H7 in (A) PW350, (B) PB350, and E. coli K12 in (C) PW350, and (D) PB350. Error bars indicate the standard deviation of triplicates.

347

3.4 Effects of High Temperature Biochar on E. coli Transport

348

As can be seen from Figure 5, when unoxidized PW600 was mixed with QS at rate of 20wt.%,

349

the attenuation of E. coli O157:H7 and K12 increased by 82% and 22%, respectively, compared

350

to the control. Similarly, the addition of PB600 to QS at 20wt% increased the attenuation of E.

351

coli O157:H7 by 62% compared to QS control. However, when the transport of K12 was



Page 20 of 36

352

assessed in PW600 or PB600, only slight improvements were observed in the retention of cells.

353

Figure 5 also shows that mixing the oxidized biochars, irrespective of their feedstock type, into

354

QS promoted the transport of both E. coli strains by 5% to 65% compared with their unoxidized

355

counterparts.

1.2

1.2

A) O157:H7

QS/Control

1

QS/Control

B) O157:H7

QS+PW-600-UO

QS+PB-600-UO

1

QS+PW-600-OX

QS+PB-600-OX

0.8 C/Co

C/Co

0.8 0.6

0.6

0.4

0.4

0.2

0.2

0

0 0

2

4

6

8

10 12 14 16 18 PV

0

1.2

10

12

14

16

QS/Control QS+PB-600-UO QS+PB-600-OX

0.8

0.8 C/Co

C/Co

8 PV

1

QS+PW-600-OX

0.6

0.6

0.4

0.4

0.2

0.2

0

0 0

356 357 358 359

6

D) K12

QS+PW-600-UO

1

4

1.2

QS/Control

C) K12

2

2

4

6

8

10 12 14 16 18 PV

0

2

4

6

8

10 12 14 16 18 PV

Figure 5. Bacterial breakthrough curves (BTCs) of column experiments run with E. coli O157:H7 in (A) PW600, (B) PB600 (B), and E. coli K12 in (C) PW600, and (D) PB600. Error bars indicate the standard deviation measured from triplicates.


Page 21 of 36


360

3.5 Collision Efficiencies

361

To quantify the influence of biochar addition to QS on bacterial retention, values of the collision

362

efficiencies of bacterial adsorption on QS or QS/biochar were calculated using the blocking

363

model (equations 1-3) (Table S4). The ability of the blocking model to fit the pseudo-steady state

364

portion of our data was judged by the R2 values which were on average (0.934±0.043, n=17).

365

When the blocking model was considered, the highest collision efficiency was found for E. coli

366

O157:H7 (α=7.4×10-4) when transported through QS mixed with PW600-OX biochar. The

367

lowest collision efficiency (α=8.22 ×10-6) was obtained when QS was used alone indicating that

368

amending sand with biochar is effective for bacterial attenuation. However, the highest collision

369

efficiency (α=4.03×10-4) for E. coli K12 was observed when PB350-UO biochar was added to

370

QS. The collision efficiencies estimated for E. coli O157:H7 were, on average, 1.7 times higher

371

than those estimated for E. coli K12. Observing higher collision efficiencies for the pathogenic

372

strain compared to the nonpathogenic strain in most of the cases can be justified by the fact that

373

E. coli K12 is more negatively charged than E. coli O157:H7 at the experimental conditions

374

(pH>7) (Figure 2). As such, electrostatic repulsion forces will be higher to biochar-amended

375

sand or pure sand and as such a higher energy barrier is to be overcome in order for E. coli K12

376

to attach to the porous media compared to E. coli O157:H7 as our data demonstrates.

377

4. Discussion

378

4.1 Effect of PW600-UO Biochar Concentration on Bacterial Transport in

379

Biochar Amended Sand Columns

380

Our results indicate that the collision efficiencies of both bacterial strain are dependent on

381

biochar concentration with collision efficiencies of the pathogenic strain, on average, being 1.7



382

fold higher than those calculated for the nonpathogenic strain. The collision efficiencies

383

estimated for both bacterial types were linearly increasing with increase in PW600-UO biochar

384

application rates (Figure 6). As more biochar gets added, more sites will be available for

385

bacterial attachment. At low application rates of biochar, the surface area as well as pores

386

available for bacterial attachment are not sufficient. Even though biochar is characterized by a

387

high surface area that can be available for adsorption22, bacterial cells can’t access the area

388

provided by the micro-pores of the biochar due to their large size32. In comparison, when the

389

PW600-UO biochar concentration increased from 5% to 20%, the collision efficiencies increased

390

by 9.4 fold for E. coli O157:H7 and by 4.1 fold for E. coli K12 (Figure 6). This increase in

391

collision efficiencies can be attributed, in part, to additional sites available on the biochar surface

392

for bacterial attachment. However, if surface area was the only attributing factor to enhanced

393

attachment, we should have seen a similar fold increase in the attachment of E. coli K12 to that

394

observed for O157:H7, which was not the case. Attachment is affected by surface area,

395

physiochemical properties of QS, biochar and bacteria as well as by environmental conditions. It

396

has to be noted here that E. coli K12 are characterized by higher negative charge compared to E.

397

coli O157:H7 (Figure 2F). The higher negative charge is expected to result in repulsive forces

398

and lower attachments for E. coli K12 compared to E. coli O157:H7.


Page 22 of 36

Page 23 of 36


0.0006

Collision efficiency (α)

0.0005

K12

0.0004

O157:H7

0.0003 0.0002 0.0001 0 0

5

10

15

20

25

Biochar Concentration (wt.%) 399 400 401 402 403 404 405 406 407 408

Figure 6 The relationship between the collision efficiencies calculated using a blocking model (equations 1-3) and biochar (unoxidized PW-600) concentration for E. coli O157:H7 (blackfilled circles) and E. coli K12 (yellow-filled circles). The dashed lines are the linear fits to the data with the gray and blue lines having slopes of 3×10-5 and 1 ×10-5, respectively and intercepts of zero. The R2 values are 0.986 and 0.934 for the E. coli O157:H7 and E. coli K12 strains, respectively.

409

Bacterial retention was strongly influenced by the biochar type investigated. Biochar preparation

410

conditions including feedstock origin, pyrolysis temperature, and post-pyrolysis oxidation all

411

affected the biochar type produced and its surface physiochemical properties and as such

412

bacterial attachment to it (Figure 2-6, Tables S1-S2). Different biochar preparations led to

413

variations in biochar surface charge, degree of hydrophobicity, surface roughness, hydrogen-

414

bonding capacity, and porous structure (Table 1). Below, the roles of biochar bulk and surface

415

properties on bacterial retention will be discussed.

4.2 Effect of Biochar on Bacterial Retention

416

The accumulation of ash content and mineral elements increased with the increase in

417

pyrolysis temperature and varied in accordance with the feedstock origin (Table 1). Our results 23 ACS Paragon Plus Environment


Page 24 of 36

418

indicate that cations, such as Ca2+, Mg2+, K+, and Na+, were much higher in PB biochars

419

compared to PW biochars22. The reduction in E. coli O157:H7 transport through the columns of

420

QS mixed with PB-UO biochars irrespective of their pyrolytic temperature (Figures 4B and 5B)

421

can be attributed to electrostatic repulsive forces being screened by the positive increased metal

422

content of the biochar. Rivera-Utrilla et. al. (2001)33 observed that bacterial adsorption on

423

activated carbon increased in the presence of Ca2+ and Mg2+ and the adsorption was reduced

424

considerably after demineralization of carbon. The authors also suggested that bacterial cell

425

surface hydrophobicity, which is known to have a positive influence on cell-substrate adsorption,

426

is much higher in the presence of soluble metal cations due to the binding of metallic ions to the

427

outer membrane of bacteria. In particular, Ferris et al. (1986)34 explained the linkage between

428

cell hydrophobicity and soluble metals in terms of the diffuse-double layer theory which predicts

429

that water molecules become coordinated to site-bound counter-ions at charged surfaces. They

430

suggested that the increase in the degree of local order of liquid water results in an increase in the

431

hydrophobicity of the surface and a decrease of solvent entropy. Furthermore, several

432

researchers34–38 found that cations, especially divalent cations, neutralized the anionic charge of a

433

bacterial cell and made its planar charge distribution heterogeneous, thus decreasing repulsive

434

electrostatic forces. Note that aqueous cations may neutralize the surface charge of either the

435

bacterial cell or the biochar surface. Our results suggest that the high retention of E.

436

coli O157:H7 in PB-UO biochars amended sand columns compared to the QS control can be

437

linked to their high contents of divalent and monovalent cations.

438

A common source of the inhomogeneity of surface functionality of biochar that has been

439

recognized in the literature is the presence of various functional groups on the surface of biochar,

440

along with metal impurities. Our data indicate that mixing oxidized biochars, regardless of their


Page 25 of 36


441

type and production temperature, into QS increased the collision efficiencies (α) by an average

442

48.9 and 4.6 for E. coli O157:H7 and E. coli K12 compared to QS alone, respectively. Results

443

were statistically significant (P

Quantitative Effects of Biochar Oxidation and Pyrolysis Temperature

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