Escuela Agrícola Panamericana, Zamorano 

Food Science and Technology Department 

Food Science and Technology Major 

 

 

 

 

 

 

Special Graduation Project 

Survival of E. coli O157:H7 on hydroponically grown traditional and 

microgreen Genovese and Lemon basil (Ocimum basilicum) under 

home-scale conditions 

 

 

 Presented by 

Gleydi Lisseth Espinoza Aguirre 

Advisors 

Raúl Espinal, Ph.D.  

Angela Walla, Ph.D. 

 

 

 

 

Honduras, September 2024 



2 

 

 

Authorities 

 

 

 

SERGIO ANDRÉS RODRÍGUEZ ROYO 

President 

 

 

 

 

ANA M. MAIER ACOSTA 

Vice President and Academic Dean 

 

 

 

 

ADELA M. ACOSTA MARCHETTI 

Head of Food Science and Technology Department 

 

 

 

 

JULIO NAVARRO 

Secretary General 

 

 



3 

 

 

Content 

List of Tables ........................................................................................................................................... 4 

List of Figures .......................................................................................................................................... 5 

Abstract ................................................................................................................................................... 7 

Resumen ................................................................................................................................................. 8 

Introduction ............................................................................................................................................ 9 

Materials and Methods ......................................................................................................................... 12 

Location ................................................................................................................................................. 12 

Inoculum Preparation ........................................................................................................................... 12 

Traditional Varieties .............................................................................................................................. 12 

Phase 1 – Germination Stage ................................................................................................................ 12 

Inoculation of Nutrient Solution ........................................................................................................... 13 

Phase 2 – Sampling process .................................................................................................................. 13 

Microgreens Varieties ........................................................................................................................... 14 

Phase 1 – Germination and Growth Process ........................................................................................ 14 

Inoculation of Microgreens ................................................................................................................... 14 

Phase 2 .................................................................................................................................................. 15 

Results and Discussion .......................................................................................................................... 18 

Traditional System ................................................................................................................................ 18 

Conclusions ........................................................................................................................................... 39 

Recommendations ................................................................................................................................ 40 

References ............................................................................................................................................ 41 

Appendices ............................................................................................................................................ 44 

 

 



4 

 

 

List of Tables 

Table 1 Sampling design for microgreen cultivar: roots and nutrient solution across timepoints (days).

 .............................................................................................................................................................. 16 

Table 2 Sampling design for traditional cultivar: roots and nutrient solution across timepoints (days).

 .............................................................................................................................................................. 17 

Table 3 Differences between the growth of E. coli O157:H7 in nutrient solution between the Genovese 

and Lemon varieties in the traditional system over twenty-seven days. ............................................. 19 

Table 4 Differences between the growth of E. coli O157:H7 in roots between the Genovese and Lemon 

varieties in the traditional system over twenty-seven days. ................................................................ 20 

Table 5 Growth of E. coli O157:H7 in nutrient solution and roots between the Genovese and Lemon 

varieties in the traditional system over twenty-seven days. ................................................................ 23 

Table 6 Differences between the growth of E. coli O157:H7 in mat between the Genovese and Lemon 

varieties in the microgreens system over fourteen days. .................................................................... 29 

Table 7 Differences between the growth of E. coli O157:H7 in roots between the Genovese and Lemon 

varieties in the microgreens system over fourteen days. .................................................................... 30 

Table 8 Growth of E. coli O157:H7 in mat and Roots between the Genovese and Lemon varieties in the 

microgreens system over fourteen days. ............................................................................................. 33 

 

 

 

 

 

 

 

 



5 

 

 

List of Figures 

Figure 1 Growth of E. coli O157:H7 in nutrient solution samples between the Genovese and Lemon 

varieties in the traditional system over twenty-seven days. ................................................................ 21 

Figure 2 Growth of E. coli O157:H7 in roots samples between the Genovese and Lemon varieties in 

the traditional system over twenty-seven days. .................................................................................. 22 

Figure 3 Growth behavior of E. coli O157:H7 in relation to the type of samples in nutrient solution and 

roots of Genovese basil in the traditional system over twenty-seven days. ........................................ 24 

Figure 4 Growth behavior of E. coli O157:H7 in relation to the type of samples in nutrient solution and 

roots of Lemon basil in the traditional system over twenty-seven days. ............................................. 25 

Figure 5 Growth pattern of E. coli O157:H7 in nutrient solution and roots of traditional cultivars of 

Genovese and Lemon basil over a 27-day production cycle. ............................................................... 26 

Figure 6 Growth of E. coli O157:H7 in mat samples between the Genovese and Lemon varieties in the 

microgreens system over fourteen days. ............................................................................................. 31 

Figure 7 Growth of E. coli O157:H7 in roots samples between the Genovese and Lemon varieties in 

the microgreens system over fourteen days. ....................................................................................... 32 

Figure 8 Growth behavior of E. coli O157:H7 in relation to the type of samples in mat and roots of 

Genovese basil in the microgreens system over fourteen days. .......................................................... 34 

Figure 9 Growth behavior of E. coli O157:H7 in relation to the type of samples in mat and roots of 

Lemon basil in the microgreens system over fourteen days. ............................................................... 35 

Figure 10 Growth pattern of E. coli O157:H7 in media (mat) and roots of microgreen cultivars of 

Genovese and Lemon basil over a 14-day production cycle. ............................................................... 36 

 

 

 

 



6 

 

 

List of Appendices 

Appendix A Ampicillin requirement ...................................................................................................... 44 

Appendix B Sampling schedule for traditional system and microgreens ............................................. 45 

Appendix C Box plot of E. coli O157:H7 growth pattern in nutrient solution and roots of the traditional 

Genovese basil cultivar over a 27-day production cycle ...................................................................... 46 

Appendix D Box plot of E. coli O157:H7 growth pattern in nutrient solution and roots of the traditional 

Lemon basil cultivar over a 27-day production cycle ........................................................................... 47 

Appendix F Box plot of E. coli O157:H7 growth pattern in nutrient mat and roots of the microgreen 

Lemon basil cultivar over a 27-day production cycle ........................................................................... 49 

Appendix G Hydroponic units used for Genovese and Lemon basil varieties in the traditional system

 .............................................................................................................................................................. 50 

Appendix H Hydroponic units used for Genovese and Lemon basil varieties in the microgreens system

 .............................................................................................................................................................. 51 

 

 

 

 

 

 

 

 

 

 

 

 



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Abstract 

The consumption of fresh culinary herbs, especially basil, has increased due to their health benefits. 

Consequently, their production has surged, particularly in hydroponic systems at home. Since culinary 

herbs are often consumed raw, they pose a food safety risk if contaminated. Recent outbreaks in 

hydroponic production have shown that foodborne pathogens can survive in these systems, including 

components such as nutrient solutions, growth mats, and roots. For this reason, this study determined 

the survival rates of E. coli O157:H7 within hydroponic units of Genovese and Lemon basil cultivars 

under home-scale conditions. The study compared the survival of E. coli O157:H7 over 27 days and 14 

days in the traditional and microgreen systems, respectively. Bacterial counts were monitored in the 

nutrient solution, growth mat, and root samples to evaluate each variety's microbial interactions and 

possible inhibitory effects. Results indicated a significant reduction in bacterial counts over time (p < 

0.05). In the traditional system, E. coli O157:H7 decreased from 6 Log CFU/mL to 0 Log CFU/mL over 

27 days, while in the microgreens system, it reduced from 5 Log CFU/mL to 1 Log CFU/mL over 14 

days. No significant differences were observed between the two basil varieties within each system (p 

> 0.05). Both varieties showed similar bacterial reduction in all samples, suggesting that bacterial 

persistence may depend more on factors other than variety influence in bacterial survival. These 

findings highlight the potential of growing basil in traditional and microgreen systems to mitigate 

bacterial contamination, with implications for ensuring food safety and improving agricultural 

practices. 

Keywords: cultivars, food safety, hydroponic system, traditional system 

 

 

 

 



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Resumen 

El consumo de hierbas culinarias frescas, como la albahaca, ha aumentado debido a sus beneficios 

para la salud, impulsando su producción en sistemas hidropónicos caseros. Sin embargo, dado que se 

consumen crudas, representan un riesgo de seguridad alimentaria si están contaminadas. Brotes 

recientes han demostrado que patógenos como E. coli O157:H7 pueden sobrevivir en sistemas 

hidropónicos, incluidos componentes como soluciones nutritivas, substratos y raíces. Este estudio 

evaluó la supervivencia de E. coli O157:H7 en unidades hidropónicas de cultivares albahaca genovesa 

y albahaca limón bajo condiciones a pequeña escala. Se comparó la supervivencia de E. coli O157:H7 

durante 27 días en el sistema tradicional y 14 días en el sistema de microvegetales. Se monitorearon 

los conteos bacterianos en la solución nutritiva, substrato de crecimiento y muestras de raíces para 

analizar las interacciones microbianas de cada variedad y posibles efectos inhibitorios. Los resultados 

mostraron una reducción significativa en los conteos bacterianos (p < 0.05). En el sistema tradicional, 

E. coli O157:H7 disminuyó de 6 Log UFC/mL a 0 Log UFC/mL en 27 días, y en microvegetales de 5 Log 

UFC/mL a 1 Log UFC/mL en 14 días. No se observaron diferencias significativas entre las variedades 

en cada sistema (p > 0.05). Ambas variedades mostraron reducciones bacterianas similares, sugiriendo 

que la persistencia bacteriana depende más de otros factores que de la variedad. Estos resultados 

destacan el potencial de cultivar albahaca en sistemas tradicionales y de microvegetales para mitigar 

la contaminación bacteriana, con implicaciones para la seguridad alimentaria y la mejora de prácticas 

agrícolas. 

Palabras clave: cultivares, seguridad alimentaria, sistema hidropónico, sistema tradicional 

 

 

 

 

 



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Introduction 

Culinary herbs have long been characterized by their aromatic flavors and medicinal 

properties, tracing back to ancient civilizations where they were vital for their culinary and therapeutic 

benefits. Nowadays, the cultivation and consumption of culinary herbs have evolved into a global 

phenomenon, with the United States and Italy standing as significant players in both production and 

consumption as part of their cultures (Spence, 2024). Among the myriad herbs, basil holds a 

prominent place in American cuisine, adding depth and freshness to various dishes. In addition, due 

to its typical pungent and composite fragrance, it is employed in Mediterranean cuisine and is 

commonly used as a food garnish and preservative, being considered a valuable source of antioxidants 

(Corrado et al., 2020).  

While traditional cultivation methods have been the norm for centuries, the emergence of 

microgreen varietals has introduced new dimensions to herb cultivation, offering intensified flavors 

and enhanced nutritional profiles. Concurrently, there is a growing trend toward home cultivation of 

fresh culinary herbs, driven by a desire for sustainability, convenience, and flavor due to the high 

concentration of nutrients mostly found in leaves. According to recent studies, while herb production 

in the United States is primarily conducted within controlled environment agriculture systems, 

including greenhouses, there is also a growing trend of consumers cultivating fresh culinary herbs at 

home (Dohlman et al., 2024). 

Home hydroponic production, although efficient and space-saving, can inadvertently 

predispose to microbial contamination and growth if proper precautions are not observed, and if 

humid conditions favor microbial growth (Dankwa et al., 2020). Given the recent outbreaks and recalls 

related to foodborne pathogens in fresh vegetables and culinary herbs, such as E. coli O157:H7, there 

is a pressing need to address the safety concerns surrounding home hydroponic production. Despite 

multiple efforts and measures to prevent and reduce contamination, most Shiga toxin-producing E. 

coli (STEC) cases have been linked to the consumption of leafy greens, particularly lettuce, with 40 



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outbreaks reported in the United States and Canada between 2009 and 2018 (Marshall et al., 2020). 

Although basil has had fewer outbreaks compared to lettuce, recent cases have still been reported. 

Between 1996 and 2015, nine outbreaks involving basil, parsley, and cilantro were documented, 

resulting in 2,699 illnesses. Specifically, four outbreaks were related to basil, three to cilantro, and two 

to parsley where Cyclospora cayetanensis caused seven outbreaks, while E. coli O157:H7 and Shigella 

sonnei each caused one (Food and Drug Administration [FDA], 2022). 

Also, more recently, epidemiological and tracing investigations have identified additional 

outbreaks related to basil. According to the Centers for Disease Control and Prevention (2024), an 

outbreak that concluded on June 18th, 2024, was linked to Salmonella contamination in organic basil 

from the Infinite Herbs brand, affecting 36 people and resulting in four hospitalizations across 14 

states. 

E. coli O157:H7, a particularly virulent strain of Escherichia coli, poses significant risks to 

human health when present in fresh vegetables due to its potential to cause severe illness, including 

hemolytic uremic syndrome. Understanding the factors contributing to its proliferation in fresh 

produce, including herbs, is paramount to ensuring food safety. Every year, it is estimated by the U.S. 

Centers for Disease Control and Prevention (CDC) that 48 million Americans are affected by foodborne 

illnesses annually, resulting, unfortunately, in 128,000 hospitalizations and 3,000 deaths (K. Turner et 

al., 2019). Recent literature has demonstrated extensive production and consumption of herbs, with 

the United States producing around 200 billion pounds of herbs and spices annually (Sanchez, 2023). 

These quantities are closely related to the healthy and cultural consumption trend worldwide. 

Regarding basil, global production is estimated at 42.7 million tons, with China covering the highest 

production rate at 48% (Yitbarek & Wendimu, 2023). 

Although cultivating plants in the hydroponic system represents a solution for increasing food 

production and reducing the environmental footprint, there are still aspects that need to be studied 

since the survival of pathogens changes (Dobrin et al., 2018). In particular, the varieties of basil play 



11 

 

 

an important role in bacterial survival. In general, basil plants can be classified into four major essential 

oil (EO) chemotypes based on their EO composition: those rich in methyl chavicol, linalool, methyl 

eugenol, and methyl cinnamate, along with numerous additional subtypes (Verrillo et al., 2021). In 

addition, it is important to highlight that understanding basil's compound profiles and the influence 

of different varieties on their concentration is crucial for determining effective methods of bacterial 

inhibition. Specifically, the presence of phenolic components in the essential oil can contribute to 

antimicrobial activity by causing the leakage of intracellular ATP and potassium ions, which leads to 

cell death (Joshi, 2014). Understanding the mechanism of action of basil’s compounds against 

different bacteria is vital, as is studying the various production systems in which basil is grown, in 

developing bacterial inhibition strategies to improve food safety practices. Therefore, studying the 

different dynamics of pathogens has become vital in understanding the extent to which a production 

system can be exposed depending on the conditions in which it is found. 

Therefore, this study aims to determine the survival rates of E. coli O157:H7 in traditional and 

microgreen Genovese and Lemon basil. Secondly, to compare the survival of E. coli O157:H7 in 

nutrient solution and roots of Lemon and Genovese cultivars. Lastly, aims to investigate where within 

the hydroponic system E. coli O157:H7 survives best, including nutrient solution, substrate, water, and 

roots. 

 

 

 

 

 

 

 

 



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Materials and Methods  

Location 

The entire experiment was conducted at the International Center for Food Industry Excellence 

(ICFIE), located in the Experimental Sciences Building at Texas Tech University in Lubbock, Texas, United 

States of America. 

Inoculum Preparation  

For this process, to resuscitate E. coli O157:H7 35150 GFP, isolates were picked from one vial 

of the ampicillin-resistant bacteria stored in a -80 °C freezer. One loop was transferred into four 10 

mL of Brain Heart Infusion (BHI) broth and incubated at 37 °C overnight. After confirming the starting 

inoculum concentration, the four 10 mL BHI tubes were poured into two sterile 30 mL high-strength 

centrifuge tubes, balancing 20 mL per tube in the centrifuge. The tubes containing the inoculum were 

centrifuged at 4000 rpm for a total of 10 minutes. Subsequently, 20 mL of sterile water/PBS was added 

to each centrifuge tube. The contents of both centrifuge tubes were then thoroughly mixed by 

vortexing. The final inoculum concentration was plated to confirm it had reached 10^7 CFU/mL. 

Traditional Varieties 

Phase 1 – Germination Stage  

Basil was produced using the Deep-Water Culture (DWC) method for the traditional method. 

Two varieties were used: Genovese basil and Lemon basil, each with three replicates and one control 

per variety. Genovese (Lot: 80185) and Lemon (Lot: 74089) basil seeds were obtained from Johnny's 

Selected Seeds supplier, and the nutrient solution was acquired from JR Peters Inc. (Jack’s Nutrients) 

using two formulations denominated as Part A and Part B. For the germination process, since it was 

needed to work with basil under homegrown conditions, basil seeds germinated for fourteen days in 

a plant growth chamber from Percival Scientific with Controlled Environment Agriculture (CEA). First, 

the growth chamber trays, and hydroponic units were disinfected to ensure aseptic conditions for the 

start of germination. A volume of 4 liters of nutrient solution per unit was prepared to meet the 



13 

 

 

optimum requirements for basil (100 to 150 ppm N), and the rockwool cubes with the seeds were 

saturated. The temperature was set at 20 °C (68 °F) with 70% relative humidity and a constant CO2 

concentration of 1000 ppm. Once the units were ready, the healthiest-looking seedlings that showed 

three true leaves were transplanted into the Deep-Water Culture (DWC) hydroponic system, and the 

units were programmed to run in vegetative mode. In this mode, a cycle of 16 hours of light and 8 

hours of darkness is run with color LED lights, including red, white, and blue lights. 

Inoculation of Nutrient Solution  

Once the desired concentration was obtained, and after two days of transplanting and 

acclimatizing the plants, 4 mL of the inoculum was transferred from the tube containing 10^7 CFU/mL. 

This inoculum was then introduced into the 4-liter nutrient solution of the Deep-Water Culture (DWC) 

hydroponic unit for the traditional system through one of the holes on the top of the unit. This process 

resulted in a final concentration of 10^4 CFU/mL, as one log reduction was expected. To sample, the 

inoculum was circulated by the internal pump for 30 minutes in the nutrient solution; this first 

sampling was Day 0, and 10 mL of the nutrient solution was taken into a sterile empty tube. 

Phase 2 – Sampling process 

For 45 days, the Electrical Conductivity (EC) and pH were adjusted daily according to the plant 

requirements to maintain ideal conditions for each cultivar, within the range of 5.8-6.2 for pH and 1.0-

1.4 mS/cm for EC. During this period, samples were taken every three days, including both nutrient 

solution and root samples, totaling 24 samples across both types. 

Nutrient Solution Samples. 

 Samples of the nutrient solution were collected by extracting 10 mL from the reservoir into a 

sterile empty tube. Depending on the sampling day, these samples were diluted with 9 mL of Buffered 

Peptone Water (BPW) and directly plated onto MacConkey agar with 200 µg/mL ampicillin using the 

spread plating method. Several dilutions were performed to ensure countable plates and allow for 

reporting of colony-forming units (CFU) within the range of 25-250 colonies. 



14 

 

 

Root Samples. 

Root samples were obtained by removing the rock wool with the respective plant and cutting 

the roots into a stomacher bag at 230 rpm. Additionally, the roots were weighed, and the 

corresponding amount of Buffered Peptone Water (BPW) was added by multiplying the weight by 9 

to dilute the sample in a relation of 1:10. The samples were homogenized for 1 minute using a 

stomacher at 230 rpm before being directly plated onto MacConkey agar with 200 µg/mL ampicillin 

using the spread plating method. 

Microgreens Varieties 

Phase 1 – Germination and Growth Process 

For the microgreen method, two varieties were used: Genovese basil and Lemon basil, each 

with three replicates and one control per variety. Genovese (Lot: 100147) and Lemon (Lot: 61205) 

basil seeds were obtained from Johnny's Selected Seeds supplier, and the nutrient solution was 

acquired from JR Peters Inc. (Jack’s Nutrients) using two formulations denoted as Part A and Part B. 

The basil seeds were germinated for seven days, as their germination period is shorter compared to 

traditional methods. Initially, the plant growth chamber from Percival Scientific with Controlled 

Environment Agriculture (CEA) and the growing trays were disinfected to maintain aseptic conditions 

for germination. To begin the germination process, 5 g of seeds were placed on grow mats and sprayed 

with 100 mL of deionized sterile water and nutrient solution. To ensure optimal germination, the grow 

trays were covered with domes and placed in a growth chamber set at a constant temperature of 20 

°C (68 °F) with 70% relative humidity, complete darkness, and a constant CO2 concentration of 1000 

ppm. The trays with the seeds were kept in darkness until day 5 to simulate industry practices. 

Inoculation of Microgreens 

Once the desired concentration of 10^6 CFU/mL was achieved, on Day 6, 1 mL was transferred 

from the 10^6 CFU/mL tube and inoculated into 100 mL of water used to submerge the microgreen 

growth mat placed above the draining tray. This resulted in a concentration of 10^4 CFU/mL, as one 



15 

 

 

log reduction was expected. To sample the inoculum, a 1-inch square of the grow mat was placed into 

a stomacher bag at 230 rpm for both mat and root samples; this initial sampling occurred on Day 0. 

Phase 2 

For 21 days, the microgreens were grown in the plant growth chamber from Percival Scientific 

with Controlled Environment Agriculture (CEA), under ideal plant conditions. Each day, every grow 

mat was irrigated with 50 mL of deionized water and nutrient solution. Throughout this period, 

samples were collected on predetermined days for both the growth mat (media) and root samples, 

totaling 8 samples for each. Samples were collected by cutting a 1-inch square of the growth mat into 

a stomacher bag at 230 rpm for both the media and root samples. It was necessary to weigh the grow 

mat cubes for both sample types, and for the root samples, only the roots were weighed after cutting 

the plants. After sampling, the respective amount of Buffered Peptone Water (BPW) was added by 

multiplying the weight by 9 to dilute the sample. The samples were homogenized for one minute using 

a stomacher at 230 rpm before being directly plated onto MacConkey agar with 200 µg/mL ampicillin 

using the spread plating method. 

Microbial Enumeration. 

To enumerate bacterial colonies, the selected dilutions were plated in duplicate on 

MacConkey agar supplemented with 200 µg/mL ampicillin to reduce background microflora. Plates 

were then incubated at 37 °C for 24 hours. After incubation, colonies were counted within the range 

of 25-250 colonies per plate. In cases where plates showed an excessive number of colonies, additional 

dilutions were plated to achieve counts within the manageable range. 

Colonies were counted based on the characteristics exhibited by the bacteria on the medium. 

The selected medium, MacConkey agar with 200 µg/mL ampicillin, resulted in pink colonies because 

E. coli O157:H7 ferments lactose, which decreases the pH of the agar. Additionally, in cases where no 

bacterial colonies were observed, an enrichment with Brain Heart Infusion (BHI) broth was performed 

to verify the presence of the bacteria in the system. 



16 

 

 

Experimental Design 

A 2x2 factorial Randomized Complete Block design (Factorial RBD) was employed to assess 

measures over time. The experiment comprised four distinct treatment combinations, each defined 

by the interaction between two factors: type of cultivation system (traditional and microgreen) and 

variety of basil (Genovese and Lemon). For each treatment combination, there were three replicates, 

resulting in a total of 16 experimental units. For the microgreen system, the samplings were carried 

out over a period of 14 days with five sampling points on days 0, 3, 6, 9, and 14 (Table 1). For the 

traditional system, the samplings were carried out for 27 days with 10 sampling points on days 0, 3, 6, 

9, 12, 15, 18, 21, 24, and 27 (Table 2). Data analysis was conducted using the statistical software R 

Studio 4.3.1, utilizing ANOVA alongside the non-parametric Pairwise Wilcoxon Test to identify 

significant differences between treatments (p < 0.05). 

Table 1 

Sampling design for microgreen cultivar: roots and nutrient solution across timepoints (days). 

Replicate Variety Treatment Sample type 
Timepoint (Days)/ Sample number 

0 3 6 9 14 

Control Genovese Control 
Roots 1 2 3 4 5 

Nutrient solution 6 7 8 9 10 

1 Genovese E. coli O157:H7 
Roots 11 12 13 14 15 

Nutrient solution 16 17 18 19 20 

2 Genovese E. coli O157:H7 
Roots 21 22 23 24 25 

Nutrient solution 26 27 28 29 30 

3 Genovese E. coli O157:H7 
Roots 31 32 33 34 35 

Nutrient solution 36 37 38 39 40 

Control Lemon Control 
Roots 41 42 43 44 45 

Nutrient solution 46 47 48 49 50 

1 Lemon E. coli O157:H7 
Roots 51 52 53 54 55 

Nutrient solution 56 57 58 59 60 

2 Lemon E. coli O157:H7 
Roots 61 62 63 64 65 

Nutrient solution 66 67 68 69 70 

3 Lemon E. coli O157:H7 
Roots 71 72 73 74 75 

Nutrient solution 76 77 78 79 80 
Note. Treatments applied in 3 replicates for Genovese and Lemon basil varieties. Control: non-inoculated hydroponic units. E. coli O157:H7: 

inoculated units. Timepoint indicates sampling days (0, 3, 6, 9, 14). 



17 

 

 

 

Table 2 

Sampling design for traditional cultivar: roots and nutrient solution across timepoints (days). 

Replicat
e 

Variety 
Treatme

nt 
Sample type 

Timepoint (Days)/ Sample number 

0 3 6 9 12 15 18 21 24 27 

Control Genovese Control 
Roots 1 2 3 4 5 6 7 8 9 10 

Nutrient solution 11 12 13 14 15 16 17 18 19 20 

1 Genovese 
E. coli 

O157:H7 

Roots 21 22 23 24 25 26 27 28 29 30 

Nutrient solution 31 32 33 34 35 36 37 38 38 40 

2 Genovese 
E. coli 

O157:H7 

Roots 41 42 43 44 45 46 47 48 49 50 

Nutrient solution 51 52 53 54 55 56 57 58 59 60 

3 Genovese 
E. coli 

O157:H7 

Roots 61 62 63 64 65 66 67 68 69 70 

Nutrient solution 71 72 73 74 75 76 77 78 79 80 

Control Lemon Control 
Roots 81 82 83 84 85 86 87 88 89 90 

Nutrient solution 91 92 93 94 95 96 97 98 99 100 

1 Lemon 
E. coli 

O157:H7 

Roots 101 102 103 104 105 106 107 108 109 110 

Nutrient solution 111 112 113 114 115 116 117 118 119 120 

2 Lemon 
E. coli 

O157:H7 

Roots 121 122 123 124 125 126 127 128 129 130 

Nutrient solution 131 132 133 134 135 136 137 138 139 140 

3 Lemon 
E. coli 

O157:H7 

Roots 141 142 143 144 145 146 147 148 149 150 
Nutrient solution 151 152 153 154 155 156 157 158 159 160 

Note. Treatments applied in 3 replicates for Genovese and Lemon basil varieties. Control: non-inoculated hydroponic units. E. coli O157:H7: 

inoculated units. Timepoint indicates sampling days (0, 3, 6, 9, 15, 18, 21, 24, 27). 

 

 

 

 

  



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Results and Discussion 

Traditional System 

According to Dhulappanavar and Gibson (2023), one of the largest sources of contamination 

in hydroponic systems is the water used in the preparation of nutrient solutions. They found that 

water is the entry route for human pathogens through the roots of plants submerged in a nutrient 

solution within the hydroponic system (Dhulappanavar & Gibson, 2023). For this reason, this section 

focuses on first determining the growth of E. coli O157:H7 in the nutrient solution for each of the two 

varieties in the study (Table 3). 

Table 1 provides data on the differences in the growth of E. coli O157:H7 in nutrient solution 

for the Genovese and Lemon varieties over a 27-day sampling period. Both varieties showed a 

decrease in E. coli O157:H7 counts, indicating reduced bacterial growth. Notably, on Day 3, both 

varieties experienced a significant decrease in E. coli counts compared to Day 0, with a reduction of 

2.6 Log CFU/mL for Genovese and 2.19 Log CFU/mL for Lemon. However, the decrease was slightly 

more pronounced in the Genovese variety compared to the Lemon variety. Despite higher counts on 

other sampling days, it was determined that there was no statistically significant difference between 

the two varieties (p > 0.05).  

The behavior of bacterial growth in the nutrient solution could have been influenced by the 

compounds of the two varieties of basil. The basil varieties in this study, Genovese and Lemon share 

many of the same essential oils; however, the concentration of these oils varies significantly between 

the two varieties. Lemon basil is distinguished by its dominant aromatic profile of α-citral (33.34%) 

and β-citral (27.29%), compounds responsible for its strong citrus aroma, while Genovese basil has a 

markedly different aromatic composition, with high levels of linalool (40.77%), eucalyptol (29.37%), 

and trans-α-bergamotene (4.27%) (Ciriello et al., 2023). It is important to emphasize that the 

concentration of basil compounds is influenced not only by the variety and the plant's genetics but 

also by the cultivation methods employed. This is particularly relevant in the context of hydroponic 



19 

 

 

systems. In addition, there are exudates released by the plants, which may inhibit bacterial growth, 

such as various organic acids, phenolic compounds, and flavonoids. This is supported by the results of 

a study by Shaw et al. (2016), which found that although E. coli O157: H7 was able to outcompete the 

background flora in the hydroponic system, the absorption of nutrients by plants and the release of 

ions from the roots could affect the survival and growth of pathogens at a lower rate. Despite the 

composition of the compounds for each variety in the nutrient solution, a notable decrease in bacterial 

counts was observed, though there were no significant differences between the varieties evaluated. 

Table 3 

Differences between the growth of E. coli O157:H7 in nutrient solution between the Genovese and 

Lemon varieties in the traditional system over twenty-seven days. 

 Variety 

Time (Day) 

Genovese Lemon  

Nutrient solution 
Log CFU/mL ± SD 

Nutrient solution 
Log CFU/mL ± SD 

 
CV (%) 

0 6.17 ± 0.05a 6.00 ± 0.06a 0.92 
3 3.57 ± 0.12a 3.81 ± 0.31a 5.78 
6 2.15 ± 0.32a 2.54 ± 0.94a 25.91 
9 1.43 ± 0.74a 1.97 ± 0.91a 49.12 
12 1.18 ± 0.31a 1.00 ± 0.00a 13.29 
15 1.13 ± 0.23a 1.00 ± 0.00a 10.14 
18 1.00 ± 0.00a 1.00 ± 0.00a 0.00 
21 1.00 ± 0.00a 1.00 ± 0.00a 0.00 
24 0.00 ± 0.00a 0.00 ± 0.00a 0.00 
27 0.00 ± 0.00a 0.00 ± 0.00a 0.00 

Note. SD: Standard deviation. CV (%): Coefficient of variation. 
a-b Means with the same lowercase letters for E. Coli log CFU/mL indicate no 

significant differences (p > 0.05) in each column. Each Nutrient Solution Log CFU/mL microbiological count per variety represents the mean 

of 3 repetitions.  

 

Table 4 shows the bacterial counts in Log CFU/mL for both Genovese and Lemon varieties, 

where no significant differences were observed in the root system of the plants for each variety (p > 

0.05). Comparing the groups between varieties, the bacterial reduction for each sampling day was 

statistically the same, so the same letter was attributed to each mean. Although no significant 

differences were found between varieties, there was a logarithmic reduction over time. Unlike in the 



20 

 

 

nutrient solution, bacterial counts were no longer reported until day 24 of sampling, and the presence 

of bacteria was still detected in roots on day 27 of sampling. 

Table 4 

Differences between the growth of E. coli O157:H7 in roots between the Genovese and Lemon 

varieties in the traditional system over twenty-seven days. 

                                      Variety 

Time (Day) 

Genovese Lemon   

Roots 
Log CFU/mL ± SD 

Roots 
Log CFU/mL ± SD 

CV (%) 

0 3.64 ± 0.66a 3.59 ± 0.23a 12.22 
3 3.01 ± 0.72a 3.23 ± 0.40a 18.09 
6 1.35 ± 0.16a 2.43 ± 0.90a 24.32 
9 1.55 ± 0.64a 2.30 ± 0.50a 33.51 
12 1.38 ± 0.34a 1.39 ± 0.39a 26.25 
15 1.10 ± 0.17a 1.06 ± 0.10a 12.70 
18 1.00 ± 0.00a 1.00 ± 0.00a 0.00 
21 1.00 ± 0.00a 1.00 ± 0.00a 0.00 
24 1.00 ± 0.00a 1.00 ± 0.00a 0.00 
27 1.00 ± 0.00a 1.00 ± 0.00a 0.00 

Note. SD: Standard deviation. CV (%): Coefficient of variation. 
a-b Means with the same lowercase letters for E. Coli log CFU/mL indicate no 

significant differences (p > 0.05) in each column. Each microbiological count of Roots Log CFU/mL per variety represents the mean of 3 

repetitions.  

 

Figures 1 and 2 reveal the growth of E. coli O157:H7 in the traditional system for both nutrient 

solution and roots, considering both varieties of basil. It can be seen how, over time, for a sampling 

period of 27 days, the effect of the variety variable was not significant since the Wilcoxon test and T-

test analysis in the comparison of the two paired variety samples did not reveal a significant difference 

(p > 0.05) between treatments in both nutrient solution and roots samples. It should be noted that in 

both varieties, a similar survival curve of E. coli O157:H7 was obtained without statistical differences 

in terms of the data. This similarity may be attributed to both varieties having similar physiological 

characteristics, such as similar leaf morphology and chemical profiles. Both varieties possess essential 

oils like eugenol and linalool, and comparable levels of flavonoids and phenolic compounds that 



21 

 

 

conferred comparable resistance mechanisms causing them to respond similarly to the bacteria in the 

experimental hydroponic environment with uniform crop conditions (Zote et al., 2024). 

Figure 1 

Growth of E. coli O157:H7 in nutrient solution samples between the Genovese and Lemon varieties in 

the traditional system over twenty-seven days. 

 

 

The data obtained showed that no significant differences were observed between both 

varieties in the nutrient solution and root samples. This finding may be attributed to the type of 

sampling conducted and the specific part of the plant analyzed, as each part contains varying amounts 

of essential oils that inhibit bacteria based on their content. This observation is supported by a study 

conducted by Kamelnia et al. (2023), which determined that basil leaves and stems predominantly 

contain essential oil constituents such as Eugenol (42.74%), Linalool (20.54%), and Eucalyptol (15.27%) 

of the overall essential oil composition of the basil plant, reflecting these percentages in different 

proportions. Additionally, many of these compounds are released through root exudates, which can 

lead to reduced bacterial presence in the rhizosphere due to their antimicrobial properties. These 



22 

 

 

constituents are known for their biological activities, including the antifungal and anti-aflatoxin 

properties of the plant. Basil’s properties disrupt ergosterol biosynthesis in fungal plasma membranes 

and alter mitochondrial membrane potential, impeding in that way fungal growth and inhibiting 

aflatoxin B1 production (Kumar et al., 2020). 

Figure 2 

Growth of E. coli O157:H7 in roots samples between the Genovese and Lemon varieties in the 

traditional system over twenty-seven days. 

 

Table 5 shows the growth of O157:H7 in nutrient solution and roots of the two varieties in the 

traditional system, with sampling carried out over twenty-seven days. At first, on day 0, a difference 

was detected between nutrient solution and roots (a-b), which indicated that despite the system 

having been inoculated at the same time and with the same logarithmic concentration (6 Log CFU/mL), 

a lower percentage of bacteria was attached to the roots compared to nutrient, which can be 

supported by the counts obtained of 6 Log CFU/mL in nutrient solution and 3.60 Log CFU/mL in roots. 

Additionally, the data reflect significant differences (p < 0.05) both in the comparison of nutrient 

solution and roots and for the comparison of each case throughout the sampling time with significant 

differences in each of the days. 



23 

 

 

Table 5 

Growth of E. coli O157:H7 in nutrient solution and roots between the Genovese and Lemon varieties 

in the traditional system over twenty-seven days. 

Note. SD: Standard deviation. CV (%): Coefficient of variation. 
a-b Means with different lowercase letters for E. Coli log CFU/mL indicate 

significant differences between treatments in each variety (p < 0.05) in each column; 
u-z 

Means with different lowercase letters for E. Coli log 

CFU/mL indicate significant differences between the evaluated times (p < 0.05) in each row. Each microbiological count of Nutrient Solution 

and Roots Log CFU/mL per variety represents the mean of 3 repetitions.  

 

The growth behavior of E. coli O157:H7 in relation to the type of sampling of the two varieties 

is displayed in Figure 3 and Figure 4. After analyzing the incorporation of the two varieties with each 

type of sample and finding no differences between them, it was shown that the variable of the sample 

type presented significant differences (p < 0.05) between nutrient solution and roots. These 

differences were observed on days 0, 6, 24, and 27 for the Genovese variety analysis (a-d). 

Subsequently, days with the same letter designation (a-a) showed no significant difference (p > 0.05) 

for the other sampling days. This explains the growth behavior of the bacteria in the traditional system 

over time. Despite an initial inoculation concentration of 10^7 CFU/mL, the nutrient solution exhibited 

a concentration of 6 Log CFU/mL on day 0 of sampling, while root samples showed 3.60 Log CFU/mL, 

indicating a higher concentration in the nutrient solution initially. However, an increase in the bacteria 

was not observed, as both concentrations exhibited an exponential decrease over the sampling 

period, attributed to the essential oils of basil. 

                                                                Variety 

Time (Day) 

Genovese Lemon  

Nutrient solution  
Log CFU/mL ± SD 

Roots 
Log CFU/mL ± SD 

Nutrient solution  
Log CFU/mL ± SD 

Roots 
Log CFU/mL ± SD 

CV (%) 
 

0 6.17 ± 0.05au 3.64 ± 0.66bu 6.00 ± 0.06au 3.59 ± 0.23bu 6.57 
3 3.57 ± 0.12av 3.01 ± 0.72au 3.81 ± 0.31av 3.23 ± 0.40au 11.94 
6 2.15 ± 0.32aw 1.35 ± 0.16av 2.54 ± 0.94aw 2.43 ± 0.90av 25.12 
9 1.43 ± 0.74ax 1.55 ± 0.64avw 1.97 ± 0.91ax 2.30 ± 0.50avw 41.32 
12 1.18 ± 0.31ay 1.38 ± 0.34awx 1.00 ± 0.00ay 1.39 ± 0.39awx 19.77 
15 1.13 ± 0.23ay 1.10 ± 0.17ax 1.00 ± 0.00ay 1.06 ± 0.10ax 11.42 
18 1.00 ± 0.00ay 1.00 ± 0.00ax 1.00 ± 0.00ay 1.00 ± 0.00ax 0.00 
21 1.00 ± 0.00ay 1.00 ± 0.00ax 1.00 ± 0.00ay 1.00 ± 0.00ax 0.00 
24 0.00 ± 0.00bz 1.00 ± 0.00ax 0.00 ± 0.00bz 1.00 ± 0.00ax 0.00 
27 0.00 ± 0.00bz 1.00 ± 0.00ax 0.00 ± 0.00bz 1.00 ± 0.00ax 0.00 



24 

 

 

Figure 3 

Growth behavior of E. coli O157:H7 in relation to the type of samples in nutrient solution and roots of 

Genovese basil in the traditional system over twenty-seven days. 

 

 

For the Lemon variety analysis (Figure 4), significant differences (p < 0.05) in the growth 

behavior of E. coli O157 were observed on days 0, 24, and 27 (a-d). Subsequently, days with the same 

letter designation (a-a) showed no significant differences (p > 0.05) for the other sampling days. 

Although the specific days of bacterial reduction varied between the Genovese and Lemon varieties, 

the overall trend remained consistent. This illustrates the similar growth behavior of the bacteria 

when comparing nutrient solution and root samples. 

 

 

 

 

 

 



25 

 

 

Figure 4 

Growth behavior of E. coli O157:H7 in relation to the type of samples in nutrient solution and roots of 

Lemon basil in the traditional system over twenty-seven days. 

 

 

In Figure 5, the growth behavior of E. coli O157:H7 is presented as a function of time. The 

Wilcoxon Test was utilized to compare two groups, nutrient solution and roots, in relation to the 

dependent variable of bacterial counts in Log CFU/mL. This test revealed significant differences (p < 

0.05) in bacterial counts between these two sample types. Additionally, the Kruskal-Wallis Test was 

applied to compare independent samples across different sampling days, demonstrating significant 

differences (P < 0.05) in bacterial counts over time. Each letter (a-f) in the figure denoted differences 

between sampling days. The results showed that, although the Nutrient Solution initially had a higher 

concentration of bacteria, the rate of bacterial decrease over time was greater compared to the Roots. 

Specifically, by day 24 in the traditional system, E. coli O157:H7 was no longer detectable in the Roots 

samples. This absence was further confirmed through an enrichment process, which also indicated no 



26 

 

 

presence of the bacteria. These findings highlight the differential impact of the sample type on the 

survival and reduction of E. coli O157:H7 over the study period. 

Figure 5 

Growth pattern of E. coli O157:H7 in nutrient solution and roots of traditional cultivars of Genovese 

and Lemon basil over a 27-day production cycle. 

 

 

The behavior of bacterial growth in aquatic environments is influenced by various factors, 

from the system's temperature to the nutrient solution used. In aquatic environments, bacterial 

survival is strongly dependent on the source and presence of nutrients in the system since they allow 

bacterial cells to accumulate essential material for motility functions, energy production, and cellular 

biomass, allowing bacteria to reduce their disappearance rate (Ozkanca, 1993). This explains why, in 

the traditional system in nutrient solution, a higher concentration of E. coli O157:H7 was observed 

due to the nutrient-rich medium. Likewise, despite being in a rich medium, the bacteria would be 

exposed to certain components of the plant that inhibit bacterial growth. On the other hand, although 

bacteria can lose the ability to grow in an environment when they enter a viable but non-culturable 

state, they can continue absorbing nutrients and maintaining a reserve of metabolites (Dhulappanavar 

& Gibson, 2023). 



27 

 

 

In the case of roots samples in the system during the sampling days, the bacteria prevailed 

differently than in nutrient solutions, which suggests that the bacteria attached to the roots and 

presented reduced but longer survival rates because it was able to provide an environment most 

favorable for survival such as an anaerobic environment with less exposure to environmental 

stressors. These findings are similar to a study conducted by Dankwa et al., (2020) which found that 

in hydroponic systems the highest bacterial counts were found in the substrate for coliforms 

associated with the fact that some substrates retain nutrients on their surfaces serving as a nutrient 

reservoir for plant use and providing a favorable environment for microorganisms to thrive.  Likewise, 

the bacteria decrease in root samples over time could have been due to the internalization of the 

bacteria in other parts of the plant not sampled. Studies have shown that E. coli O157:H7 can 

internalize hydroponically grown plants through the root system's injury, allowing the bacteria to 

disperse to the stem and leaves (Moriarty et al., 2019). 

The reduction of E. coli O157:H7 counts in the traditional system from 6 to 1 Log CFU/mL in 

the roots over a 27-day period demonstrates that, although E. coli O157:H7 was present, no bacterial 

counts were reported in the nutrient solution by day 24. This reduction could be attributed to 

compounds released by basil through its roots, specifically the plant's exudates. Root exudates 

released in the rhizosphere primarily consist of carbon-based compounds but also include released 

ions (such as H+), inorganic acids, oxygen, and water; among these compounds are secondary 

metabolites and phenols, which have been shown to have antibacterial mechanisms (Badri y Vivanco, 

2009). Similarly, in the traditional system, the reduction in bacterial counts in the roots could be 

attributed to the concentration of exudates in the system, influenced by environmental factors and 

light intensity. In the traditional system, a 16-hour light cycle with red, white, and blue lights was used. 

Studies have shown that light intensity affects the exudation of secondary metabolites due to changes 

in photosynthesis, as the root exudation process follows diurnal rhythms, increasing during light 

periods (Badri & Vivanco, 2009). 



28 

 

 

The reduction of bacteria in the roots of both varieties is attributed to a compound present in 

both, specifically rosmarinic acid (RA). Research has shown that rosmarinic acid (RA) is one of the 

major compounds exuded by basil roots in response to pathogen attack or stress conditions. This 

compound has been demonstrated to cause morphological changes in the cells of the affected 

organisms (Bais et al., 2002). 

The reduction of E. coli O157:H7 observed over the 27 days of sampling demonstrated the 

efficacy of different compounds in both Genovese and Lemon basil varieties. Lemon basil contains 

60.63% citral, with α-citral (33.34%) and β-citral (27.29%), whereas Genovese basil has high levels of 

linalool (40.77%), eucalyptol (29.37%), and trans-α-bergamotene (4.27%), with eugenol (2.63%) 

present in lower concentrations compared to Lemon basil (Ciriello et al., 2023). Essential oils (EOs) 

from each variety contain various active compounds that affect bacterial cells. For instance, eugenol 

and linalool increase cell membrane permeability, leading to cell viability loss by disrupting ionic 

homeostasis in the electron transport chain, decreasing intracellular potassium levels while increasing 

extracellular levels, ultimately resulting in a loss of vital functions and cell death (Stan et al., 2022). 

The mechanism of action against bacteria is determined by the active molecules of each compound 

and their structures, such as the phenolic and aldehyde compounds in basil, which are responsible for 

the cytotoxicity of essential oils. These compounds lead to the denaturation of cytoplasmic proteins 

and the inactivation of cellular enzymes, resulting in bacterial cell death (Raut & Karuppayil, 2014). 

Microgreens System 

The production method of microgreens is similar to that used for sprouted seeds, creating 

favorable conditions for the growth of microbial pathogens since they are typically grown in closed 

facilities. Due to this reason, this section will focus on determining the growth of E. coli O157:H7 in 

Mat Samples and Roots Samples over a shorter period, considering two varieties of basil in the study. 

Table 6 shows the results for the growth of E. coli O157:H7 for mat samples, where no 

significant differences (p>0.05) were observed in the concentration of the bacteria between the 



29 

 

 

Genovese and Lemon varieties in mat samples throughout the fourteen days of study. This could 

suggest that, in this specific system, the two plant varieties did not significantly influence the growth 

of the bacteria in the microgreen system, in which the variety variable did not significantly affect the 

dependent variable of the study. 

Table 6 

Differences between the growth of E. coli O157:H7 in mat between the Genovese and Lemon 

varieties in the microgreens system over fourteen days. 

                                      Variety 

Time (Day) 

Genovese Lemon   

Mat 
Log CFU/mL ± SD 

Mat 
Log CFU/mL ± SD  

CV (%) 

0 5.08 ± 0.16a 5.26 ± 0.06a 2.19 
3 3.39 ± 0.36a 3.61 ± 0.37a 10.35 
6 2.65 ± 0.44a 2.57 ± 0.40a 16.16 
9 2.17 ± 0.42a 1.83 ± 0.26a 16.67 
14 1.00 ± 0.00a 1.59 ± 0.36a 11.27 

Note. SD: Standard deviation. CV (%): Coefficient of variation. 
a-b Means with the same lowercase letters for E. Coli log CFU/mL indicate no 

significant differences (p > 0.05) in each column. Each Mat Log CFU/mL microbiological count per variety represents the mean of 3 

repetitions.  

 

The growth of E. coli O157:H7 in the roots of both basil varieties within the microgreens 

system is presented in Table 7. The data indicates that there are no significant differences (p > 0.05) 

in bacterial counts between the two varieties' roots, as the daily counts are statistically similar 

throughout the fourteen-day period. In both cases, the logarithmic bacterial counts remained 

comparable. 

 

 

 



30 

 

 

Table 7 

Differences between the growth of E. coli O157:H7 in roots between the Genovese and Lemon 

varieties in the microgreens system over fourteen days. 

                                      Variety 

Time (Day) 

Genovese Lemon   

Roots 
Log CFU/mL ± SD 

Roots 
Log CFU/mL ± SD 

CV (%) 

0 5.07 ± 0.18a 5.18 ± 0.24a 4.12 
3 3.12 ± 0.19a 3.05 ± 0.11a 4.72 
6 2.61 ± 0.23a 2.34 ± 0.29a 10.47 
9 1.30 ± 0.52a 1.74 ± 0.23a 26.79 
14 1.06 ± 0.10a 1.43 ± 0.15a 10.16 

Note. SD: Standard deviation. CV (%): Coefficient of variation. 
a-b Means with different lowercase letters for E. Coli log CFU/mL indicate 

significant differences (p < 0.05) in each column. Each microbiological count of Roots Log CFU/mL per variety represents the mean of 3 

repetitions.  

 

Figures 6 and 7 show how the variety variable did not have a significant effect after the 

analysis with the Wilcoxon test and T-test (p > 0.05) in the comparison of the Genovese and Lemon 

varieties. The reduction of the bacteria decreased in the sampling period similarly, which is why the 

same letters (a-a) are assigned between groups. In relation to the data obtained, the effect of both 

varieties did not show differences, although antimicrobial compounds are attributed to them. The 

Genovese and Lemon varieties belong to the wide range of basil that is produced by the properties 

and preferences of the market; however, for microgreens both varieties have a similar concentration 

of polyphenols, flavonoids, anthocyanins, and nitrates in which their antioxidant capacity is the one 

that differs, being 19.6% for Genovese variety and 11.8% for Lemon Variety (Fayezizadeh et al., 2023). 

 

 

 



31 

 

 

Figure 6 

Growth of E. coli O157:H7 in mat samples between the Genovese and Lemon varieties in the 

microgreens system over fourteen days. 

 

 

The data obtained showed that no significant differences (p > 0.05) were observed between 

the two varieties (Genovese and Lemon basil) in the mat and roots samples on each of the sampling 

days. A reduction in bacterial count was observed over time for both the mat and roots samples. 

Although this reduction was not statistically significant as indicated by the different letters (a-e) 

denoting time points, there was an evident decline in bacterial concentration from approximately 5 

Log CFU/mL to 1 Log CFU/mL. This reduction was more pronounced in the root samples over time, 

which can be attributed to the components present in the roots. 

 

 

 

 

 



32 

 

 

Figure 7 

Growth of E. coli O157:H7 in roots samples between the Genovese and Lemon varieties in the 

microgreens system over fourteen days. 

 

 

Table 8 presents the bacterial counts of E. coli O157:H7 for both basil varieties, categorized 

by sample type and time over the fourteen-day sampling period. The data shows a logarithmic 

reduction over time, with significant differences (p < 0.05) observed on day 3, where counts were 3.50 

Log CFU/mL for mat samples and 3.00 Log CFU/mL for roots samples, as indicated by different letters 

(a-b) between samplings. No statistical differences (p > 0.05) were observed on days 0, 6, 9, and 14, 

which can be attributed to the bacterial inhibition properties of the microgreen compounds. However, 

significant differences (p < 0.05) were noted across different sampling days, with each day being 

statistically distinct, as represented by the letters (u-y). 

 



33 

 

 

Table 8 

Growth of E. coli O157:H7 in mat and Roots between the Genovese and Lemon varieties in the 

microgreens system over fourteen days. 

Note. SD: Standard deviation. CV (%): Coefficient of variation. 
a-b Means with different lowercase letters for E. Coli log CFU/mL indicate 

significant differences between treatments in each variety (p < 0.05) in each column; 
u-y 

Means with different lowercase letters for E. Coli 

log CFU/mL indicate significant differences between the evaluated times (p < 0.05) in each row. Each Mat and Roots Log CFU/mL 

microbiological count per variety represents the mean of 3 repetitions.  

 

The bacterial growth behavior of E. coli O157:H7 depending on the type of sampling over time, 

is shown in Figures 8 and 9. Over time, an exponential logarithmic reduction was observed. Although 

significant differences (p < 0.05) were observed on day 14, the bacterial reduction was similar in both 

mat samples and root samples at the beginning. However, by the last day of sampling, a difference 

was evident between mat Samples and roots samples. Additionally, in contrast to the traditional 

system, in microgreens, both samples after inoculation did not present significant differences on the 

first day, as was observed in the nutrient solution of the traditional system. This behavior may be due 

to the bacteria in the mat not taking advantage of a nutrient-rich environment since they are attached 

to a mat rather than being in a submerged water system. This finding is supported by an investigation 

conducted by Wright and Holden (2018), which found higher levels of bacteria in the mat samples in 

the absence of plants. Despite having an initial similar concentration of bacteria, the bacterial 

concentrations in the plants were lower due to the presence of plant compounds. 

 

                                                                Variety 

Time (Day) 

Genovese Lemon  

Mat  
Log CFU/mL ± SD 

Roots 
Log CFU/mL ± SD 

Mat  
Log CFU/mL ± SD 

Roots 
Log CFU/mL ± SD 

CV (%) 
 

0 5.08 ± 0.16au 5.07 ± 0.18au 5.26 ± 0.06au 5.18 ± 0.24au 3.15 
3 3.39 ± 0.36av 3.12 ± 0.19bv 3.61 ± 0.37av 3.05 ± 0.11bv 7.53 
6 2.65 ± 0.44aw 2.61 ± 0.23aw 2.57 ± 0.40aw 2.34 ± 0.29aw 13.31 
9 2.17 ± 0.42ax 1.3 ± 0.52ax 1.83 ± 0.26ax 1.74 ± 0.23ax 21.73 
14 1.00 ± 0.00ay 1.06 ± 0.1ax 1.59 ± 0.36ay 1.43 ± 0.15ax 10.71 



34 

 

 

Figure 8 

Growth behavior of E. coli O157:H7 in relation to the type of samples in mat and roots of Genovese 

basil in the microgreens system over fourteen days. 

 

 

Similarly, the Lemon variety showed a significant reduction in bacterial counts to day 14, as 

did the Genovese variety, demonstrating the reduction effect these basil varieties possess due to their 

essential oils. Despite not showing significant differences between them, this effect can be attributed 

to both varieties sharing the same environmental conditions and being subject to the same changes. 

Additionally, for the Lemon variety, during the initial sampling days, the reduction in bacterial counts 

between mat samples and roots samples was not significantly different until day 9 (p > 0.05). 

 

 

 

 

 

 

 

 



35 

 

 

Figure 9 

Growth behavior of E. coli O157:H7 in relation to the type of samples in mat and roots of Lemon basil 

in the microgreens system over fourteen days. 

 

 

Figure 10 shows the growth behavior of E. coli O157:H7 as a function of time, where for the 

time variable, significant differences were reported (p < 0.05) using Krystal-Wallis and Wilcoxon test, 

having a significant and statistically different reduction as they progressed in the sampling days. The 

different letters symbolize differences between the days evaluated (a-e), where on day 9 for roots, 

the bacterial counts are reduced further until reaching day 14 with the same rate of reduction, so both 

days did not present differences. Statistics (p > 0.05). 

 

 

 

 

 

 



36 

 

 

Figure 10 

Growth pattern of E. coli O157:H7 in media (mat) and roots of microgreen cultivars of Genovese and 

Lemon basil over a 14-day production cycle. 

 

The growth behavior of E. coli O157:H7 in the microgreens system is directly related to the 

environmental conditions that affect the colonization of bacteria, one of the main factors being the 

availability of water and local humidity (Xiao et al., 2015). Environmental factors in the study could 

have impacted bacterial survival since the microgreens were grown in a controlled environment 

chamber. Temperature, along with pH influences E. coli enzyme activity where the ideal temperature 

for E. coli is 37 °C, with a minimum of 7 °C and a maximum of 46 °C (Bryant, 2017). Deviations from 

this range can cause enzyme denaturation and disrupt cellular functions. It is worth noting that the 

microgreens were maintained at a temperature of 20 °C within the controlled environment chamber, 

which is below the optimal temperature of 37 °C for the bacteria. Consequently, this lower 

temperature likely resulted in slower growth or could have been attributed to reduced metabolic 

activity. 



37 

 

 

Based on the results obtained, it was determined that both Genovese and Lemon varieties did 

not present significant differences in the logarithmic decrease of the bacteria; this could be because 

the properties of both varieties had a similar effect on bacterial inhibition. However, the effect of time 

in the study determined significant differences in the reduction of E. coli O157:H7 for both mat 

samples and roots samples, with a greater reduction in the last two days of sampling. This can be 

reinforced by research carried out by Wright and Holden (2018) where in a study they quantified 

bacterial colonies of E. coli O157:H7 in microgreens they determined that viable counts decreased on 

day 12. In addition, on days 9 and 14, no significant differences were shown, which may be due to a 

lower reduction in bacteria on day 14 compared to the other days. 

The bacterial counts for the present system reduced throughout the 14 days of sampling, in 

which the initial inoculum concentration of 10^6 CFU/mL decreased to low logarithmic 

concentrations. It should be noted that microgreens are more vulnerable to bacterial internalization 

than mature plants since, in immature plants, the structures that protect the plant system are not 

completely formed, which allows bacteria to enter the Xylem, unlike mature plants that protect them 

with the Casparian strip: a cell wall made up of insoluble substances (E. R. Turner et al., 2020). In the 

case of mat samples, a reduction was also observed, though it was less pronounced compared to the 

roots. This could be attributed to the inert and porous substrate, which helps retain the nutrient 

solution for the bacteria without directly interacting with the inhibitory compounds emitted by the 

basil roots (Rusu et al., 2021). 

The reduction in the concentration of E. coli O157:H7 in the microgreens system could be 

attributed to the essential properties of the basil varieties, as well as the different conditions of the 

system that impacted the development of the plants and facilitated the potential internalization of 

the bacteria. The roots of microgreens, being smaller and more exposed, present more damage 

compared to other systems; this allows pathogens to adhere to the roots and be internalized into the 

plant within approximately seven days (Wang et al., 2021).  



38 

 

 

In general basil microgreens contain volatile oils at concentrations ranging from 0.2% to 1.7% 

with the main compounds including estragole 15-80% linalool 35-50% and eugenol as well as 

flavonoids and phenolic acids such as chicoric acid (Rusu et al., 2021). Due to their rapid and active 

growth stage, microgreens typically have higher concentrations of nutrients and antioxidants 

compared to mature plants. The antioxidant capacity, as previously mentioned, being 19.6% for the 

Genovese variety and 11.8% for the Lemon Basil variety, in relation to the presence of linalool, 

estragole, and phenolic acids, leads to a decrease in pH, which inhibits bacterial growth (Fayezizadeh 

et al., 2023). Based on the findings, bacterial inhibition may be attributed not only to the structure of 

the compounds but also to stress factors affecting bacteria, such as pH. It is important to note that 

the optimal pH range for E. coli growth is between 6.0 and 7.0, with a minimum tolerance of 4.0 to 4.5 

(Montville et al., 2012). Consequently, changes in pH can affect E. coli growth and survival, as low pH 

can lead to membrane denaturation and inhibit growth. Furthermore, organic acids such as phenolic 

acids, once dissociated inside the cell, can penetrate the cell membrane, causing the release of 

protons and subsequently reducing intracellular pH (Bryant, 2017). 

 

 

 



39 

 

 

Conclusions 

The study revealed that survival rates of E. coli O157:H7 varied significantly between the 

traditional system and microgreens, indicating that the growth environment was influential in 

bacterial survival. 

It was found that the selection of the basil variety was not a determining factor in the effectiveness of 

pathogen control, but rather the conditions in each system. 

Genevese and Lemon basil varieties did not present significant differences in the inhibition of 

the bacteria, however, both varieties allowed E coli O157:H7 to attach to their roots, leading to longer 

persistence in the hydroponic system. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



40 

 

 

Recommendations 

Increase the sample size, as well as the number of repetitions, and consider the different 

growing conditions to reduce variability in the data.  

Perform an analysis of phenolic compounds, essential oils and nutrient solution in the plant 

of interest to identify components with antimicrobial properties.  

Optimize crop conditions that influence the inhibition of bacterial growth, such as nutrient 

solution, pH, electrical conductivity, temperature, and light spectrum.  

Explore and apply new strategies in the cultivation system, such as more controlled conditions 

that affect the production of bioactive compounds.  

Designate ideal spaces during sampling to avoid risks and cross-contamination, ensuring 

safety and promoting an ideal work environment.  

Work with different crops and varieties that are suitable for the hydroponic system to gather 

more information and determine differences for research improvement. 

Perform a cost-benefit analysis to determine the profitability of scaling hydroponic units 

versus implementing alternative methods. 

 

 

 

 

 

 

 

 

 

 



41 

 

 

References 

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44 

 

 

Appendices 

Appendix A 

Ampicillin requirement 

 

Ampicillin requirement 
 

Stock Concentration ampicillin 50μg/mL 
 

4mg/mL using sterile distilled water and stored at 4 °C. 
 

Added 12.5 mL of stock to 1 L sterile distilled water to achieve a final concentration of 50μg/mL. 
 

50μg/mL of ampicillin in 1 L of sterile distilled water 

4𝑚𝑔

𝑚𝑙
𝑥 12.5 𝑚𝐿 = 50 μg/mL 

25 μg /mL of ampicillin in 1 L of sterile distilled water 

12.5 𝑚𝐿 𝑥 
250μg

𝑚𝐿
 𝑥 

𝑚𝐿

50 μg
= 62.5  

 

 

 

 

 

 

 

 

 

 

 

 

 



45 

 

 

Appendix B 

Sampling schedule for traditional system and microgreens 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Traditional System 0 3 6 9 12 15 18 21 24 27

Germination

24-Feb 10-Mar 11-Mar 12-Mar 15-Mar 18-Mar 21-Mar 24-Mar 27-Mar 30-Mar 2-Apr 5-Apr 8-Apr

Germination

Transfer to units

Prepare inoculum

Inoculation

Water sampling

Plant sampling (Roots)

Microgreens System Germination 0 1 3 6 9 14

18-Mar 22-Mar 23-Mar 24-Mar 26-Mar 29-Mar 1-Apr 6-Apr

Celanining and sanitizing of trays

Sowed

Expose the trays to light

Prepare inocolum

Inoculate

Media sampling

Plant sampling (Roots)



46 

 

 

Appendix C 

Box plot of E. coli O157:H7 growth pattern in nutrient solution and roots of the traditional Genovese 

basil cultivar over a 27-day production cycle 

 

 

 

 

 

 

 

 

 

 

 

 



47 

 

 

Appendix D 

Box plot of E. coli O157:H7 growth pattern in nutrient solution and roots of the traditional Lemon 

basil cultivar over a 27-day production cycle 

 

 



48 

 

 

Appendix E 

Box plot of E. coli O157:H7 growth pattern in nutrient mat and roots of the microgreen Genovese 

basil cultivar over a 27-day production cycle 

 

 

 

 

 

 

 

 

 

 

 

 



49 

 

 

Appendix E 

Box plot of E. coli O157:H7 growth pattern in nutrient mat and roots of the microgreen Lemon basil 

cultivar over a 27-day production cycle 

 

 

 

 



50 

 

 

Appendix F 

Hydroponic units used for Genovese and Lemon basil varieties in the traditional system 

 

 

 

 

 

 

 



51 

 

 

Appendix G 

Hydroponic units used for Genovese and Lemon basil varieties in the microgreens system