Effectiveness of the Aeration Process Using Radiant Catalytic Ionization (RCI) in the Elimination of Selected Pathogenic Microorganisms in Municipal Wastewater and Slurry—A Pilot Laboratory-Scale Study

Abstract

(1) Background: Improper disinfection of slurry and municipal wastewater poses a serious threat to public health. These fluids are reservoirs of viruses, bacteria, fungi and parasites. (2) Methods: This study aimed to evaluate, on a laboratory scale, the disinfection effectiveness of fine bubble aeration with air activated by radiant catalytic ionization (RCI) against Enterococcus faecalis, Escherichia coli, Salmonella Senftenberg W775, Listeria monocytogenes, Clostridioides difficile, Aspergillus niger and Ascaris suum eggs in comparison to conventional atmospheric air aeration. The inactivation kinetics was calculated on the basis of Weibull and first-order models. (3) Results: The final number of microorganisms on the last day in the slurry disinfected with RCI ranged from 1.14 × 10² for L. monocytogenes to 1.91 × 10⁷ CFU (colony-forming unit) × mL⁻¹ for C. difficile. After using atmospheric air aeration, the bacteria number ranged from 2.82 × 10³ for L. monocytogenes to 2.24 × 10⁷ CFU × mL⁻¹ for C. difficile. In the case of aeration using RCI technology, the maximum time required to eliminate 99.9% of the microorganisms population was 20.84 days in slurry and 16.40 days in wastewater and was determined for A. niger. In the case of atmospheric air, this time was 47.76 days in slurry and 28.74 days in wastewater and was determined for C. difficile. In turn, the time to inactivate the number of invasive A. suum eggs by 90% was 20.70 and 24.61 weeks for RCI and 21.33 and 27.82 weeks for atmospheric air, respectively. Both in the case of slurry and municipal wastewater, disinfection with RCI was more effective than aeration with atmospheric air. (4) Conclusions: Our study, for the first time, exploits the possibility of using RCI in aeration to improve the efficiency of pathogen elimination from wastewater and slurry.

1. Introduction

The problem of proper slurry management is present primarily during intensive livestock production, especially in the case of industrialized livestock farms. Treated wastewater or slurry is most often used for the irrigation and fertilization of agricultural fields, which is an attractive solution due to the replenishment of water deficits (which are increasingly observed in many regions of the world), as well as by enriching the soil with nutrients [1,2]. A high-value, fast-acting natural fertilizer readily used in agriculture is slurry, which, in addition to its high nutrient content for plants, has a high biodiversity of resident microorganisms [3]. The danger associated with such management of slurry and treated wastewater is the presence of potentially pathogenic microorganisms, which can get into the soil and water and on the surface of plants [1,2,4,5]. Phenol present in sewage also poses a risk to human and animal health, hence the need to remove it using physicochemical and biological methods, including those using biochar-immobilized functional microorganisms [6].

Microorganisms isolated from raw wastewater and slurry can come directly from human and animal feces or enter from the external environment. In addition to saprophytes, both untreated wastewater and slurry can contain pathogenic microorganisms, including Enterococcus spp., Staphylococcus spp., Salmonella spp., Escherichia coli, Campylobacter spp., Listeria monocytogenes, Yersinia enterocolitica, Brucella spp., Bacillus anthracis and Mycobacterium spp. [7,8,9]. Viruses, including African swine fever virus, parasite eggs and oocysts, have also been detected in slurry and raw wastewater [10,11,12]. Most of these pathogens are associated with acute gastrointestinal symptoms, which can occur as a result of contact with raw or inadequately treated wastewater and slurry. Moreover, inadequately treated wastewater and slurry can lead to the production of contaminated food and the spread of contaminants in the environment through leaching and water runoff.

The microbiological quality of treated wastewater depends primarily on the proper execution of the water treatment process. Technologies commonly used for secondary and tertiary treatment seem to guarantee microbiological safety for the soil environment into which treated slurry or wastewater is introduced [13]. The level of risk of environmental contamination from irrigation is also related to the irrigation strategy adopted (mixing with other water irrigation systems) [14]. Pathogens isolated from irrigation water samples include Salmonella spp., E. coli O157:H7, Campylobacter spp. and the cysts and oocysts of Giardia and Cryptosporidium [15]. In the case of slurry, the least complicated method of disinfection through storage does not have a clearly defined effectiveness in reducing indicator bacteria and pathogens and leads to the formation and emission of ammonia (NH₃) and methane (CH₄) into the atmosphere [16,17,18]. In contrast, anaerobic fermentation, which, depending on the thermal conditions (mesophilic or thermophilic), makes it possible to achieve a satisfactory level of pathogen reduction in a relatively short time [19,20]. The application of Fe²⁺/Fe²⁺/periodate (PI) coupled with the polyoxometalate (POM) sludge conditioning method leads to the intensive damage of cellular structures, which may contribute to reducing the risk of transferring pathogenic microorganisms in slurry and treated sewage [21].

The stabilization and disinfection of potentially microbiologically contaminated liquids used for fertilization or irrigation can also be achieved by aerating them. Most often, this process consists of aerating the liquid using mechanical agitators and rotors or devices, forcing air bubbles into the liquid [22]. Depending on the system, the aeration reactor can be filled once and emptied at the end of the process (a closed system), or the liquid can be supplied continuously, guaranteeing a higher stability of changes in the aerial material (continuous system) compared to a closed system. Aeration can take place under extremely different thermal conditions, from temperatures below 20 °C to values exceeding 70 °C [3,23].

The results of many studies confirm the effectiveness of aeration as a method for reducing the number of intestinal bacterial pathogens, e.g., E. coli or Salmonella, but also prove that the rate of this reduction largely depends, for example, on the intensity of aeration [24,25]. Considering the fact that the increase in temperature of the liquid subjected to the process of aeration, resulting from the life processes of indigenous microorganisms, is relatively small, it may not be a sufficiently effective factor with a hygienic function. The lack of a system for additional heating of the aerated liquid may also extend the time necessary to obtain a satisfactory reduction in the number of pathogens to values that are practically unreasonable. This allows us to predict with high probability that the disinfection efficiency of the aeration process of, e.g., wastewater or slurry, will be difficult to estimate and different for individual installations.

This problem may be solved by combining liquid slurry aeration with another disinfection method to achieve higher and more stable success rates for reducing pathogenic microorganisms. One technology whose bactericidal effect has been confirmed in air and on various surfaces is radiant catalytic ionization (RCI) [26,27,28]. RCI is classified on the borderline between physical and chemical disinfection methods. This method is based on the phenomenon of photocatalysis and the production of reactive oxygen species (ROS) which react with cellular proteins, lipids and nucleic acids, leading to cell death [29]. The device enabling the use of this technology consists of a matrix covered with a coating, which includes, in addition to TiO₂ acting as a catalyst, rhodium, silver and copper. Its next necessary component is a unit emitting UV radiation, which, in contact with the hydrophilic matrix coating, initiates or stimulates the processes of peroxide and hydroxide formation [30]. The RCI method has been shown to be effective in eliminating microorganisms in air [31,32,33] and on surfaces [29]. Reactive oxygen species (ROS) are the main product of RCI devices. ROS exhibit bactericidal activity through the following mechanisms: by affecting the main cellular components of microorganisms (polysaccharides, proteins and nucleic acids), leading to cell death [34]. The main ROS emitted by the RCI device is the hydroxyl radical, leading to the inactivation of microorganisms [29,35]. The hydroxyl radical can cause irreversible damage to the cell wall and changes in cell membrane permeability, ultimately leading to the leakage of internal elements and cell death [36].

The application of RCI technology to reduce the level of fluid contamination represents innovative research on a global scale. However, given that the use of photocatalysis with UV light and a TiO₂ catalyst has demonstrated inhibitory effects on various species of bacteria and parasites in water and effectively reduced the survival rate of E. coli in fresh and salt water, an attempt to use RCI-activated air for slurry aeration may be warranted [37,38,39]. An attempt to use this method for wastewater treatment was proposed by Zhao and Yang [40].

The use of so-called hybrid techniques for wastewater treatment, combining oxidative methods with UV radiation or ultrasound action, creates a synergistic effect in generating chemically active compounds, including free radicals. Their multiplied destructive effect on the cellular structures of microorganisms increases the disinfection efficiency of hybrid techniques. In addition, the combination of certain techniques may minimize the limitations and disadvantages of the performance of the methods used individually. This has a positive impact on the technological parameters and efficiency of individual liquid waste management [41,42].

The aim of this study was to conduct a laboratory-scale evaluation of the disinfection effectiveness of the fine-bubble aeration process using RCI-activated air against selected bacteria, mold fungi and parasite eggs and compare it with the effectiveness of conventional aeration atmospheric air. Specifically, the research was designed to demonstrate the potential of this novel technology in eliminating pathogens from slurry and wastewater. It is important to emphasize that this work represents fundamental research, not the development of a ready-to-implement technological solution or a finalized process scheme. The schematic presented was created solely for laboratory purposes. This study marks the world’s first attempt to utilize RCI in this manner and is intended to stimulate interest among decision-makers in the wastewater treatment sector.

2. Materials and Methods

2.1. Research Material

The research material consisted of samples of pig slurry and raw municipal wastewater. The collected samples were separated for the solid and liquid fractions by centrifugation (4500 rpm for 30 min). The separated fractions were then mixed together to obtain thin (2% dry matter), medium (5% dry matter) and thick (10% dry matter) slurry and wastewater. The samples prepared in this way were poured into sterile bags with a string closure and were subjected to radiation sterilization with a beam of high-energy electrons at a dose rate of 20 kGy.

2.2. Physical and Chemical Determinations

In each batch of pig slurry and municipal wastewater, a number of physical and chemical determinations were performed immediately after its delivery to the laboratory. The pH was determined by the potentiometric method [43], the REDOX potential by the potentiometric method, BOD₅ by the electrochemical method [44], COD by the spectrophotometric method [45], the dry matter content by the weight-drying oven method, total nitrogen content by the spectrophotometric method and total potassium and total phosphorus by inductively coupled plasma atomic emission spectrometry (ICP-OES) [46]. Additionally, for each sample, the temperature, pH, REDOX potential and dry matter content were determined at the beginning and the end of aeration.

2.3. Microorganisms and Parasite Egg Suspension

The following environmental microorganisms were used in the study: Enterococcus faecalis, Escherichia coli, Salmonella Senftenberg W775, Listeria monocytogenes and Clostridioides difficile, mold fungi Aspergillus niger and Ascaris suum eggs. Bacteria and fungi came from the collection of the Department of Microbiology of L. Rydygier Collegium Medicum in Bydgoszcz of the Nicolaus Copernicus University in Toruń. In turn, A. suum eggs were obtained from sexually mature females. The uteri were dissected, and at a later stage, their fragments, about 2 cm long from the bifurcation, were cut in order to avoid the presence of unfertilized eggs in them.

The tested methods should ensure the effectiveness of disinfection and the safety of wastewater, even in the case of exceptionally high levels of microbiological contamination, which can occur as a result of various types of failures or discharge of wastewater from healthcare facilities. For this reason, we used suspensions containing large numbers of the tested microorganisms. Bacterial suspensions of 5 McFarland scale (approx. 10¹¹ CFU (colony-forming unit)/mL) were prepared in sterile saline (Polpharma, Starogard Gdański, Poland) from colonies grown (after 24 h incubation) on CAB agar (Biomerieux, Marcy-l’Étoile, France). Mold fungi suspensions of 10 McFarland were prepared in sterile 0.05% aqueous Tween 80 (Biomaxima, Lublin, Poland). The fungi were cultured on Sabouraud agar (Biomaxima, Lublin, Poland) for 48 h. In order to obtain the A. suum suspension, the eggs were pressed from pieces of the uterus with a glass rod into a Petri dish filled with 0.1 M H₂SO₄ (Pol-Aura, Zawroty, Poland) solution in sterile water.

2.4. Inoculation of Wastewater and Slurry with Microorganisms and Parasite Eggs

For the contamination of wastewater and slurry, suspensions prepared as described in Section 2.3 were used in triplicate. Based on the previous tests, the volume of the added suspension was determined for 25 mL/L of the sample. To avoid changes in the parameters of wastewater and slurry, the suspensions prepared in physiological saline were centrifuged at 10,000 rpm for 5 min, and the supernatant was removed. Then, the sediment of bacteria or fungi was hydrated by adding slurry or wastewater of the appropriate density in a volume equal to the volume of saline removed. The prepared suspensions were added to the samples in the aeration chamber.

The egg suspension was then introduced in a volume of 1 mL into perlon pouches with holes 28 µm in diameter. Then, the pouches filled with the suspension were tightly tied and placed in the volume of sewage or slurry intended for aeration.

The aeration process started 1 h after the contamination of the tested samples with microorganisms.

2.5. Aeration Process

Separately for each microorganism and each density of wastewater and slurry, fine-bubble (0.3–0.5 mm) aeration was carried out using an aeration set constructed according to the diagram shown in Figure 1.

The aeration process was performed in a 25 L tank in a closed system without access to fresh wastewater/slurry. The maximum working pressure of the pump was 0.05 MPa, with a capacity of 240 L/min and a maximum aeration intensity of 9.6 vvm. Aeration was carried out in two variants: (1) with the use of atmospheric air (open valve 4, closed valve 3 and turned-off RCI device) and (2) with air “activated” using RCI technology (valve 3 open, RCI device on and valve 4 closed). The efficiency of the RCI device was 1/5 higher than that of the air pump; therefore, a buffer tank (2) was used, which was a temporary store of “activated” air. The aeration intensity is described as the oxygen transfer rate (OTR), defined as the rate at which oxygen is transferred from the gas phase (atmospheric air/RCI air) into the 1 m³ of slurry or wastewater. The values of the OTR were determined by monitoring the increase in the dissolved oxygen concentration in the liquid upon aeration and adjusted to the three levels of 1, 2 or 3 gO₂/m³/h, regardless of the wastewater/slurry dry matter content. These values were controlled by the settings of a commercially available aeration controller used in domestic wastewater treatment plants, in conjunction with a flow meter (6) and a dissolved oxygen sensor (9), which allowed for the precise control of the process conditions. The foam generated during aeration was recirculated back to the aeration chamber, and the released gases were discharged through the filter (10) outside the chamber. Aeration was carried out for 12 days, with sampling after 0, 1, 2, 4, 6, 8, 10 and 12 days. The temperature and dissolved oxygen concentration were monitored throughout the aeration period.

2.6. Detailed Description of RCI System

The structure of the RCI cell allows the flow of purified air (Figure 1). The RCI device consists of two matrices, with a structure of elongated tubular elements made of polycarbonate arranged in parallel to resemble a honeycomb. Each tube has a diameter of 4 mm and a length of 15 mm. The combined active area of both matrices, representing the total inner surface of all the tubes coated with TiO₂ and metal clusters (rhodium, silver and copper), is 763.28 cm². The coating of the basic matrix elements exhibits hydrophilic properties. In addition, the RCI unit is equipped with an 8 W UV lamp. The UV lamp uses argon gas with mercury and carbide fibers with a spectrum of 100 and 367 nm. It also generates UV rays with wavelengths of 185 and 254 nm. As a result of catalytic oxidation, stimulated by UV radiation, reactive oxygen species are generated at the heterogeneous (gas–solid) interface: hydroxyl radicals, superoxide ions and hydroxide ions. The total number of negative air ions (NAIs: O₂⁻, CO₃⁻, NO₃⁻, OH⁻, NO₂⁻, HCO₃⁻ and their water clusters) generated is about 5.0 × 10⁴ ions × cm⁻³ of air determined using an air ion counter. The device does not emit ozone. Details of the RCI technology are contained in Patent No. US 8,585,979 B2.

2.7. Determining the Number of Microorganisms and the Percentage of Invasive Eggs Recovered from the Test Samples

Before sampling, the entire volume was thoroughly mixed. Ten ml of slurry or wastewater was added to 90 mL of sterile saline, and a serial 10-fold dilution was prepared and plated on CAB (bacteria) or Sabouraud agar (fungi). After incubation for 24 h at 37 °C (for C. difficile, under anaerobic conditions; for fungi, followed by 48 h at 25 °C), the grown colonies were counted, and the number of microorganisms in 1 mL of aerated sample was determined (CFU/mL).

Perlon bags with A. suum eggs under the subsequent sampling terms were removed from the aeration chamber. They were then rinsed with sterile water with the addition of gentamicin and nystatin, and after dissection, the contents were placed in a Petri dish filled with 0.1 M H₂SO₄ solution in sterile water and incubated for 28 days at 28 °C. After the end of incubation, the percentage of invasive eggs was determined by microscopic counting. In each of the three replicates, 100 eggs were counted, simultaneously designating eggs with an amorphous “granular” filling as dead and those with visible motile L3 larvae as invasive.

2.8. Statistical Analysis

The obtained results of microbiological and parasitological determinations were analyzed and visualized using R Statistical software (v. 4.3.1) [47].

For each of the considered variants of aeration, microbiological and parasitological determinations were performed in 3 repetitions. In order to describe the kinetics of inactivation, the first-order model and the Weibull model were utilized [48,49]. The first-order model is expressed as:

$${\log }_{10}{\displaystyle \frac{N}{N_0}}=-kt$$

(1)

where N is the number of bacteria or fungi at any time t (with $N_0$ being the number of bacteria/fungi at t = 0), and k is the rate constant. The ${\displaystyle \frac{N}{N_0}}$ fraction was further presented as the survival ratio and denoted as $S\left(t\right)$. For each combination of evaluated factors, a decimal reduction time was calculated using

D = 1/k

(2)

indicating the time to observe a 1-log reduction in the number of microorganisms. Consistently, the time to observe a 3-log reduction in this number was obtained and denoted as D3.

The Weibull model was built according to the following equation:

$${\log }_{10}S\left(t\right)=-b{t}^n$$

(3)

where the scale parameter is denoted as b and the shape parameter as n. The value of the scale parameter determines the concave ($n<1$) or convex $(n>1)$ appearance of the survival curve. For $n=1,$ the Weibull model and the first-order model are equal. Similar to the first-order model, the time required for the 1-log (t₁) and 3-log (t₃) reduction in the number of bacteria or fungi is calculated using

$$t_i={\left({\displaystyle \frac{i}{b}}\right)}^{{\displaystyle \frac{1}{n}}}$$

(4)

where i is the number of desired log reductions.

The number of A. suum invasive eggs (per 100 eggs) was determined for each sampling date. The outcome (1) could not be expressed as (2) differs from the survival ratio introduced above; therefore, a simple linear regression model with a fixed intercept was fitted. The time to observe a reduction in the number of invasive eggs by 10 and 90 (per 100 eggs) was estimated using the reciprocal of the rate parameter represented by the slope of each linear model.

In order to determine the goodness of fit of the presented models, the root mean squared error (RMSE) was calculated, with smaller values corresponding to a better fit. The estimated parameters from each of the fitted models were presented as the value ± standard error (SE) unless stated otherwise.

3. Results

The tested physical and chemical parameters for the municipal wastewater and the pig slurry were within the normal range (

Table 1. Physico-chemical parameters of fresh pig slurry and raw municipal wastewater.

PARAMETERS	MATERIAL
	Pig Slurry	Raw Municipal Wastewater
pH	7.36	7.85
REDOX potential [mV]	−82	−64
Dry matter content [%]	4.96	2.17
COD [mg/L]	21,265	1954
BOD5 [mg/L]	10,239	827
Total nitrogen [mg N/L]	3196	96
Total phosphorus [mg P/L]	732	13
Total potassium [mg K/L]	1085	15

).

During the aeration process, an increase in batch temperature was observed (

Table 2. Changes in the temperature values in pig slurry and municipal wastewater during the aeration process.

PARAMETER	TYPE OF AERATION	MATERIAL	INTENSITY OF AERATION (OTR)
			1 gO2/m3/h		2 gO2/m3/h		3 gO2/m3/h
			0 d	12 d	0 d	12 d	0 d	12 d
Temperature [°C]	ATMOSPHERIC AIR	Thin slurry	20.0	25.9	20.4	35.2	19.8	36.6
		Medium slurry	20.1	27.1	19.8	36.7	20.6	38.2
		Thick slurry	19.7	29.3	20.2	39.3	20.1	40.8
		Thin wastewater	19.6	24.8	19.9	34.2	20.4	35.3
		Medium wastewater	20.3	26.9	20.1	35.4	19.7	37.4
		Thick wastewater	20.2	28.0	20.3	38.6	19.9	39.3
	RCI	Thin slurry	19.8	26.7	20.2	36.9	20.5	37.5
		Medium slurry	19.6	28.9	20.4	38.3	19.9	39.8
		Thick slurry	20.0	31.1	19.8	40.1	20.2	41.2
		Thin wastewater	20.2	26.0	19.7	36.5	20.3	36.9
		Medium wastewater	20.4	28.7	20.5	37.8	19.7	38.0
		Thick wastewater	19.9	30.4	20.1	38.7	20.4	39.9

Note: RCI—radiant catalytic ionization.

). Within 12 days, the temperature increase ranged from 5.2 °C to 21 °C for thin wastewater aerated with atmospheric air (OTR of 1 gO₂/m³/h) and for thick slurry aerated with RCI-“activated” air (OTR of 3 gO₂/m³/h), respectively. The temperature rise correlated with the dry matter content in the batch. Also, the aeration intensification increased the final batch temperature, with noticeably greater differences for 1 gO₂/m³/h vs. 2 gO₂/m³/h and 3 gO₂/m³/h than for 2 gO₂/m³/h vs. 3 gO₂/m³/h. The use of RCI-“activated” air resulted in 1–2 °C higher temperatures at the end of the aeration process compared to the application of atmospheric air.

Aeration, regardless of the process variant, increased the pH value of both pig slurry and municipal wastewater (

Table 3. Changes in the pH values in pig slurry and municipal wastewater during the aeration process.

PARAMETER	TYPE OF AERATION	MATERIAL	INTENSITY OF AERATION (OTR)
			1 gO2/m3/h		2 gO2/m3/h		3 gO2/m3/h
			0 d	12 d	0 d	12 d	0 d	12 d
pH	ATMOSPHERIC AIR	Thin slurry	7.74	8.36	7.74	8.67	7.74	8.78
		Medium slurry	7.36	8.13	7.36	8.47	7.36	8.64
		Thick slurry	7.01	8.10	7.01	8.42	7.01	8.60
		Thin wastewater	7.99	8.54	7.99	8.88	7.99	8.96
		Medium wastewater	7.85	8.52	7.85	8.86	7.85	8.93
		Thick wastewater	7.38	8.50	7.38	8.81	7.38	8.88
	RCI	Thin slurry	7.74	8.48	7.74	8.76	7.74	8.83
		Medium slurry	7.36	8.20	7.36	8.54	7.36	8.71
		Thick slurry	7.01	8.16	7.01	8.51	7.01	8.69
		Thin wastewater	7.99	8.62	7.99	8.92	7.99	9.00
		Medium wastewater	7.85	8.59	7.85	8.92	7.85	8.99
		Thick wastewater	7.38	8.54	7.38	8.88	7.38	8.90

Note: RCI—radiant catalytic ionization.

). After 12 days of aeration, the lowest pH value of 8.10 was measured for air-treated thick slurry (OTR of 1 gO₂/m³/h) and the highest value of 9.00 for thin wastewater aerated with RCI-“activated” air (OTR of 3 gO₂/m³/h). The pH of the slurry and the wastewater increased, together with the density of the charge and the intensity of aeration.

The observed changes in the REDOX potential clearly indicate the advantage of oxidizing substances in the slurry and wastewater after their aeration and the creation of oxygen conditions. The lower the dry matter content, the higher the final REDOX potential of the batch (

Table 4. Changes in the redox potential values in pig slurry and municipal wastewater during the aeration process.

PARAMETER	TYPE OF AERATION	MATERIAL	INTENSITY OF AERATION (OTR)
			1 gO2/m3/h		2 gO2/m3/h		3 gO2/m3/h
			0 d	12 d	0 d	12 d	0 d	12 d
REDOX potential [mV]	ATMOSPHERIC AIR	Thin slurry	−71	+82	−71	+124	−71	+137
		Medium slurry	−82	+70	−82	+102	−82	+110
		Thick slurry	−102	+63	−102	+97	−102	+100
		Thin wastewater	−51	+91	−51	+127	−51	+141
		Medium wastewater	−64	+81	−64	+110	−64	+119
		Thick wastewater	−88	+70	−88	+99	−88	+103
	RCI	Thin slurry	−71	+121	−71	+162	−71	+188
		Medium slurry	−82	+105	−82	+149	−82	+164
		Thick slurry	−102	+94	−102	+126	−102	+145
		Thin wastewater	−51	+131	−51	+174	−51	+201
		Medium wastewater	−64	+112	−64	+153	−64	+176
		Thick wastewater	−88	+101	−88	+132	−88	+155

Note: RCI—radiant catalytic ionization.

). Also, the intensification of aeration elevated the REDOX potential. The highest REDOX potential (+201 mV) was recorded in the thin wastewater sample aerated with RCI-“activated” air (OTR of 3 gO₂/m³/h) and the lowest (+63 mV) in the air-aerated thick slurry sample (OTR of 1 gO₂/m³/h). The REDOX potential values were significantly higher for the RCI-“activated” air treatment than the atmospheric air treatment.

The obtained results show that the dry matter content in the slurry and municipal wastewater decreased during aeration (

Table 5. Changes in the dry matter content in pig slurry and municipal wastewater during the aeration process.

PARAMETER	TYPE OF AERATION	MATERIAL	INTENSITY OF AERATION (OTR)
			1 gO2/m3/h		2 gO2/m3/h		3 gO2/m3/h
			0 d	12 d	0 d	12 d	0 d	12 d
Dry matter content [%]	ATMOSPHERIC AIR	Thin slurry	1.98	1.15	1.98	0.97	1.98	0.87
		Medium slurry	5.08	3.15	5.08	2.76	5.08	2.55
		thick slurry	10.04	6.53	10.04	5.92	10.04	4.94
		Thin wastewater	2.05	1.17	2.05	0.94	2.05	0.86
		Medium wastewater	4.93	2.96	4.93	2.72	4.93	2.36
		Thick wastewater	9.98	6.03	9.98	5.61	9.98	5.25
	RCI	Thin slurry	1.98	1.01	1.98	0.75	1.98	0.61
		Medium slurry	5.08	2.69	5.08	2.08	5.08	1.68
		Thick slurry	10.04	6.30	10.04	5.78	10.04	4.77
		Thin wastewater	2.05	0.96	2.05	0.70	2.05	0.62
		Medium wastewater	4.93	2.66	4.93	2.06	4.93	1.66
		Thick wastewater	9.98	5.83	9.98	5.30	9.98	4.94

Note: RCI—radiant catalytic ionization.

). In most cases, a decrease of 40–60% was noted. The dry matter content reduction correlated with aeration intensity.

The initial number of bacteria in the slurry and municipal wastewater ranged from 7.41 × 10⁸ to 9.12 × 10⁹ CFU/mL. The initial number of A. niger fungi ranged from 9.54 × 10⁷ to 9.33 × 10⁹ CFU/mL. The initial percentage of A. suum invasive eggs was 98%.

Regardless of all other experimental factors, air activated with RCI technology better eliminated the tested microorganisms and inactivated A. suum eggs than aeration with atmospheric air. The final number of bacteria in the slurry subjected to disinfection with RCI-activated air ranged from 1.14 × 10² CFU/mL for L. monocytogenes to 1.91 × 10⁷ CFU/mL for C. difficile. For the disinfection with atmospheric air, the number of bacteria ranged from 2.82 × 10³ CFU/mL for L. monocytogenes to 2.24 × 10⁷ CFU/mL for C. difficile.

We have presented the results using the Weibull model and the classical first-order kinetics approach (

Table 6. Parameters of the Weibull and first-order models for the survival curves of tested bacteria for different experiment variants.

	Aeration Type	Bacteria	Variant	Weibull Model Estimated Parameters					First-Order Estimated Parameters
				b + SE	n + SE	RMSE	t1 [days]	t3 [days]	Rate ± SE [1/days]	D1 [days]	D3 [days]	RMSE
SLURRY
Maximal effectiveness	Atmospheric	Listeria monocytogenes	Thick; 3 gO2/m3/h	1.191 ± 0.155	0.6 ± 0.061	0.255	0.75	4.67	0.494 ± 0.036	2.02	6.07	0.639
	RCI	Escherichia coli	Thick; 3 gO2/m3/h	1.333 ± 0.23	0.711 ± 0.079	0.416	0.67	3.13	0.705 ± 0.039	1.42	4.26	0.706
Minimal effectiveness	Atmospheric	Clostridioides dificile	Thin; 1 gO2/m3/h	0.309 ± 0.056	0.588 ± 0.085	0.09	7.36	47.76	0.125 ± 0.01	8.00	23.99	0.179
	RCI	Clostridioides dificile	Thin; 1 gO2/m3/h	1.355 ± 0.245	0.319 ± 0.09	0.318	0.39	12.10	0.312 ± 0.04	3.21	9.63	0.716
WASTEWATER
Maximal effectiveness	Atmospheric	Listeria monocytogenes	Thin; 3 gO2/m3/h	1.853 ± 0.247	0.359 ± 0.066	0.371	0.18	3.83	0.455 ± 0.064	2.20	6.60	1.143
	RCI	Escherichia coli	Thick; 3 gO2/m3/h	1.173 ± 0.244	0.805 ± 0.094	0.489	0.82	3.21	0.762 ± 0.035	1.31	3.94	0.629
Minimal effectiveness	Atmospheric	Clostridioides dificile	Thin; 3 gO2/m3/h	0.488 ± 0.123	0.541 ± 0.119	0.195	3.77	28.74	0.178 ± 0.018	5.62	16.85	0.323
	RCI	Clostridioides dificile	Thin; 1 gO2/m3/h	0.501 ± 0.17	0.81 ± 0.154	0.342	2.35	9.11	0.329 ± 0.021	3.04	9.11	0.381

Notes: RCI—radiant catalytic ionization, SE—standard error, RMSE—root mean square error, t1/D1—time needed for 90% elimination, t3/D3—time needed for 99.9% elimination.

,

Table 7. Parameters of the Weibull and first-order models for the survival curves of Aspergillus spp. for different experiment variants.

	Aeration Type	Variant	Weibull Model Estimated Parameters					First-Order Estimated Parameters
			b + SE	n + SE	RMSE	t1 [days]	t3 [days]	Rate ± SE [1/days]	D1 [days]	D3 [days]	RMSE
SLURRY
Maximal effectiveness	Atmospheric	Thick; 3 gO2/m3/h	0.431 ± 0.106	0.745 ± 0.112	0.2	3.09	13.52	0.246 ± 0.014	4.07	12.20	0.251
	RCI	Thick; 2 gO2/m3/h	1.421 ± 0.232	0.366 ± 0.08	0.31	0.38	7.72	0.36 ± 0.042	2.78	8.34	0.751
Minimal effectiveness	Atmospheric	Thin; 1 gO2/m3/h	0.48 ± 0.058	0.564 ± 0.057	0.093	3.68	25.78	0.184 ± 0.014	5.42	16.27	0.258
	RCI	Thin; 1 gO2/m3/h	0.676 ± 0.226	0.491 ± 0.16	0.333	2.22	20.84	0.224 ± 0.024	4.47	13.40	0.434
WASTEWATER
Maximal effectiveness	Atmospheric	Thick; 3 gO2/m3/h	1.061 ± 0.23	0.466 ± 0.104	0.35	0.88	9.29	0.329 ± 0.039	3.04	9.12	0.694
	RCI	Thick; 3 gO2/m3/h	1.462 ± 0.089	0.43 ± 0.03	0.125	0.41	5.33	0.421 ± 0.042	2.37	7.12	0.755
Minimal effectiveness	Atmospheric	Thin; 3 gO2/m3/h	0.887 ± 0.169	0.425 ± 0.092	0.254	1.33	17.57	0.253 ± 0.03	3.96	11.87	0.536
	RCI	Thin; 1 gO2/m3/h	1.713 ± 0.144	0.2 ± 0.044	0.174	0.07	16.40	0.307 ± 0.048	3.26	9.77	0.853

Notes: RCI—radiant catalytic ionization, SE—standard error, RMSE—root mean square error, t1/D1—time needed for 90% elimination, t3/D3—time needed for 99.9% elimination.

and Table S1). For bacteria, in the Weibull and first-order models, the shortest times to reduce the number of bacteria by 99.9% were shown for L. monocytogenes and E. coli in atmospheric air and RCI aeration, respectively. The longest time to reduction was always for C. difficile, regardless of the experience variant (Table 6). The results show that the Weibull model fitted better to the experimental data than the first-order model for all pathogens and all variants of the experiments, which consequently indicate a nonlinear trend.

In the slurry, for the variant with air activated by RCI technology aeration, the shortest time required to achieve a 99.9% reduction in bacteria ranged from 3.13 days (2 gO₂/m³/h, thick slurry) to 6.12 days (2 g gO₂/m³/h, thin slurry) for E. coli and from 4.43 days (3 gO₂/m³/h, thick slurry) to 6.94 days (3 gO₂/m³/h, thin slurry) for L. monocytogenes (Figure 2 and Figure 3). The longest time required for a 99.9% bacterial reduction with RCI in slurry was 12.10 days (1 gO₂/m³/h, thin slurry) for C. difficile (Figure 4).

In the atmospheric air variant, the reduction times ranged from 4.67 days (3 gO₂/m³/h, thick slurry) to 11.21 days (1 gO₂/m³/h, thin slurry) for L. monocytogenes and from 6.92 days (3 gO₂/m³/h, thin slurry) to 14.59 days (1 gO₂/m³/h, thin slurry) for E. coli (Figure 2 and Figure 3). For C. difficile, the required time ranged from 17.66 days (3 gO₂/m³/h, thick slurry) to 47.76 days (1 gO₂/m³/h, thin slurry) (Figure 4).

For Aspergillus, the time required for a 99.9% reduction ranged from 7.72 days (2 gO₂/m³, thick slurry) to 20.84 days (1 gO₂/m³/h, thin slurry) for aeration with RCI and from 13.52 days (3 gO₂/m³/h, thick slurry) to 25.78 days (1 gO₂/m³/h, thin slurry) for aeration with atmospheric air (Table 7, Figure 5).

In the slurry aerated with air activated with RCI technology, the number of A. suum invasive eggs on the last day of the experiment ranged from 82.33 to 91.33 for each 100 tested A. suum eggs, while in slurry aerated with atmospheric air, it ranged from 88.33 to 92.33 eggs.

For A. suum, linear regression was determined. The shortest estimated time to reach a 90% decrease in the initial invasive egg count was 10.76 and 15.32 weeks in slurry (3 gO₂/m³/h, thick slurry) aerated with RCI and atmospheric air, respectively. The longest time was determined as 20.70 weeks for the RCI variant and 24.61 weeks for atmospheric air (1 gO₂/m³/h, thin slurry) (

Table 8. Parameters of the linear model for the survival curves of A. suum eggs for different experiment variants.

	Aeration Type	Variant	First-Order Estimated Parameters
			Rate ± SE [1/weeks]	D10 [weeks]	D90 [weeks]	RMSE
SLURRY
Maximal effectiveness	Atmospheric	Thick; 3 gO2/m3/h	5.875 ± 0.413	1.70	15.32	1.055
	RCI	Thick; 2 gO2/m3/h	7.281 ± 0.287	1.37	12.36	0.733
Minimal effectiveness	Atmospheric	Thin; 1 gO2/m3/h	3.657 ± 0.264	2.74	24.61	0.673
	RCI	Thin; 1 gO2/m3/h	4.347 ± 0.281	2.30	20.70	0.718
WASTEWATER
Maximal effectiveness	Atmospheric	Thick; 3 gO2/m3/h	6.201 ± 0.358	1.61	14.51	0.914
	RCI	Thick; 3 gO2/m3/h	9.557 ± 0.171	1.04	9.42	0.435
Minimal effectiveness	Atmospheric	Medium; 1 gO2/m3/h	3.235 ± 0.199	3.09	27.82	0.507
	RCI	Medium; 1 gO2/m3/h	4.219 ± 0.182	2.37	21.33	0.465

Notes: RCI—radiant catalytic ionization, SE—standard error, RMSE—root mean square error, D10—time needed for 10% elimination, D90—time needed for 90% elimination.

, Figure 6).

In wastewater, the final bacterial count ranged from 2.11 × 10¹ to 5.01 × 10⁶ CFU/mL for RCI technology and from 4.90 × 10³ to 9.77 × 10⁶ CFU/mL for atmospheric air. The shortest reduction time needed for 99.9% elimination was observed for L. monocytogenes and E. coli, while the longest was observed for C. difficile. Depending on the disinfection method, reduction times for these bacteria ranged, in the case of using RCI, from 3.21 days (3 gO₂/m³/h, thick wastewater, E. coli) to 9.11 days (2 gO₂/m³/h, thin wastewater, C. difficile) and from 3.83 days (3 gO₂/m³/h, thin wastewater, L. monocytogenes) to 28.74 days (3 gO₂/m³/h, thin wastewater, C. difficile) for atmospheric air (Figure 7, Figure 8 and Figure 9).

For Aspergillus spp. in wastewater, the time to reduction ranged from 9.29 days (3 gO₂/m³, thick wastewater) to 17.57 days (3 gO₂/m³/h, thin wastewater) for aeration with atmospheric air, while this time was shorter during RCI aeration and ranged from 5.33 days (3 gO₂/m³/h, thick wastewater) to 16.40 days (1 gO₂/m³/h, thin wastewater) (Table 7, Figure 10).

The number of A. suum invasive eggs on the 12th day of the experiment ranged between 82.33–90.67 and 87.33–92.33 per each 100 eggs for RCI technology and atmospheric air, respectively.

The theoretical times to reduce 90% of A. suum eggs were from 9.42 weeks (3 gO₂/m³/h, thick wastewater) to 21.33 weeks (1 gO₂/m³/h, medium wastewater) and from 14.51 weeks (3 gO₂/m³/h, thick wastewater) to 27.82 weeks (1 gO₂/m³/h, medium wastewater) for RCI and atmospheric air, respectively (Table 8, Figure 11).

The aeration of slurry and wastewater with RCI-activated air increased the number of bacteria and fungi on the second and third days of the process, while the disinfection with atmospheric air systematically decreased these numbers (Tables S1 and S2).

Also, other experimental factors, i.e., the content of dry matter in the slurry and municipal wastewater, as well as the intensity of aeration, resulting in the concentration of dissolved oxygen in the tested material, influenced the effectiveness of disinfection methods. As the density of both slurry and municipal wastewater increased, other experimental factors, i.e., the content of dry matter in the slurry and municipal wastewater, as well as the intensity of aeration, resulted in the concentration of dissolved oxygen in the tested material. The elimination of the tested microorganisms and the inactivation of parasite eggs, regardless of the disinfection method, was connected with the dry matter and dissolved oxygen contents (Tables S1 and S2). In general, the final number of bacteria and time to the reduction of 99.9% of microorganisms in the slurry and the municipal wastewater were lower in the case of aeration with RCI-activated air than atmospheric air.

4. Discussion

Failure to properly manage raw wastewater by discharging it into water can endanger the health of humans, animals and the environment. To prevent the spread of pathogenic microorganisms present in raw wastewater or slurry, it is necessary to subject these fluids to an effective method of disinfection. Researchers are still searching for new efficient technologies that do not require the use of a thermal factor. One may be RCI technology, the inhibitory effect of which has already been demonstrated against pathogens in the air and on various types of abiotic surfaces and food [26,27,28,30]. While there are reports on the use of an RCI-related photolysis mechanism to remove pollutants (hormones and dyes) from wastewater, research on RCI application to reduce the number of pathogens in liquid animal feces or wastewater is limited [50,51,52].

In our research, we evaluated, for the first time, the effectiveness of fine-bubble aeration using air activated by RCI technology. The study shows that aeration with RCI-“activated” air more effectively eliminates pathogens from wastewater and slurry compared to the traditional aeration method. RCI leads to the production of ROS and the generation of hydrogen peroxide in the gas plasma, which may improve the germicidal effect of the aerated liquid. We have shown that the effectiveness of RCI technology depends primarily on the species of microorganism and the density of the liquid undergoing the process. We observed shorter times to reduce 99.9% of bacteria after RCI-activated air disinfection for all bacterial pathogens, except C. difficile, in both tested liquids at their highest OTR (3 gO₂/m³/h). In the available literature, there are studies on the use of photocatalysis in wastewater disinfection by adding TiO₂ in the form of a suspension [53] or placing solid carriers with immobilized TiO₂ [54]. The immobilization of the photocatalyst on solid structures is more expensive and seems to reduce the photocatalysis effectiveness, but it ensures greater stability of the water treatment system. Scientists have shown the immobilized photocatalyst is more resistant to the inhibitory effect of organic pollutants, making this disinfection method more suitable for the treatment of wastewater containing significant amounts of these compounds [54,55]. However, there are no data indicating the efficiency of the aeration of wastewater or slurry with air subjected to the photocatalysis process.

The gradual reduction in the number of microorganisms during the aeration was not related to the lethal effect of the thermal conditions prevailing during the process. The temperatures in the tested substances, regardless of the aeration variant used, in only a few cases slightly exceeded 40 °C. This classifies the analyzed disinfection processes in the mesophilic category, with relatively low efficiency of microorganism elimination [56]. A study by Venglovsky et al. [8] showed that after 12 days of slurry storage, the number of E. coli was higher at 42 °C than at 4 °C and 20 °C. Based on the conducted research, we believe that the temperature during the aeration process did not affect its course. Both variants of aeration are associated with the depletion of readily available nutrients as a result of the intensive decomposition of organic matter. In addition, RCI technology generates ROS that limit the number of microorganisms. Nevertheless, on the second and third days of the aeration with RCI, we observed an increase in the number of microorganisms. We assume that the production of large amounts of ROS, the non-selective mineralization capacity of which is used to degrade organic pollutants in wastewater, may also intensify the decomposition rate of organic matter contained in slurry or wastewater [57,58]. In turn, the increase in easily digestible nutrient amounts in the initial stage of the process may contribute to the faster multiplication of microorganisms. We did not notice a similar phenomenon in the conventional aeration process.

The alkalization of the environment observed during aeration could also have an impact on the reduction in the number of pathogens. Heinonen-Tanski et al. [59] recognized an increase in pH from 7.6 to 9.0 as the main cause of the complete elimination of Salmonella introduced into slurry aerated for 2–5 weeks at a temperature below 40 °C. In the present study, we noted an increase in pH to a maximum value of 9 in aerated slurry and wastewater. Also, Venglovsky et al. [8] confirmed the statistically significant relationship between the pH of the slurry and the number of microorganisms.

It was also found that the amount of oxygen generated during the process and the dry matter content affected disinfection effectiveness. The increase in aeration intensity and dissolved oxygen content generally shortened the reduction in the bacteria. Hanajima et al. [25] revealed the slowest inactivation rate of E. coli in slurry at low aeration levels. In contrast, Baez and Shiloach [60] showed that increased oxygen concentration promotes the formation of ROS, causing the impaired growth of microbial cells and increased mutagenesis. The reduction in the pathogen number could also result directly from the rapid mineralization of organic matter, leading to a rapid depletion of nutrients [25].

We observed the longest time to the reduction of 99.9% of all bacterial pathogens and A. suum eggs at the lowest dry matter content. However, earlier studies by Skowron et al. [61] showed the shortest time to the reduction of Salmonella Dublin, E. coli and Enterococcus spp. in stored pig slurry with the lowest density. Also, Sudlitz and Chmielewski [62] reported that the more concentrated the municipal sediment, the higher the dose of the electron beam used for their irradiation was necessary for the elimination of E. coli, Salmonella spp., Clostridium perfringens, eggs Toxocara spp., Trichuris spp. and Ascaris spp. Our study shows that in liquids with a lower dry matter content, RCI more efficiently reduced the number of pathogens compared to classical aeration.

The resistance of bacterial pathogens to stressors is related to the structure of their cell walls [63]. Gram-positive bacteria are usually more resistant to high temperatures, toxic chemicals and photocatalysis-based disinfection [64]. Lin et al. [63] found that Enterococus spp. survived longer than E. coli during anaerobic fermentation under thermophilic conditions. The results of our research confirm these observations. The shortest times to the elimination of 99.9% of bacteria were for L. monocytogenes in the variant with aeration with atmospheric air and for E. coli in RCI-activated air. In the vast majority of experimental combinations (type of liquid, density and air entrainment), RCI technology better reduced the number of bacteria compared to the conventional method in all variants of the experiment. The earlier results of Skowron et al. [61] showed that depending on the thermal conditions and the density of aerated slurry, the theoretical survival rate for S. Dublin, E. coli and Enterococcus spp. ranged from 81.85 to 220.80 days, from 74.93 to 199.36 days and from 118.67 days to 335.84 days, respectively. The longest theoretical survival time was recorded for C. difficile. Also, other authors have confirmed the resistance of these microorganisms to various disinfection factors [8,63,65,66].

A preliminary and simplified cost analysis of the described solution indicates that implementing fine-bubble aeration with air activated using RCI technology requires only the installation of two additional components. The first is the RCI module set (20 connected devices), and the second is a storage tank for the activated air. The estimated cost of such an upgrade for a municipal wastewater treatment plant serving approximately 300,000 population equivalents would be about EUR 65,000 and an additional energy consumption of approximately 1.2 kWh. The lifespan of the RCI modules used is estimated at 24 months. After this period, they will need to be replaced, resulting in a cost of around EUR 53,000.

Our study has some limitations. The first limitation is that we used one strain from each bacterial and fungal species. Our study was conducted to show which microorganisms are most resistant to aeration with RCI and take further steps to improve the efficiency of this device. The second limitation is the design of the device in a closed system, which allowed us to perform this pilot study, but in the future, we want to work on a flow system.

5. Conclusions

This laboratory-scale study allowed us to estimate the disinfection potential of RCI technology to reduce the number of pathogens and the viability of parasite eggs in slurry and municipal sewage. The obtained results allow us to conclude that the applied technology can be an alternative to the traditional aeration method because it reduces organic matter faster, although, at the same time, it can increase the cost of the process itself. The innovative use of activated air by RCI can help to reduce the number of bacteria in sewage and slurry in the future and minimize the risk of contamination of the soil environment. Since there are very few studies on RCI-like technology, it is difficult to compare the phenomena occurring in our own research with those observed by other researchers. To our knowledge, this is the first laboratory-scale study about the possibility of using RCI in the aeration of slurry and wastewater. In addition, many factors, such as the dry matter content, can modify the antimicrobial effect of RCI. Therefore, further research on optimizing this technology and applying it in practice is certainly warranted.