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Article

Pilot-Scale Evaluation of a Filter Prototype for Bacterial Inactivation in Agro-Food Processing Wastewater

by
Piotr Kanarek
1,*,
Barbara Breza-Boruta
1 and
Wojciech Poćwiardowski
2
1
Department of Microbiology and Plant Ecology, Faculty of Agriculture and Biotechnology, Bydgoszcz University of Science and Technology, 6 Bernardyńska Street, 85-029 Bydgoszcz, Poland
2
Department of Food Industry Technology and Engineering, Faculty of Chemical Technology and Engineering, Bydgoszcz University of Science and Technology, 85-326 Bydgoszcz, Poland
*
Author to whom correspondence should be addressed.
Water 2025, 17(17), 2631; https://doi.org/10.3390/w17172631
Submission received: 18 July 2025 / Revised: 22 August 2025 / Accepted: 28 August 2025 / Published: 5 September 2025
(This article belongs to the Section Wastewater Treatment and Reuse)
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Abstract

The processing of freshly cut fruits and vegetables represents an important niche for implementing circular economy principles, particularly through the reuse of washing water. This is especially relevant as post-wash water is often treated as wastewater and discarded without reuse. One promising research avenue is the use of plant-derived extracts in water sanitation processes. Their antimicrobial properties offer a natural alternative to conventional disinfectants while reducing the formation of harmful disinfection by-products. The aim of this study was to evaluate the effectiveness of different filter bed configurations in removing pathogens from water. These configurations included a hydrogel saturated with natural plant extracts, an ion exchange resin layer, and an activated carbon layer. The most effective composite was also tested using real process water from a fruit washing line. The test materials included concentrated extracts from oak bark (Quercus robur), willow (Salix alba), birch (Betula pendula), raspberry shoots (Rubus idaeus), tea leaves (Camellia sinensis), and linden flowers (Tilia cordata), all immobilized in hydrogel, along with activated carbon and ion-exchange resin. Water samples were artificially inoculated with six opportunistic pathogens and collected process water was also used. Samples were analyzed microbiologically at six time intervals. The composite filter (hydrogel–resin–carbon) achieved a reduction of over 2 log10 in heavily inoculated water (~108 CFU mL−1) and maintained at least a 1 log10 reduction in real process effluents. The proposed solution supports blue water footprint reduction strategies (as the system aims to decrease the demand for freshwater resources through the reuse of treated wastewater) and aligns with the principles of green processing.

1. Introduction

The integration of circular economy principles into zero-waste policies contributes directly to mitigating the adverse effects of anthropogenic climate change [1]. Particularly important in this context is the need to intensify efforts aimed at water conservation and recovery, as both water scarcity, and the disruption of hydrological systems are becoming increasingly apparent worldwide—not only in the Global South, but also across the Northern Hemisphere, affecting both rural areas and expanding urban centers [2,3]. The expanding awareness of environmental issues among the public not only shapes consumer behavior but can also facilitate the adoption of legal frameworks aimed at lowering climate stress over time [4,5].
Agriculture is one of the most water-intensive sectors of the economy and plays a crucial role in the design, planning, and implementation of measures aimed at water conservation [6,7]. According to data from the European Environment Agency, the average annual water use for agricultural purposes across the EEA-39 countries between 2008 and 2017 was 92 billion m3. In 2017 alone, the EU-28 member states used an average of 50 billion m3 of water per year, while Turkey reported usage of approximately 40 billion m3 [8]. In the European Union, the adoption of diverse water-saving solutions has been facilitated through the Common Agricultural Policy, including measures such as irrigation automation, precision farming, the reuse of treated water, the promotion of less invasive cultivation methods, and the use of crops with increased resilience to water stress [9].
The processing of freshly harvested fruits and vegetables is recognized as a relevant niche for implementing circular economy solutions, particularly through the reuse of water employed in raw material washing [10,11]. This issue is particularly important given that wash water is often discharged as wastewater and not reused. However, this approach is primarily driven by concerns over preventing microbiological cross-contamination, which may lead to outbreaks that pose risks to public health and compromise the quality of the final product. An alternative strategy involves the use of conventional chemical disinfection, typically based on chlorine compounds such as sodium and calcium hypochlorite [12]. However, the use of this conventional approach may lead to the formation of harmful disinfectant byproducts (DBPs), some of which exhibit carcinogenic, cytotoxic, teratogenic, or neurotoxic properties (Table 1). In the early cleaning stages at agri-food-processing facilities, fruits and vegetables often carry soil particles, dust, and remnants of dead organic matter. These materials can react with disinfectants, which raises concerns about the potential formation of toxic or otherwise undesirable compounds [13].
Due to the wide availability and convenience of chemical disinfection, alternative methods—such as mechanical filtration using carbon, sand, or silica filters, UV irradiation, or cold plasma—tend to attract less interest among producers [11,21]. The technical problem addressed in this study is the lack of alternative, non-invasive methods for the effective disinfection of wastewater generated in the fresh-cut industry.
In recent years, there has been growing interest in the use of less invasive methods involving natural plant extracts, which may offer a more appealing alternative [22]. Research on the use of plant-based raw materials as natural preservatives, and antimicrobials has gained importance in response to the growing demand for safe and environmentally friendly alternatives to synthetic additives [23,24]. Plant extracts, due to their antibacterial and antifungal properties, are used not only in the food industry, but also in pharmaceuticals and cosmetology [25,26,27,28,29]. The active compounds found in plant extracts—such as flavonoids, alkaloids, and tannins—exhibit diverse antimicrobial mechanisms. Flavonoids, for example, can inhibit DNA gyrase activity, destabilize bacterial cell membranes, and interfere with cellular energy metabolism [30]. Alkaloids, on the other hand, have been shown to inhibit bacterial cell wall formation, alter membrane permeability, suppress the synthesis of proteins and nucleic acids, and disrupt various metabolic pathways [31]. Tannins exert antibacterial activity through several mechanisms, including iron chelation, disruption of the bacterial cell membrane, inhibition of cell wall synthesis and fatty acid biosynthesis. The compounds also demonstrate antivirulence effects, such as the suppression of quorum sensing and biofilm formation [32]. In light of the above, the use of plant extracts in water sanitization processes represents a promising area of research, as their antimicrobial properties may serve as a natural alternative to conventional disinfectants while simultaneously reducing the formation of harmful disinfection byproducts [33].
The research hypothesis assumes that the use of a multilayer filtration bed composed of hydrogel infused with natural plant extracts, ion-exchange resin, and activated carbon significantly enhances the efficiency of pathogen removal from wastewater compared to single-layer filtration systems.
The aim of this study was to experimentally evaluate the efficiency of a pilot-scale filtration bed designed for the treatment of wastewater under semi-technical conditions. The research focused on assessing the effectiveness of pathogen removal using different bed configurations, which included hydrogel infused with natural plant extracts, an ion-exchange resin layer, and an activated carbon layer. The objective was to determine which configuration offers the highest microbiological purification efficiency while maintaining operational stability and demonstrating potential for application in environmental engineering practice. The second phase of the study involved preliminary validation on an industrial scale. The most effective configuration identified during the pilot tests was applied to the treatment of real process water collected from a fruit processing facility.

2. Materials and Methods

2.1. Preparation of Filter Material

Based on previous research, the selected plant material included bark of Quercus robur, Salix alba, and Betula pendula, stems of Rubus idaeus, leaves of Camellia sinensis, and flowers of Tilia cordata [34].
Hydroethanolic extracts (70%) were prepared from the selected plant material. For this purpose, 25 g of cleaned and dried plant material (pre-dried for 12 h at 30 °C) were ground into a fine powder and then soaked in 250 mL of solvent. The mixtures were shaken for 24 h at 150 rpm, then filtered using standard laboratory-grade filter paper. The filtered extracts (150 mL) were concentrated threefold to 50 mL using a high-efficiency rotary evaporator (Laborota 4000, Heidolph, Schwabach, Germany) at 65 °C and 90 rpm. To ensure sterility, the extracts were further filtered through 25 mm syringe filters with a pore size of 0.22 µm.
Subsequently, the extracts were applied for hydrogel hydration to prepare the material for further analysis (Figure 1).
The remaining filtration materials included ion-exchange resin and activated carbon. According to the manufacturer Formaster S.A., Kielce, Poland), the macroporous resin removes calcium and magnesium ions from water through ion exchange, while the activated carbon, derived from coconut shells, effectively adsorbs chlorine and other contaminants due to its high porosity, thereby improving the taste and odor of the water. According to the manufacturer, the service life of activated carbon cartridges is estimated at 4–6 months, while ion-exchange resin cartridges lose efficiency once their exchange capacity is exhausted. For medium-hard water this corresponds to approximately 700–1000 L, with higher capacities for lower hardness. In this study, these values were considered as reference information, while the experimental evaluation focused on microbiological performance.

2.2. Filtration System

The filtration system (Figure 2) consisted of an initial tank containing 25 L of inoculated water with bacteria at a specified concentration, a water pump ensuring constant flow, control valves regulating both the direction and intensity of flow, manometers monitoring pressure at various stages, and a main flow-through filter filled with a suitable filtration medium—activated carbon with ion-exchange resin, hydrogel, or a mixture of these, depending on the setup. Filtered water was collected in a receiving tank for further analysis.

2.3. Bacterial Inoculum

As identified in prior studies, the test strains comprised opportunistic pathogens isolated from wash waters in agri-food processing facilities. These included Pseudomonas aeruginosa, Klebsiella oxytoca, Klebsiella pneumoniae, Proteus vulgaris, Serratia marcescens, and Enterococcus faecalis [34,35]. To prepare the bacterial inoculum, strains stored in Brain Heart Infusion (BHI) broth (Merck, Darmstadt, Germany) with glycerol at −20 °C were revived on general-purpose medium (Tryptic Soy Agar, TSA; Merck, Germany) and subcultured twice. Bacteria were subsequently collected with a sterile inoculation loop and suspended in 9 mL of sterile 0.9% sodium chloride solution. The suspension was adjusted to a concentration of 1.0–2.0 × 108 CFU/mL, consistent with the cell density typically applied in antimicrobial susceptibility assays. A 1 mL aliquot from each bacterial suspension was inoculated into 250 mL of sterile Nutrient Broth (Merck, Germany) and mixed thoroughly to achieve uniform dispersion of the cells. The same procedure was carried out for each strain tested.
To prepare the inoculum for further testing, a total of 1.5 L of bacterial culture (6 × 250 mL), each at a concentration of 2 × 108 CFU/mL, was added to 25 L of sterile water. This resulted in a final bacterial concentration of approximately 1.2 × 107 CFU/mL. The actual concentration was confirmed by determining colony-forming units (CFU/mL) in samples collected immediately after inoculation.

2.4. Testing Procedure

Twenty-five liters of sterile water were inoculated with 1.5 L of bacterial suspensions containing the test strains (6 × 250 mL) and transferred into the preliminary tank. After opening the control valves and activating the pump, the water was passed through filtration columns in one of the following three configurations: (1) a filter filled with activated carbon and ion-exchange resin; (2) a filter containing hydrogel; and (3) a filter composed of a mixture of activated carbon, ion-exchange resin, and hydrogel (Figure 3).
Water samples were collected and analyzed at six intervals: 1 h, 2 h, 3 h, 6 h, and 12 h. Approximately 4 L of water were circulated through the system during each test. Following filtration, microbiological analyses were performed in duplicate: 1 mL of filtered water was plated on appropriate agar medium and spread using a sterile spreader. The results were compared across the different filter configurations.

2.5. Log Reduction and Statistical Analysis

Filter performance was evaluated by monitoring the decrease in bacterial counts in the outflow over time. Results were described as a log10 reduction in colony-forming units (CFU), calculated using the following equation:
l o g r e d u c t i o n = l o g 10   C s t a r t − l o g 10   ( C a f t e r   f i l t a r t i o n )
C s t a r t —initial bacterial concentration in water before filtration (CFU/mL).
C a f t e r   f i l t a r t i o n —bacterial concentration in the sample collected from the filter outflow at a given time point (CFU/mL)
The concentration of CFU/mL was determined using colony counts from serial dilution plates, in accordance with standard microbiological procedures. When the number of colonies exceeded 300 (designated as “TNTC”—too numerous to count), the result was estimated using data from the next highest dilution that produced countable growth.
The obtained microbiological data were subjected to statistical analysis using two-way analysis of variance (ANOVA). The first factor (A) corresponded to the type of filter medium: H—hydrogel; C-IER—activated carbon with ion-exchange resin; H-C-IER—a combination of hydrogel, activated carbon, and ion-exchange resin. The second factor represented the sampling time interval (1, 2, 3, 6, 12, and 24 h). To assess the significance of the individual effects and their interaction, Tukey’s post hoc test with a 95% confidence interval was performed, allowing for comparison of the mean values of the analyzed parameters.

2.6. Validation of the System on Water from the Agri-Food Industry

The facility studied operates under an integrated food safety management system, with certification covering all stages of the production chain—from field cultivation to delivery of fruit to the end consumer. Annually, approximately 10,000 metric tons of fruit are produced, mainly apples and pears. Process water samples were collected from the main drainage channel of a fruit processing plant located in the Kuyavian-Pomeranian Voivodeship (northern Poland).

3. Results

3.1. Results for Inoculated Water

Results demonstrated that the composite filter (H-C-IER) achieved a reduction by approximately one order of magnitude in microbial load within the first hour, outperforming both the single- and double-layer filters, and continued to do so over the entire observation period. The resin–carbon configuration (C-IER) demonstrated moderate yet steady performance, while the hydrogel-only filter (H) had the highest CFU/mL levels at all time points (Figure 4).
For the H configuration, bacterial reduction after 1 h did not exceed 0.8 log10. During the 24 h operation, an increase in bacterial counts in the outflow was observed—from approximately 2 × 106 to nearly 2 × 107 CFU/mL. At the two last time points (12–24 h), colony counts were higher than in samples collected during the first hour of filtration (Table 2).
The filter setup based on C-IER layer exhibited moderate effectiveness in reducing bacterial concentrations in water initially containing around 1.2 × 107 CFU/mL. The highest reduction was observed during the first filtration time point, with an average colony count of 37 at a 10−4 dilution—equivalent to 3.7 × 105 CFU/mL—and a log reduction of 1.51.
Over time, bacterial levels in the outflow gradually increased. After 3 h, the CFU/mL value rose to 9.3 × 105, yielding a log reduction of 1.11. By the sixth hour, bacterial reduction dropped to 0.97 log, and after 24 h, the system’s effectiveness stabilized below 1 log, with a final concentration of 1.39 × 106 CFU/mL (Table 3).
The composite filter H-C-IER demonstrated the highest efficiency among all tested configurations. At the first time point, a log reduction of 2.23 was recorded, corresponding to a decrease in bacterial count to 7.0 × 104 CFU/mL (an average of 7 colonies at a 10−4 dilution).
In subsequent sampling points, retention remained high—during the first three time points, the log reduction exceeded 1.7 log10. Despite a slight increase in CFU/mL in later samples (reaching a maximum of 1.18 × 106 CFU/mL after 24 h), the filter’s effectiveness remained stable. At the final time point, the log reduction was 1.01, indicating removal of over 90% of microorganisms from the flowing water (Table 4).
Changes in filter performance over time were evaluated using linear regression based on the log reduction in bacterial counts (log10(CFU/mL)). The hydrogel filter provided a low but consistent level of bacterial removal. Its regression slope (−0.0009) indicated that operating time had little effect on performance. However, the inactivation efficiency remained limited (around 0.8 log). The C-IER filter demonstrated higher initial performance. Over time, its effectiveness decreased slightly, with a slope of −0.0177. The composite filter delivered the strongest reduction at the start of the test (1.51 log). Its efficiency declined more noticeably, as reflected by a steeper slope (−0.0487). This drop was likely caused by gradual saturation of the filter media (Figure 5).
In the comparative analysis of filtration efficiency between different bed configurations (C-IER and H-C-IER), it was demonstrated that the use of a filter containing a hydrogel layer enriched with plant extracts (H-C-IER) significantly reduced bacterial abundance compared to the bed lacking this layer (C-IER). The average CFU value for the H-C-IER configuration was 0.52 × 106 CFU/mL, while for C-IER it was 1.05 × 106 CFU/mL. These differences were statistically significant (LSD0.05 = 0.336 × 106 CFU/mL). The mean values across individual time points (levels of Factor B) also varied significantly. The lowest bacterial counts were recorded at the initial time points (B1–B3), while the highest values were observed at later stages (B5–B6), indicating a decline in filtration efficiency with prolonged exposure to biologically loaded water.
A comparison of H vs. H-C-IER configurations showed that the application of the composite filter significantly enhanced antibacterial performance at every time point. The average bacterial count for the hydrogel-only filter was 1.85 × 106 CFU/mL, compared to 0.52 × 106 CFU/mL for the variant with plant extracts (LSD0.05 = 0.340 × 106 CFU/mL) (Table 5).

3.2. Results for Agro-Industrial Process Water

Microbiological analysis of the process water samples exhibited a lower overall bacterial concentration compared to the laboratory-inoculated model. Simple staining with crystal violet applied to selected colonies indicated the presence of bacilli, cocci, and clustered cells characteristic of staphylococci, suggesting a mixed microbial community. This composition is typical for wastewater derived from vegetable processing lines (Figure 6).
The data indicate that the composite H-C-IER filter reduced bacterial concentrations in process water from 1.07 × 104 CFU mL−1 (control) to 1.1 × 102–1.0 × 103 CFU mL−1 within the first 6 timepoints of operation, corresponding to a reduction of 1.2–2.0 log10 (Table 6). The highest efficiency (≈2 log10) was observed after the first hour, followed by a gradual decline in performance, stabilizing at approximately 0.8–1.2 log10 from the third test interval onward. This trend suggests that while the filter retains over 90% of its retention capacity its efficiency declines over time.
During the first two sampling intervals, the composite removed about 11% (1 h) and 17% (2 h) more bacteria from the artificially contaminated matrix than from the actual rinse water. After three hours the difference rose to roughly 30%, suggesting that suspended solids and organic matter in the plant effluent had begun to block active bed surfaces, thereby diminishing performance. By the sixth interval, the log-reductions were virtually the same (≈1% difference), indicating that with longer contact times the composite provides a comparable bacterial barrier regardless of water-matrix complexity, although its advantage over the process water declines as the contaminant load increases.

4. Discussion

Increasing efforts to implement a circular economy and to minimize losses—particularly in preserving and conserving valuable water resources—have become a prominent research focus [36,37]. The main driver of this trend is increasing anthropogenic pressure, along with progressing climate change, which adversely affects water resources through prolonged droughts and short periods of intense rainfall. These phenomena disrupt the hydrological balance in natural reservoirs and watercourses, as well as in groundwater systems [38,39]. In this context, the fresh-cut fruit and vegetable industry is receiving growing attention due to its high water consumption, low water recycling rates, and the associated potential microbiological risks [40].
According to the FDA Produce Safety Rule, water used during and after harvest must contain no detectable E. coli in 100 mL of water, reflecting the use of this bacterium as the primary indicator of microbiological safety [41]. In the European Union, by contrast, water intended for contact with food must meet the criteria of potable water, as defined by Directive (EU) 2020/2184, which requires the absence of E. coli and Enterococcus spp. in 100 mL [42]. However, our earlier field observations indicated that in some processing facilities, the wash water was not always replaced between product batches, which significantly increases the risk of microbial accumulation and highlights the practical relevance of developing effective treatment strategies [35].
In our study, we designed and evaluated the performance of multilayer filters composed of activated carbon, ion exchange resin, and hydrogel hydrated with natural plant extracts. The system was subsequently validated using industrial process water. To the best of the authors’ knowledge, this is the first report to apply a filtration approach incorporating natural plant extracts immobilized within the filter bed for use in the fresh-cut industry. Conventional disinfection techniques are typically employed, including chlorine-based compounds (which are predominant), ozonation, peracetic acid, ultraviolet (UV) irradiation, hydrogen peroxide, as well as combined or hybrid methods [11]. In addition, the study was carried out using environmental bacterial isolates obtained from food-processing facilities and real industrial process water, rather than the reference strains or model systems employed in most previous studies [43,44,45]. This choice reflects real operating conditions and provides a preliminary indication of the applicability of the proposed approach in an industrial context.
The study was carried out under several assumptions that informed both its design and the interpretation of the findings. It was assumed that the bacterial isolates obtained from food-processing facilities are representative of the microbial contaminants typically present in wastewater from the fresh-cut industry. The multilayer filtration bed was considered to act through the complementary effects of hydrogel with natural extracts, ion-exchange resin, and activated carbon. The pilot-scale system was regarded as sufficiently representative of industrial conditions, although the experiments were conducted over limited operating intervals. Finally, the industrial process water used for validation was assumed to reflect the composition of effluents commonly generated in agro-food processing environments.
The study by Ignat et al. [46] on the disinfection of wash water using acidic electrolyzed water (30 mg Cl2 L−1) demonstrated a reduction in mesophilic bacteria of approximately 1.0–1.2 log10 CFU mL−1 after 10 min of contact. In contrast, the composite filter proposed in the present study reduced the number of inoculated bacteria by 2.2 log10 CFU mL−1 in the initial interval, without the addition of chlorine. This suggests that the multilayer system saturated with natural plant extracts may offer higher microbiological efficacy while remaining free of chlorine-based compounds. A similar level of effectiveness was reported by López-Gálvez et al. [47], who applied 2–3 mg L−1 ClO2 in the wash water recirculation system at a tomato-sorting facility, achieving a reduction in mesophilic bacteria, coliforms, and E. coli in process water by slightly more than 1 log10 CFU mL−1. Importantly, the authors also did not detect any disinfection by-products in the final product. This highlights the critical importance of selecting an appropriate dosage when using conventional disinfection methods. Higher levels of microbial reduction were achieved by Banach et al. [48] in a pilot-scale setup at an iceberg lettuce sorting facility. The study confirmed the high effectiveness of adding a low-chlorine oxidant (5 mg ClO2 L−1 or 3 mg ClO2 L−1) to the recirculating process water, resulting in a ≥5-log10 reduction in inoculated E. coli in less than 1 min.
Another example of disinfectants used in agri-food processing facilities is ozone. Compared to chlorine, its stability in water is significantly lower due to its spontaneous decomposition, which is initiated by reactions with hydroxyl ions or reduced substances [49]. Nevertheless, studies conducted by Selma et al. [50] demonstrated that 60 min of exposure to O3, or its combination with UV-C, resulted in a 5.9–6.6 log10 reduction in microbial load in wastewater from vegetable processing. It is important to note, however, that ozonation generates a wide range of secondary inorganic and organic compounds. Ozonation by-products can be classified into two main groups: oxidation by-products, formed from naturally occurring constituents of the water itself (e.g., bromide ions), and transformation products, resulting from reactions between ozone and trace contaminants present in the solution [51].
In contrast, our study was based on the use of non-invasive methods involving natural filter components—namely, activated carbon and natural plant extracts—supported by an ion exchange resin. In tests using inoculated water, this system demonstrated greater stability and a higher level of bacterial reduction. Activated carbon filters are widely used as a medium for water purification. While their primary function is the removal of contaminants through adsorption onto a highly porous surface, they may also exert effects on microbial populations [52]. Microorganisms can become immobilized within the pores of activated carbon, which consequently leads to a reduction in their abundance in the flowing water [53]. As noted by Sbardella et al. [54], over time, colonization of the filter bed occurs, along with the stabilization of interactions between microorganisms and the carbon phase. This leads to the phenomenon of biological activated carbon (BAC) filtration, in which sessile microorganisms degrade immobilized contaminants. While this process can be considered beneficial in the case of natural colonization or the use of microbial inoculants, it may be highly undesirable in the context of our study, due to the use of opportunistic pathogens in the inoculated water. Therefore, a subsequent step in the development of the system should include a detailed analysis of biofilm formation within the filtration unit, together with the implementation of risk management strategies aimed at minimizing potential adverse effects. Many authors highlight one of the major limitations associated with the use of activated carbon filters—their potential to become reservoirs of antibiotic resistance genes and multidrug-resistant bacteria, which may pose a significant risk to public health safety [55,56,57]. Nevertheless, activated carbon remains one of the longest-used and most thoroughly documented sorbents in environmental engineering, with its industrial application dating back to the early 20th century [58].
Ion exchange resins, traditionally used in water softening and demineralization processes, have in recent years gained increasing attention as components of active antibacterial barriers [59]. However, in our study, they were employed as a conventional component complementing the composite filter bed, with the aim of enhancing its functionality by improving sorption capacity and potentially contributing to overall water quality improvement.
The final component of the filter bed was a hydrogel hydrated with natural plant extracts—an approach that has been scarcely described in the scientific literature to date. Previous in vitro studies on the extracts [34] demonstrated their effectiveness under laboratory conditions; however, the present work represents the first report in which this solution has been implemented in the context of process water treatment. Importantly, most available publications concerning hydrogels enriched with plant extracts primarily relate to biomedical applications, particularly wound healing and localized therapy [60,61,62]. Compounds identified in the earlier GC-MS analysis—such as epigallocatechin gallate, shikimic, ellagic, and gallic acids, as well as betulin, salicin, and procyanidins—may be responsible for the observed antimicrobial activity of the extracts.
Filtration is commonly employed in environmental engineering as a final polishing step to lower particle-associated microbial loads and enhance the effectiveness of subsequent disinfection [63,64]. An example of effective bacterial removal is the study by Zhang et al., which showed that adding biochar to sand columns enhances the elimination of E. coli and B. subtilis under both slow and rapid filtration, mainly due to the material’s higher adsorption capacity [65]. Other studies have shown that the depth of the sand bed influences both filter performance and flow rate. Deeper beds improve the removal of suspended solids, turbidity and coliform bacteria, while dissolved contaminants are less effectively reduced [66]. These findings demonstrate the effectiveness of conventional granular media mainly through physical straining and adsorption. By contrast, our multilayer bed integrates these mechanisms with the antimicrobial activity of plant-extract-infused hydrogel, offering an additional mode of bacterial reduction beyond polishing alone.
Sodium polyacrylate-based hydrogels are superabsorbents that undergo extensive swelling in aqueous environments. As a result of this process, a porous structure is formed within the polymer network. This feature enables the gradual release of immobilized compounds into the surrounding medium, both in medical applications and in our own studies with plant extracts [67,68]. The release of active substances (e.g., in medicines) from hydrogels can occur through different mechanisms: diffusion of the active substance with the absorbed liquid, erosion of the polymer matrix leading to pore enlargement, or swelling of the matrix due to water uptake [67]. Although we did not perform a kinetic analysis of the release process in our study, the literature clearly indicates that sodium polyacrylate-based systems typically exhibit non-Fickian diffusion behavior and therefore require models that account for both diffusive transport and polymer network relaxation [69,70].
With an initial load of approximately 107 CFU mL−1, the laboratory-inoculated water represented an extremely challenging test scenario, significantly exceeding the level of contamination observed in the wastewater collected from the fruit processing facility. Despite this high microbial burden, the H-C-IER composite filter achieved a reduction in more than 2 log10 within the first three hours, and after 24 h, it still maintained an effectiveness greater than 1 log10. The bacterial concentrations used in our study are consistent with common research practice. For example, Faith et al. employed an inoculum concentration of 107 CFU of Salmonella enterica in their experimental design. An alternative approach was presented by Yesil et al. [71], who applied a broader range of inoculum concentrations—108, 107, and 105 CFU/g—for the inoculation of spinach leaves. In real process water—characterized by a lower bacterial concentration but a higher content of organic matter following apple-washing—the observed log reductions were lower (≈1–1.3 log10), indicating that such contaminants may partially affect the performance of the composite. Nevertheless, the filter met the minimum efficiency criterion of 90%. As noted in the study by López-Gálvez et al., the physicochemical quality of process water in a fresh produce facility varied between processing lines, which was attributed to differences in the types of raw materials being washed as well as the uneven efficiency of the washing systems themselves [72]. This, in turn, can lead to differences in the microbial load of the process water itself.
A limitation of the present study lies in the barrier properties of the hydrogel. The observed antibacterial effect is primarily attributable to the diffusion of plant extracts from the hydrogel matrix into the surrounding medium. While this release mechanism is intrinsic to the functioning of the system, it makes it difficult to assess the potential barrier effect of the hydrogel itself. A comprehensive characterization of both the release kinetics and the barrier properties will be required in future studies to optimize the hydrogel formulation and to gain a clearer understanding of its role in microbial inactivation. In this preliminary study, the operational time of the prototype was limited to 24 h, during which a marked decrease in the efficiency of the C and IER components was observed. Although this time frame allowed us to obtain a first evaluation of the system, further investigations are needed to assess its performance over longer operating periods. Future optimization of the filter will focus not only on testing extended operational times but also on increasing the effective filtration surface, with the aim of improving both efficiency and durability. In this context, the H component appears particularly promising, and further studies will be directed toward refining its performance and stability.
An interesting approach aimed at enhancing the efficiency of bacterial inactivation in process water could involve the integration of additional modules into the proposed system—for example, a sedimentation tank, which reduces suspended solids that may serve as a habitat for microorganisms [73]. Another strategy involves increasing the filtration surface area by adding a greater number of filter tubes, in order to adjust the performance and lifespan of the filtration unit to the specific conditions of a given production facility—such as the type of processed crop, continuous vs. seasonal operation, or geographical location. In addition, future work should include long-term performance tests under continuous operation to better reflect industrial practice. Further studies will also be necessary to characterize the release kinetics of natural extracts from the hydrogel matrix and to optimize the balance between diffusion and barrier properties.
The proposed filter system in our study may serve as an alternative or complementary solution to conventional disinfection methods, which are known to generate undesirable by-products.

5. Conclusions

The results showed that the composite filter bed—comprising hydrogel, ion exchange resin, and activated carbon, saturated with natural plant extracts from tea waste, willow bark, oak bark, birch bark, raspberry shoots, and linden flowers—reduced the bacterial load by more than 2 log10 in highly inoculated water (1.2 × 107 CFU mL−1) and maintained at least a 1 log10 reduction in real wastewater effluents from fruit processing. This effect was achieved without the use of chlorine, thereby eliminating the risk of forming undesirable disinfection by products. While the filter maintained its efficacy, long-term operation requires further evaluation of durability and potential regeneration. In the future, the system could be enhanced with preliminary clarification modules (e.g., sedimentation tanks), which may further increase filter lifespan and performance. The proposed solution aligns with the development of modern methods for reducing contamination in wastewater for potential reuse in the agri-food industry—supporting blue water footprint reduction strategies and compliance with “green processing” principles.

Author Contributions

Conceptualization, P.K. and B.B.-B.; methodology, P.K.; software, P.K.; validation, P.K. and B.B.-B.; formal analysis, P.K., W.P. and B.B.-B.; investigation, P.K. and B.B.-B.; resources, B.B.-B.; data curation, P.K.; writing—original draft preparation, P.K.; writing—review and editing, P.K. and B.B.-B.; visualization, P.K.; supervision, B.B.-B.; project administration, B.B.-B.; funding acquisition, P.K. and B.B.-B. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by funds from the Ministry of Education and Higher Eductation of the Republic of Poland, No: DWD/5/0207/2021. The APC was funded by Bydgoszcz University of Science and Technology, as part of the “Działania Naukowe Młodych” program, grant number: DNM 9/2025.

Data Availability Statement

All relevant data supporting the findings of this article are included within the manuscript. Raw data, as well as any additional materials, are available upon request from the corresponding author.

Acknowledgments

During the preparation of this manuscript, the authors used ChatGPT (OpenAI, GPT-4, July 2025 version) for assistance with language editing, scientific phrasing, and improving the clarity of the English text. The authors have critically reviewed, verified, and edited all generated content, and take full responsibility for the final version of this publication.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
DBPsDisinfectant byproducts
CFUColony-forming unit
HHydrogel
C-IERCarbon-ion exchange resin filter

References

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Water 17 02631 g001
Figure 1. Graphical representation of the hydrogel in various forms used during the study: (a) dry, non-hydrated hydrogel; (b) hydrated hydrogel; (c) hydrogel hydrated with plant extracts; (d) comparison of hydrogel volume before and after hydration.
Figure 1. Graphical representation of the hydrogel in various forms used during the study: (a) dry, non-hydrated hydrogel; (b) hydrated hydrogel; (c) hydrogel hydrated with plant extracts; (d) comparison of hydrogel volume before and after hydration.
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Water 17 02631 g002
Figure 2. Scheme of the filtration system used in the study.
Figure 2. Scheme of the filtration system used in the study.
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Water 17 02631 g003
Figure 3. Schematic overview of the filter media used in the tested configurations: (1) filter filled with activated carbon and ion-exchange resin; (2) filter containing hydrogel; and (3) filter composed of a mixture of activated carbon, ion-exchange resin, and hydrogel.
Figure 3. Schematic overview of the filter media used in the tested configurations: (1) filter filled with activated carbon and ion-exchange resin; (2) filter containing hydrogel; and (3) filter composed of a mixture of activated carbon, ion-exchange resin, and hydrogel.
Water 17 02631 g003
Water 17 02631 g004
Figure 4. Summary of bacterial counts (CFU/mL) after filtration at specific intervals. Abbreviations: H—hydrogel; C-IER—activated carbon with ion-exchange resin; H-C-IER—a combination of hydrogel, activated carbon, and ion-exchange resin.
Figure 4. Summary of bacterial counts (CFU/mL) after filtration at specific intervals. Abbreviations: H—hydrogel; C-IER—activated carbon with ion-exchange resin; H-C-IER—a combination of hydrogel, activated carbon, and ion-exchange resin.
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Water 17 02631 g005
Figure 5. Analysis of the linear relationship between time and bacterial log reduction (log10(CFU/mL)).
Figure 5. Analysis of the linear relationship between time and bacterial log reduction (log10(CFU/mL)).
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Figure 6. Microscopic images showing diverse morphological forms of bacteria isolated from selected colonies cultured from process water ((A)—cocci, (B)—bacilli, and (C)—staphylococci; simple crystal violet staining, microscope: Olympus BX53, Tokyo, Japan).
Figure 6. Microscopic images showing diverse morphological forms of bacteria isolated from selected colonies cultured from process water ((A)—cocci, (B)—bacilli, and (C)—staphylococci; simple crystal violet staining, microscope: Olympus BX53, Tokyo, Japan).
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Table 1. Characteristics of the main disinfection by-products.
Table 1. Characteristics of the main disinfection by-products.
DBP TypeExamples of CompoundsSources of FormationToxicityReferences
Carbon-based DBPs Chloroform, Dichloroacetic acid, Trichloroacetic acidReaction of disinfectants with natural organic matter and anthropogenic contaminantsCytotoxicity, genotoxicity (compound-specific), some carcinogenic effects[14,15,16]
Nitrogen-based DBPs Halonitromethanes, Haloacetamides, HaloacetonitrilesReactions with nitrogen-containing compounds (e.g., proteins, amino acids)Strong genotoxicity, mutagenicity, neurotoxicity[15,17]
Inorganic DBPsBromate, Chlorite, ChlorateByproducts of ozone and chlorine dioxide disinfectionVariable; bromate with confirmed genotoxicity and carcinogenicity[15,18]
Iodinated DBPsIodoacetic acid, Iodoform, Iodo-trihalomethanesPresence of iodide in water during disinfectionVery high toxicity; strong cytotoxicity and genotoxicity[15,19]
Brominated DBPsBromodichloromethane, Bromoform, Dibromoacetic acidPresence of Bromide in waterHigher toxicity than chlorinated analogs; including genotoxic and carcinogenic[15,20]
Chlorinated DBPsChloroform, Trichloroacetic acid, Chloroacetic acidTypical products of chlorine-based disinfectionLower toxicity than Br- and I-DBPs; variable toxicological profiles[13,15]
Table 2. Summary of bacterial count reduction over time for the hydrogel-based filter with natural extracts.
Table 2. Summary of bacterial count reduction over time for the hydrogel-based filter with natural extracts.
Time Point [h]Mean Colony Count (10−4)CFU/mLlog10 CFU/mLLog-Reduction
11961.96 × 1066.290.79
21841.84 × 1066.270.81
31741.74 × 1066.240.84
61871.87 × 1066.270.81
121711.71 × 1066.230.85
241991.99 × 1066.300.78
Table 3. Summary of bacterial count reduction over time for the ion-exchange resin and activated carbon filter.
Table 3. Summary of bacterial count reduction over time for the ion-exchange resin and activated carbon filter.
Time Point [h]Mean Colony Count (10−4)CFU/mLlog10 CFU/mLLog-Reduction
1373.70 × 1055.571.51
2727.20 × 1055.861.22
3939.30 × 1055.971.11
61291.29 × 1066.110.97
121571.57 × 1066.200.88
241391.39 × 1066.140.94
Table 4. Summary of bacterial count reduction over time for the hydrogel, ion-exchange resin and activated carbon filter.
Table 4. Summary of bacterial count reduction over time for the hydrogel, ion-exchange resin and activated carbon filter.
Time Point [h]Mean Colony Count (10−4)CFU/mLlog10 CFU/mLLog-Reduction
177.00 × 1044.852.23
2101.00 × 1055.002.08
3212.10 × 1055.321.76
6707.00 × 1055.851.23
12969.60 × 1055.981.10
241181.18 × 1066.071.01
Table 5. Comparative bacterial abundance over time between experimental filter configurations.
Table 5. Comparative bacterial abundance over time between experimental filter configurations.
Level of Factor A
A1 (C-IER)A2 (H-C-IER)Mean
Level of Factor BBacterial abundance (×106 CFU mL−1)
B10.370.070.22 c
B20.720.090.41 c
B30.930.120.53 c
B41.290.701.00 b
B51.570.961.27 b
B61.391.181.29 a
Mean1.05 a0.52 b
C-IER vs. H-C-IER: LSD0.05 = 0.336 × 106 CFU mL−1, LSD0.05 (Tukey); A = 0.158 × 106, B = 0.421; B/A = n.s.; A/B = n.s.
A1 (H)A2 (H-C-IER)Mean
Level of Factor BBacterial abundance (×106 CFU mL−1)
B11.96 c, A0.07 a, B1.02 b
B21.84 c, A0.09 a, B0.97 b
B31.74 c, A0.12 a, B0.93 b
B41.87 c, A0.70 a, A1.28 b
B51.71 b, A0.96 a, B9.03 a
B61.99 b, A1.18 a, B1.59 a
Mean1.85 a0.52 b
H vs. H-C-IER: LSD0.05 = 0.340 × 106 CFU mL−1; A = 0.127; B = 0.340; B/A = 0.480; A/B = 0.311.
Notes: Different lowercase letters (a, b, c) within columns indicate statistically significant differences between means according to Tukey’s test (α = 0.05). Means followed by the same lowercase letter do not differ significantly. Capital letters within rows denote homogenous groups; values sharing the same letter are not significantly different.
Table 6. Log-reduction in bacterial counts in process water after filtration through the composite bed at selected time intervals.
Table 6. Log-reduction in bacterial counts in process water after filtration through the composite bed at selected time intervals.
Time PointCFU mL−1log10 CFU mL−1Log-Reduction *
1 h1102.041.99
2 h2002.301.73
3 h6302.801.23
6 h6402.811.22
Notes: * The control sample showed 107 colonies at the 10−2 dilution, corresponding to 1.07 × 104 CFU mL−1, or 4.03 log10 CFU mL−1.
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Kanarek, P.; Breza-Boruta, B.; Poćwiardowski, W. Pilot-Scale Evaluation of a Filter Prototype for Bacterial Inactivation in Agro-Food Processing Wastewater. Water 2025, 17, 2631. https://doi.org/10.3390/w17172631

AMA Style

Kanarek P, Breza-Boruta B, Poćwiardowski W. Pilot-Scale Evaluation of a Filter Prototype for Bacterial Inactivation in Agro-Food Processing Wastewater. Water. 2025; 17(17):2631. https://doi.org/10.3390/w17172631

Chicago/Turabian Style

Kanarek, Piotr, Barbara Breza-Boruta, and Wojciech Poćwiardowski. 2025. "Pilot-Scale Evaluation of a Filter Prototype for Bacterial Inactivation in Agro-Food Processing Wastewater" Water 17, no. 17: 2631. https://doi.org/10.3390/w17172631

APA Style

Kanarek, P., Breza-Boruta, B., & Poćwiardowski, W. (2025). Pilot-Scale Evaluation of a Filter Prototype for Bacterial Inactivation in Agro-Food Processing Wastewater. Water, 17(17), 2631. https://doi.org/10.3390/w17172631

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