Winery Wastewater Innovative Biotreatment Using an Immobilized Biomass Reactor Followed by a Sequence Batch Reactor: A Case Study in Australia

Abstract

A pilot-scale treatment system was developed to manage winery wastewater (WWW) generated by small and medium wineries. The system incorporated three stages: pre-treatment for suspended solids removal and a two-step aerobic biotreatment. The biotreatment phase utilized a bioaugmented bioreactor with encapsulated Pseudomonas putida F1, employing the Small Bioreactor Platform (SBP) technology. This innovative encapsulation method enhanced the breakdown of recalcitrant compounds and accelerated the biodegradation process. The second reactor was operated as a Sequence Batch Bioreactor (SBR) to remove the remaining organics and solids. Over the 100 days of operation, the mean WWW flow rate was 0.5 m³/d with average organic loads of 3950 mg/L COD (chemical oxygen demand) and 2220 mg/L BOD (biological oxygen demand), operating with a hydraulic retention time (HRT) of 4 days. Reductions of up to 96% in BOD and 90% in COD values were observed with stable removal rates over time. The novelty of this study is that it offers a new, effective aerobic biological treatment process, embracing bioaugmentation of encapsulated biomass followed by SBR for WWW with a relatively short HRT, high organics removal, and a stable treatment process. The effluent quality from this treatment system met the regulatory requirements for release to a municipal wastewater treatment plant and potentially also for irrigation.

1. Introduction

Grapes are the most widely grown fruit species globally. While various grape-based food products are available in the market, nearly 80% of grapes are used for wine production, around 13% are eaten fresh, and the remaining 7% are processed into fruit juice or raisins. In 2022, global grape production totaled 74.94 million tons, resulting in over 237 million hectoliters of wine by 2023 [1]. However, the volume of wastewater produced ranges from 0.7 to 1.2 times the volume of wine, leaving a significant environmental footprint [2,3]. The composition of the WWW varies daily and depends on activities within the winery throughout the year. Winery effluents contain (i) simple dissolved compounds such as organic acids, sugars, phenols, and alcohols from grapes and wine, generating a high requirement for oxygen for biological decay; (ii) moderate salinity and high concentrations of sodium relative to calcium plus magnesium as well as low concentrations of nitrogen and phosphorus relative to carbon; (iii) inorganic components from the water supply, alkali wash waters such as cleaning agents (e.g., NaOH, KOH), and processing operations; and (iv) significant amounts of sulfur. There have also been reports of micropollutants including chemical fertilizers, pesticides, and herbicides used in producing grapes [4]. The combination of the high concentration of organic and inorganic compounds and the spatiotemporal dynamics of WWW between and within wineries make WWW treatment a challenging task. Although most of the organic matter is soluble and easily biodegradable, and aerobic and anaerobic processes are commonly applied for WWW treatment [5], WWW is considered hard to treat due to the high concentration of organic matter and low pH. The low pH is due to the presence of organic acids (acetic, tartaric, and propionic) and phenols/polyphenols which inhibit the microbes needed to metabolize the organic load [2]. A recent review indicates that pH levels can vary, starting from 3.5 or, in some cases, even lower [6]. Hence, biological winery treatment systems require a long start-up time as microbial populations establish, stabilize, and grow. However, the lack of protection of established bacterial communities from sudden changes in quality, due to the seasonal nature of wine making, is challenging [7]. Ensuring a consistent, protected reservoir of microbes proven to effectively treat winery waste is therefore a critical component of the biological treatment of WWW. Anaerobic and aerobic processes are most commonly applied for WWW treatment because they can react to changes in the organic loading. A widely used form of biological treatment is based on anaerobic and aerobic lagoons and/or secondary wetlands. However, there is a need to improve the efficacy of WWW treatment, mainly, the biological process, using different technologies and treatment processes to overcome the inherent challenges, as well, making the treatment process much more intensive to reduce digestion time and consequences, as well as infrastructure.

This study introduces novel and effective treatment processes employing an aerobic biological treatment with a relatively short HRT of 4 days. After a coagulation process that primarily reduces suspended solids and colloids, it utilizes stable and selected immobilized biomass as the primary biological stage for degrading soluble recalcitrant molecules, followed by a secondary biological treatment using activated sludge in a sequencing batch (SBR-sequencing batch reactor) configuration. This biological treatment configuration is possible only if we have the ability to immobilize the introduced bacterial culture into the first bioreactor, avoiding culture loss downstream. We chose to use an innovative encapsulation method called Small Bioreactor Platform (SBP) technology to immobilize the introduced culture in the first bioreactor, while allowing for a fast biodegradation rate [8]. The SBP is a novel, biomimetic 3D confined environment for the in-situ application of microorganisms in water and soil mediums [9,10,11]. The SBP capsule is coated with a structural microfiltration membrane (0.2 µm) that prevents predators from penetrating the capsule’s internal medium, while preventing the introduced culture from escaping the capsule and allowing free diffusion of dissolved nutrients air and partials under the size of 200 nanometers. The SBP encapsulation method enables the implementation of long-term selective biomass in situ within wastewater treatment plants, avoiding downstream biomass loss by continuous flow, control of the location of the implemented culture, and relatively efficient biodegradation kinetics that can be synchronized with the HRT of the wastewater treatment plant [11,12]. Thousands of SBP capsules are immobilized within the host bioreactor using perforated cages attached to cranes [13]. This technology has already been proven to enhance the catabolism of numerous target pollutants, e.g., various phenol compounds [8], 17α-ethynylestradiol (EE2) [14], nitrate [15], BETX [16], and paracetamol [17], since the internal flora of the SBP can be selected on the basis of targeted pollutant removal. Hence, it was postulated that pre-treatment of WWW with SBP-encapsulated Pseudomonas putida F1, a well-characterized degrader of phenol and other hard biodegradable molecules, would provide sufficient biomass catabolism as a preliminary degradation step for natural flocculated biomass digestion (activated sludge) in a sequential batch reactor, facilitating a high-rate biodegradation process. To allow for a stable digestive biological process resulting from a diverse microorganism’s cultures, dual biotreatment stages were designed, built, and operated for 100 days, using the benefit of the SBP technology to digest recalcitrant compounds. The use of encapsulated biomass allowed us to operate the first bioreactor under a continuous treatment configuration, separate from the activated sludge in the secondary biological treatment process that was operated under SBR operational conditions. To the best of our knowledge, this is the first attempt to integrate a dual-stage aerobic treatment process that employs an encapsulated–immobilized selective biomass followed by activated sludge treatment operated under SBR configuration.

2. Materials and Methods

2.1. Study Design and Concept

This study was conducted for a period of 100 days during the wintertime in Melbourne, Australia and aimed to provide a treatment process that can produce high-quality effluents for irrigation. The study facility was established outdoors and fed by two reservoir tanks, with a volume of 2 m³, containing wastewater delivered from the sedimentation pond of the central WWW reservoir after a long-term sedimentation process (approximate 3–4 months). The source WWW quality fluctuated with time, generally reflecting the effects of long-term sedimentation and evaporation in open lagoons (WWW quality parameters are summarized in

Table 1. Feed winery wastewater characterization. COD—chemical oxygen demand; BOD—biological oxygen demand; TP (total phosphorus); TKN (total Kjeldahl nitrogen); TSS (total suspended solids).

Parameters
Value	pH	COD mg/L	BOD mg/L	TP mg/L	TKN mg/L	TSS mg/L
Mean	6.3 ± 0.7	3920 ± 1282	2220 ± 671.4	9.8 ± 4.6	11.5 ± 5.7	102 ± 54.4
Min	3.7	1500	780	1.2	5.1	32
Max	7.2	6265	2900	21	26	210

). Most of the organic matter is soluble and not easily biodegradable after a long period of sedimentation. Nitrogen and phosphorous are reduced and must be supplemented to support bacterial biomass growth.

The treatment facility with a daily treatment capacity of approximately 250 L (89 m³/year) was established in Melbourne, Australia. The treatment concept was to reduce organic matter through three different stages (Figure 1): (1) Pre-treatment by coagulation and sedimentation for solids removal. The aim of coagulation/flocculation is to agglomerate fine particles into flocs of large particles which can be removed to reduce turbidity, natural organic matter, and inorganic matter present in the WWW. Pre-treatment for organic matter removal is also expected to contribute to the elevation of dissolved oxygen inside the mixed liquor, due to organic matter reduction. (2) An SBP bioaugmented bioreactor that was designed to treat recalcitrant molecules, as well as further reduce the organic matter and generate better operational conditions for the next biological process. The bioaugmented bioreactor contains a perforated cage (30 cm × 30 cm × 30 cm) encasing 500 SBP capsules. The cage was positioned 30 cm below the water surface. (3) An activated sludge bioreactor that was operated under the configuration of an SBR for additional organic matter reduction. The process flow diagram is presented in Figure 1. The process description and operational activity are presented in detail in

Table 2. Operational activity protocol.

Step	Operational Activity	Aim and Treatment Position (PFD)
First treatment stage: Pre-treatment (solids removal)
1	The inflow pump (P-4) fed the system with raw wastewater at a flow rate of 9.4 L/h (225.6 L/d). An auxiliary pump (P-5) circulated the raw wastewater constantly. During the 8 d start-up phase, the raw wastewater was, in advance, diluted (1:1 v/v) with tap water and raw wastewater. Wastewater dilution was conducted to reduce organic concentration, thus allowing the development of aerobic conditions within the bioreactors.	T-4–T-5 Water preparation prior to the pre-treatment stage
2	The wastewater was fed to an equilibrium tank (T5, HRT: 12.0 h) through a 210 µm filter to reduce the concentration of suspended solids. Within the tank, pH was adjusted (P-1) with NaOH, poly-aluminium chloride (PAC) was provided (P-3) for coagulation, nutrients (P, N, Fe) were provided (P-2) to ensure microbial growth in the bioreactors, and the wastewater was stirred for homogenization.	T-5–T-6 Solids separation: filtration, coagulation, and sedimentation processes
3	From the equilibrium tank, the wastewater was transferred to a sedimentation tank for 12 h. Solids were discarded from the bottom of the tank (V-2).
Second treatment stage: encapsulated biomass reactor (bioaugmentation)
4	Wastewater was sent to the first bioreactor with an HRT of 2 d. The SBP capsules (NatiCap Hard Clean—500 units) were held within a perforated cage located approximately 15 cm below the water surface. The inflow rate capacity was 18.8 L/h (451 L/d).	T-7 Degradation of recalcitrant and toxic compounds
Third treatment stage: SBR
5	From the first bioreactor, overflow wastewater flowed to the second bioreactor (SBR) for an additional 2 d HRT. The SBR operational stages were as follows: Aeration stage (biodegradation)—47 h; Sedimentation stage—0.5 h (closing the blower); Effluent discharge—0.5 h (opening valve number 3); 500 L (half the bioreactor volume) is discharged into the effluent collector; Blower on, closing valve 1. End of cycle 1; Starting cycle number 2—repeat stages i to iv.	T-8 Organic load reduction
General terms, sampling and microscopic analysis
Overall treatment time (2 cycles, 1 m3 effluents): 96 h (4 d). The bioreactors were operated under controlled temperature (heated, 25 °C) and provided with forced air for dissolved oxygen enrichment and medium agitation. The effluents were collected by a sampling collector tank (T-9, V-20 L); they were analyzed twice a week for COD, TSS, TDS, phenols, and additional water chemical parameters and once a week for BOD. All measurements were conducted under the provision of the standard method in an external laboratory (ALS Water, Australia). Sampling: water was sampled at the sampling valves (SV): SV-1: inflow (from the feed pump) SV-2: outflow (from the outflow collector, after the SBR treatment stage). Microscopic analysis of the bioreactor (SBR) medium was conducted once a week to monitor the microorganisms and process efficacy.

.

2.2. Bacterial Cultures and SBP Encapsulation Procedure

A strain of Pseudomonas putida F1 (ATCC 700007) was encapsulated inside SBP capsules as previously described for other microbial species [8]. These capsules are made of a spheroidal cellulose acetate (CA) microfiltration membrane (0.2–0.7 µm pores) which creates a physical barrier between the pure P. putida encapsulated culture and the surrounding medium. In brief, the CA micro-filtration membrane was built on a water-soluble gelatine scaffold by a spray-coating method. The CA polymer comprises a blend of CA (Sigma, Kawasaki City, Japan) dissolved in an 80% acetone/20% methanol (v/v) solution. The solid fraction is 8% in total, comprising 88% CA and 12% castor oil as a plasticizer [10]. The SBP capsules were activated by 48 h exposure to an aquatic medium before introducing them into the reactor. Once the capsule was exposed to the aquatic medium, the microbiota became activated and proliferated within 24–48 h.

2.3. Water Analysis and Sampling

Samples for water chemical analysis were taken twice a week from the system inflow and outflow for COD (chemical oxygen demand), TSS (total suspended solids), TDS (total dissolved solids) and phenols, and once a week for BOD (biological oxygen demand), total phenols, TP (total phosphorous), TN (total nitrogen), TKN (total Kjeldahl nitrogen), EC (electrical conductivity), SAR (Sodium Adsorption Ratio), Na⁺, Ca²⁺, and Mg²⁺. All measurements were conducted under the provision of the standard method in an external laboratory (ALS Water, Australia). The effluents were collected within the sampling collector tank (T-9, volume 20 L; Figure 1), while inflow was taken directly from the middle of the wastewater storage tank (T-4, volume 1000 L; Figure 1).

2.4. Microscopic Analysis for Monitoring Floc Particles and Microorganisms Within the Mixed Liquor

Microscopic analysis of the mixed liquor, taken from the SBR medium, was conducted once a week. The aim of this analysis was to monitor the set of microorganisms and verify the presence of all trophic levels (floc particles and bacteria, ciliates, attached ciliates, and metazoans) for the evaluation of the medium ecosystem as an indicator of correct process efficacy. The microscopic analysis was made using a light microscope at a magnification of up to 1000×.

2.5. Enumeration of Culturable Heterotrophic Bacteria

Culturable heterotrophic bacteria enumeration within the SBP capsules was performed as follows: the inner medium (100 μL) of each capsule was plated in triplicate on LA (Luria agar) plates following serial dilutions (Difco, USA) and incubated for 48 h at 36 ± 1 °C.

2.6. Statistical Analysis

Data were tabulated and analyzed in JMP Pro 14.0.0 (SAS Institute, 2018). The response variables were tested for goodness of fit to the normal distribution by the Shapiro–Wilk Test. Consequently, the effect of the various treatments on the response variables meeting normality assumptions were analyzed by ANOVA, followed by pairwise comparison of means by Tukey’s HSD. Data failing the Shapiro–Wilk Test were analyzed with the non-parametric Kruskal–Wallis test followed by pairwise Wilcoxon comparison. Trends in data over time were analyzed by linear regression; R² and the F-test were tested for fit and significance.

3. Results

3.1. System Operation and Treatment Process Stability

At the system onset point, the treatment system was exposed to various conditions that affect the biological process. At the initiating stage (0–10 days), the temperature of the bioreactor mediums was low (7–10 °C), and the development of biological treatment was limited. Therefore, we stabilized the temperature of both bioreactor mediums at 25 °C using exogenous heating systems (Figure 2). Thus, the significant observed bioreactor activity start point was day 10 for bioreactor 1 (encasing the SBP capsules) and day 20 for bioreactor 2 (sequential batch reactor—SBR) to grow sufficient natural biomass. To monitor the development of the biological process over time, the microorganism set of the mixed liquor in the second bioreactor medium (SBR) was analyzed several times during the study period. The analysis aimed to determine whether the mixed liquor within the SBR activated sludge was well organized by microbial trophic levels as an indicator for biodegradation process efficacy. We monitored four major elements: particle floc shape and dimension, the presence of protozoans and metazoans as additional trophic levels to complete the ecosystem of the activated sludge, and the presence of microorganisms that can be defined as bioindicators. Large (>1000 µm), dispersed particle flocs were observed, interspersed with filaments, indicating a good biological structure matrix. Most observed filaments were identified morphologically and by Gram stain as Nocardia sp. Free-swimming ciliates were observed in addition to spirochetes, perhaps indicating a reduction in dissolved oxygen. Attached ciliates were not observed. In the upper metazoan trophic level, we found many rotifer units. In summary, it seems that the SBR mixed liquor contained a relatively good microbial community set with all three major trophic levels, indicating a typical and sustained biological process. The absence of attached ciliates can indicate high shear forces generated by the activity of the diffuser mixing the medium.

As can be seen by the COD removal rates, the biomass and biochemical activities steadily increased after 20 operational days (Figure 3). A similar phenomenon was observed for medium acidity. Winery wastewater can be acidic due to organic acids and fermentation; hence, the pH of the feed was increased to pH 6 by titration with NaOH (Figure 4). Here, as for temperature, the critical date of 20 d differentiated between start-up and full functionality. During the first 20 d of operation, the increase in pH from 6.5 to 7.5 was significant, while after this point, pH stabilized between 7.5 and 8.5 with no significant trend. We estimate that the observed rise in pH in the treated wastewater is a result of organic acids digestion by a microbial metabolic activity [18] and CO₂ stripping due to air bubbling.

3.2. System Efficacy Evaluation

Treatment efficacy was mainly measured by the organic matter (COD) reduction rate. The COD concentration was reduced from 3920 to 889 mg/L, an overall average of 80% removal (Figure 3). The efficiency of the process increased over time up to 90% in the last month of operation, even when the feed COD increased to a max of 6265 mg/L (R² = 0.26, F_1,24 = 8.30, p = 0.0082). Between day 40 and day 55, a marked decrease in feed COD was recorded due to a heavy rain event. However, the outlet COD remained stable and continued to decline slightly, though not significantly, over the next two months (Figure 3). According to our analysis (Figure 5A vs. Figure 5B), excluding the rain event does not significantly improve the regression or the tendency of COD removal of the treatment system. Over time, it is clear that COD removal improved and stabilized at over 90%.

Biological oxygen demand is a measure of bioavailable organic matter; hence, removal of BOD is one of the most important measures of bioreactor performance. In this system, BOD was reduced from 2550 mg/L in the feed to 87.5 mg/L in the effluent (Figure 6A). From the second week of operation, BOD in the effluent remained below 200 mg/L, except for during the heavy rain event when there was a significant decline in inlet BOD which caused biomass to die or be washed out of the bioreactor. Similar to COD, inlet (feed) BOD appeared to increase with time (R² = 0.25, F_1,12, p = 0.07), while outlet BOD decreased slightly, though not significantly. Hence, bioreactor efficacy was very stable in removing BOD; however, some biomass was washed away into the sample collectors and sewage, explaining the higher BOD and COD measured in the effluents. To prevent this phenomenon, a granular filter at the outlet would help to prevent suspended solids washing out; alternatively, better control over sludge removal may be required. The average BOD/COD ratio in the inflow was 0.63, indicating that most of the organic matter is biodegradable. An average BOD/COD ratio of 0.54 has been documented [6] in recent studies, indicating that the inflow BOD/COD ratios are similar to the reported average values. The BOD/COD ratio in the outflow decreased to 0.18, indicating that most of the BOD fraction was consumed during the wastewater treatment process. The system was more effective at removing BOD than COD, causing the BOD/COD ratio to drop from median 0.6 to median 0.2 (Figure 6B), meaning that the remaining organic matter is not easily degradable during the 4d HRT and poses less potential for eutrophication by oxygen-consuming species within the receiving water. In some cases (five major events), the suspended solids content of the effluent was much higher than that of the feed due to biological flocs from the system sludge escaping from bioreactor 2 to the outlet (Figure 7). This indicates a need for periodical waste sludge drainage. Furthermore, this trivial result can be prevented by a medium filter. In the last 10 d of the study, the waste sludge was drained from the system on a regular basis, resulting in a reduction in the suspended solids content in the outflow.

Organic nitrogen (TKN) exhibited a similar concentration in the inflow and outflow mediums throughout most of the experimental period (Figure 8). However, we can identify two long TKN peak episodes at 30–60 days and at 70–90 days. Those TKN peak episodes occurred in parallel to a significant reduction in the COD inflow level, suggesting either use by microbial biomass of recalcitrant molecules in the encasing proteins as a carbon source or biomass breaking down due to hunger stress, thus releasing cell contents into the medium. A similar phenomenon can be observed with the phosphorous (TP) concentration over time (Figure 8). The correlation between nitrogen and phosphorous peaks further strengthens the hypothesis that those peak events occurred due to biomass hunger and stress. During the rest of the study period, the inflow concentration of TP was higher than the outflow concentration, indicating an assimilation process.

In the case of mineral ions, TDS and EC are tightly correlated. However, in this system, the increase in EC from 500 to 2000 µs/cm was not accompanied by a significant increase in TDS (Figure 9). Hence, we suggest that the increase in EC was due to colloidal organic molecules in the wastewater that decomposed into smaller, conductive molecules which did not affect the TDS concentration. The cations and SAR in the wastewater did not change significantly from the feed to the effluent; however, the Na⁺ concentration increased with time (Figure 10). We hypothesize that the relatively high Na⁺ value is due to the cleaning solution used by the winery. The high concentration of Na⁺ in the effluent and low concentrations of divalent ions yielded a high SAR, which increased from 5 to 15 over the experimental period. This poses a serious risk for soil health and should be remediated by changing the cleaning agent in the winery from NaOH to KOH.

3.3. Scaling Up Design

The pilot treatment system was operated and monitored for almost 100 days and demonstrated that WWW can be treated at high efficacy with an HRT of 4 d in the dual-stage aerobic biotreatment system. The first stage is designed to treat recalcitrant organic matter via the implementation of selective biomass using a unique encapsulation method. This bioaugmentation treatment approach gives us the ability to selectively recognize a target molecule in a vast pool of similar molecules, which is essential for biological and chemical processes. A viable count of the SBP internal medium revealed that each SBP capsule contained 4.45 × 10⁸ CFU/mL Pseudomonas putida F1 culture. The host bioreactor contained 500 SBP capsules. Those provide 1 L of bacterial medium (each capsule contains 2 mL), creating a volume ratio of 1 to 1000 (v/v). For future system design, we can use the calculated COD reduction per day, 369 g/d (Equation (1)). Equation (1) can be used for the design of full configuration systems and provides us a predicted value of effluent COD in accordance with inflow COD values. To understand the required SBP capsule number for future system design, we used Equation (2), where each capsule affects COD reduction by 0.738 g/d (Equation (2)). This value includes all system treatment stages including pre-treatment (coagulation) and the activity of bioreactor 2. For example, the system design for reducing 10 kg COD per day requires 13,550 SBP capsules. The calculated bioreactor volume is 27 m³.

$$x=Qday\ast \Delta COD$$

(1)

$x$ = g − COD/day

Q = daily inflow capacity

ΔCOD (mg/L) = COD_Inflow − COD_Outflow

$$x={\displaystyle \frac{N}{Qday\ast \Delta COD}}$$

(2)

$x$ = 1 SBP capsule/gr-_COD/day

N = number of SBP capsules in the bioreactor

Q = daily inflow capacity

ΔCOD (mg/L) = COD_Inflow − COD_Outflow

4. Discussion

The SBP-encapsulated biomass allows us to work in a continuous configuration of two treatment stages, preventing the escape of the selective encapsulated biomass downstream into the second bioreactor (SBR) and effluents. Moreover, according to our knowledge, this is the only encapsulated bacterial formulation that allows relatively rapid transfer of contaminant molecules from the host medium into the confined medium of the SBP capsule. As long as the bacteria inside the SBP capsule are consuming the contaminant, in hard biodegradable molecules such as phenols and polyphenols, the concentration of the contaminant within the confined water body (i.e., the capsule) decreases, thus inducing the movement of contaminants from the host medium into the capsules by diffusion (i.e., to achieve equilibrium between the molecule concentrations on either side of the SBP membrane). Thus, we propose that the SBP capsule acts as a contaminant pump, thus explaining the high biodegradation rate and yield. Here, for the first time, we present a dual-stage aerobic biotreatment process that presents a stable digestion process over time: (1) the first bioreactor contains standalone selective biomass, while (2) the second bioreactor contains natural activated sludge operating under SBR conditions. The complete treatment system including the pre-treatment (coagulation and flocculation) was able to reduce up to 96% of BOD and 85–91% of COD, thus meeting the stringent requirements for effluent release to a municipal wastewater treatment plant. Compared to other treatment approaches reported in earlier studies, such as Constructed Wetlands (CWs), which integrate both physical and biological processes, COD removal efficiencies for CWs ranged from 60% to 99% [19]. In contrast, Sequential Batch Reactor (SBR) systems with a hydraulic retention time of around four days demonstrated COD removal rates exceeding 91% [20].

It is important to note that the influent used in current study had been stored for several months prior to treatment, which probably led to the degradation of most easy biodegradable BOD compounds, leaving behind more resistant organic matter for treatment. Therefore, achieving 85–91% COD removal indicates a strong treatment performance, especially given the relatively short hydraulic retention time.

From day 60 (40 days after system stabilization), BOD and COD reduction levels were stable, indicating system process stabilization and efficacy over time. A study by [21] showed that ethanol and short-chain volatile fatty acids were the main contributors to COD in WWW from waste stabilization ponds. It also showed that the total phenolic content was less than 100 mg/L, suggesting that winery effluent should always be stored in ponds prior to treatment. Regarding treatment technologies, a review by [2] reported that the COD removal efficiency of biological treatment usually reaches 80–90%, but the applied organic loads vary greatly by an order of magnitude, depending on the applied technology. The SBR configuration demonstrates high COD removal rates in comparison to other biological systems [22].

Biological treatment is recognized for its environmental sustainability and cost-efficiency. However, it demonstrates limited effectiveness in removing organic matter at high concentrations in wastewater, leading to the persistence of certain toxic or hard biodegradable compounds that degrade at such slow rates. Therefore, a relatively long HRT is required for treatment. Winery effluents are high in COD, generally acidic, and may contain phenolic and polyphenolic compounds that can inhibit biological treatment systems. In consequence, care needs to be taken in the selection of the microorganisms employed and in their adaptation to treating these effluents [18].

Generally, WWW is treated by anaerobic treatment followed by aerobic treatment. According to [5], in their review on WWW quality and treatment options in Australia, the most common include suspended biomass as free cells or flocs (anaerobic contact digesters, anaerobic sequencing batch reactors, and anaerobic lagoons), anaerobic granules (Upflow Anaerobic Sludge Blanket—UASB), or biofilms on fixed support (anaerobic filter) or on mobile support as with the fluidized bed. In the field of aerobic treatment, we can find the following treatment approaches: aerated lagoons, activated sludge, SBR, membrane bioreactor (MBR), jet-loop activated sludge, air microbubble bioreactor with or without coupling to a biological trickling filter, and wetlands [23,24].

More advanced technologies include the use of aerobic biological treatment combined with photocatalysis [25] and oxidation, mainly using Fenton’s reagents, to increase the removal of recalcitrant molecules such as aromatic and total polyphenolic compounds [26,27,28]. Fenton’s reagent, a combination of Fe²⁺ salts and hydrogen peroxide (H₂O₂), is widely used in wastewater treatment. The implementation of Fenton’s reagent is mainly due to its ability to easily oxidize organic compounds and form hydroxyl radicals [29]. Consequently, in order to achieve a high level of COD removal, a mixture of different treatment methods is recommended.

Our treatment study combines pre-treatment for suspended solids removal, followed by biodegradation of recalcitrant compounds using a bioaugmentation treatment implemented by the SBP technology and, finally, an activated sludge operating under SBR configuration. To improve our treatment process and achieve improved effluent quality, an additive oxidative pre-treatment is recommended.

Kyzas et al. [4] suggested that the composition of winery effluents varies among countries, and climate strongly influences the appropriate technology to be selected for wastewater treatment. Biological systems have been reported to have reduced rates of biological activity during cold weather, which is a concern for wineries with year-round operations [30]. Eusebio et al. [31] reported that almost all biological treatment isolates belonged to the genera Pseudomonas and Bacillus. In addition, fungi culture such as Saccharomyces cerevisiae was also found. Hence, our treatment process was efficient and stable only after a suitable temperature was attained, suggesting that temperature has a critical role in biological treatment, particularly when the biological network of digestive microorganisms is being constructed. Since a mesophilic bacterium (Pseudomonas putida F1) was used in the bioaugmentation process, our hypothesis is that bioaugmentation has a great influence on biotreatment efficacy, as seen in the COD removal values.

The average inflow organic load in our study was 3920 mg/L COD; this is considered relatively low for anaerobic treatment. The establishment of a wastewater treatment plant that contains two treatment stages (anaerobic and aerobic) necessitates massive infrastructure investment and high professional manpower for operation. This cost of establishment and operation cannot be tolerated by small to medium wineries unless a simple treatment solution can be found. Central wastewater treatment plants have already been suggested as a solution for agriculture wastewater management; however, wastewater transportation reduces the attractiveness of this solution, particularly if wineries are located far from the central treatment plant.

The cost of bioreactor technologies and the lack of surplus land for passive treatment wetlands have prompted us to engineer an integrated treatment unit that can provide a good treatment solution for small to medium wineries. Key factors in the selection of a wastewater treatment process include the financial requirement for the initial outlay (e.g., for reactors, wetlands, etc.), equipment maintenance, and the expertise required to manage the system. While there are several highly effective treatment options available, they are not practicable in many instances, especially for small to medium wineries. This is of particular relevance in Australia [5]. Our treatment process demonstrates the possibility of converting a sequential anaerobic and aerobic treatment into a dual-stage aerobic treatment, thus simplifying the treatment process and infrastructure and presenting a more suitable solution for small to medium wineries.

The issue of high Na⁺ concentration in WWW has not been extensively addressed, despite such concentrations being a concern where the water is disposed onto land and has a significant impact on plants [32]. Because of the known deleterious properties of sodium ions (introduced/mobilized via irrigation) on soil structure [33], many wineries attempt to avoid or minimize the use of sodium hydroxide (NaOH) as a cleaning agent by using KOH proprietary cleansers. The use of SAR as a measure of soil sodicity is commonly accepted, and its use in vineyard management is becoming more widespread [34]. Sodic soils generally exhibit high pH, slaking of aggregates, and dispersion and swelling of soil clays. These physical characteristics cause degradation of soil structure and are an impediment to water and root penetration [35]. Our results (Figure 9) indicate that the treatment system can slightly reduce both Ca²⁺ and Mg²⁺. Our hypothesis for the slight reduction in ion concentration is biomass accumulation and passive absorption into the cells and activated sludge floc particles.

5. Conclusions

• Winery wastewater presents a serious environmental and economical challenge due to the inability of ordinary biological processes to consistently degrade the acidic and recalcitrant organic matter in the wastewater. The potential environmental impacts of WWW include pollution of ground and surface water, soil degradation, damage to vegetation, and odors; therefore, it is critical to allocate a cost-effective solution for small to medium wineries.
• This study presents a potential cost-effective treatment solution tailored for small to medium wineries. The solution incorporates the following stages:
  • A pre-sedimentation stage to initially remove larger particles and debris.
  • pH regulation to optimize the treatment conditions for microbial activity.
  • The use of robust microbes encapsulated within the novel confined environment of the SBP process to enhance biological treatment.
  • The SBR stage as the final treatment process, effectively treating the organic matter in the wastewater.
• The performance of the pilot system was evaluated over a period of more than 80 days, consistently achieving high removal rates for both COD and BOD. Specifically, the system demonstrated removal efficiencies exceeding 90% for both parameters, indicating the effectiveness and stability of the treatment process.