Water reuse: dairy effluent treated by a hybrid anaerobic biofilm baffled reactor and its application in lettuce irrigation

There is a synergy between the large quantities of organics-rich effluents generated by the dairy industry and the continually increasing water needs for crop irrigation. In this sense, this study aimed at evaluating the effect of decreasing the hydraulic retention time (HRT) on the stability and efficiency of a hybrid anaerobic biofilm baffled reactor (HABBR) treating simulated fatand salt-rich dairy wastewater, followed by its agricultural reuse. The reactor was monitored over 328 days, during which 72, 24, and 12 h were the hydraulic detention times. After achieving steady-state, the reactor presented organic matter removal greater than 90% and produced biogas with 41± 23%, 53± 3%, and 64± 12% of methane for HRTs of 72, 24, and 12 h, respectively. The best process performance was observed for an HRT of 24 h, and thus, a lettuce culture was irrigated with the treated effluent. The irrigation was performed in five different treatments, for which the amount of treated effluent added to tap water varied from 0 to 100%. Both the effluent and the harvested vegetables were evaluated for microbial contamination. Apart from the 75% effluent supply condition, there were no losses in leaf mass or area observed; instead, there was an increase of these parameters for the 25% and 50% effluent supply treatment. The use of dairy effluent treated by the HABBR allowed for microbiologically safe food production. Therefore, the process offered both potential cost reduction for fertilizers, preservation of water resources, and a renewable energy source.


GRAPHICAL ABSTRACT INTRODUCTION
The milk processing industry is one of the most important sectors in the food industry. However, as a consequence, there is a large volume of fat-rich and salty effluents generated. Anaerobic digestion has many advantages, in addition to treating wastewater, because it also produces a methanerich biogas that can be recovered and used as a fuel, providing 20% of energy requirements from the dairy factory and reducing the total carbon footprint emissions by 13%, according to Stanchev et al. (). Although several studies have highlighted advantages of using anaerobic digestion for the treatment of dairy effluents (Demirel et al. ; Karadag et al. ), the effective treatment of such effluents remains a challenge due to its high lipid content and the inhibitory effect that long-chain fatty acids may present in certain concentrations (Alves et al. ).
The anaerobic baffled reactor (ABR) is a simple and efficient configuration for many wastewater treatments. The main advantage is the natural capability of phase separation, allowing acidogenesis and methanogenesis optimization (Cohen et al. ; Weiland & Rossi ). Consequently, different microbial groups can develop under favorable conditions. This configuration also has longer biomass retention time as an advantage, in addition to the resistance to organic and hydraulic shocks (Barker & Stuckey ). According to the authors, hybrid reactors may provide the advantages of the UASB and anaerobic film reactors, reducing their limitations. According to Karadag et al. (), hybrid biofilm reactors have been widely applied for dairy wastewaters treatment. Gomes et al. () have observed organic matter removal efficiencies greater than 90% using an hybrid UASB reactor equipped with a superior polyurethane bed for dairy effluents treatment, even with high volumetric organic loads, such as 16 kg·m À3 ·d À1 . The concept of hybrid biofilms reactors can easily be applied to ABRs, due to its natural compartmentalization. In this way, Fujihira et al. () (Ayers & Westcot ) and is a vegetable known for its high vitamin A content. It adapts well to greenhouse cultivation and has a low cost of production with a short production cycle (Filgueira ). However, the needs for lettuce irrigation are high. These characteristics make this cultivar a candidate for irrigation with agro-industrial effluents.
Besides crop yields, another paramount aspect of agricultural reuse is the microbiological security of produced vegetables. The major concern is focused on the farmer health care, as well as agricultural products consumers, especially in the case of vegetables grown on the ground and consumed raw. Most studies that consider agro-industrial reuse have been carried out to evaluate the quality of effluents and post-harvested vegetables in relation to physic-chemical parameters, and further studies are required on their microbiological quality (WHO ).
Considering a circular economy scenario implementation, through water reuse in agriculture, this research has aimed to present data on a modified ABR, a hybrid anaerobic baffled biofilm reactor (HABBR), treating simu-

Reactor operation
A bench-scale HABBR was monitored in three operational conditions, namely OC1, OC2, and OC3, which were differentiated according to the applied HRT, which were 72, 24, and 12 h, respectively. The HABBR reactor ( Figure 1) was constructed using acrylic glass, with five chambers with a total volume of 25 L. The first and second chambers contained granular biomass, and cubic polyurethane foam matrices (1 cm side length) were used as a support for biomass immobilization in chambers 3 and 4. Chamber 5 was provided only with polyurethane matrices. The granular inoculum was obtained from an efficient UASB reactor treating effluent from starch production. The reactor was kept inside a temperature-controlled chamber (37 ± 2 C) to achieve temperature stability. During OC2, a hydraulic and organic load shock experiment was carried out to verify the possibility of decreasing the HRT from 24 to 12 h. The procedure was performed three times and consisted of reducing the HRT to 12 h for 36 h, subsequently subjecting the system to 24 h of HRT for 72 h. In all conditions, the HRT was maintained by a peristaltic pump (Gilson Mini plus) with four channels connected to ports distributed equidistantly across the reactor's width.

Substrate
Formulated dairy effluent with organic matter concentration expressed as chemical oxygen demand (COD) of 3 g·L À1 , oil and grease of 310 mg·L À1 , and a conductivity of 4.5 mS·cm À1 was used to feed the HABBR. The effluent was supplemented with 1 g.L À1 sodium bicarbonate (NaHCO 3 ), and its pH was corrected with a 10% hydrochloric acid (HCl) solution to a range within pH 7.0-7.5. The formulated wastewater composition was based on the characterization previously performed by Cichello et al. (), which is presented in Table 1. The composition of macronutrient and micronutrient solutions were prepared according to Zhender et al. (). The substrate reservoir was maintained at a temperature below 4 C to minimize biochemical reactions. Before entering the reactor, the substrate was heated to 37 ± 2 C.

Reactor monitoring
The reactor was monitored twice a week. Organic matter concentration expressed as COD, Kjeldahl nitrogen (total and   Table 2).
The characteristics presented in Table 2 were defined for lettuce crop fertilization, as suggested by Raij et al. ().
The pots were filled with 3 kg of soil, which were previously adjusted adding 2.00 t·ha À1 of limestone (100%), 100  The seedlings were harvested 41 days after transplanting. The analyzed parameters were the wet and dry weight of roots and leaves and the leaf area measured by LI-Cor model LI 3100. The data were subjected to a wide analysis range. For situations in which there were significant differences, the means were subjected to regression analysis.  Sample of leaves were washed with HCl solution (0.1%) and dried in a forced circulation oven (65 C) until reaching constant weight. Subsequently, they were processed in a mill and sent of nutritional diagnosis analyses for the macronutrients N, K, Ca, S, P, Mg, and Na in accordance with Malavolta et al. ().

Effluents
Samples of 20 mL of the treated effluent were collected aseptically in a sterile container and analyzed weekly for 4 weeks. Decimal dilutions in saline peptone water were pre-

RESULTS AND DISCUSSION
Reactor startup and monitoring The reactor showed stable operation throughout all conditions studied, which could be observed through consistent bicarbonate alkalinity, as well as rising methane content in the biogas. The reactor robustness can also be stated, taking into account the low variability in the values obtained for COD removal, as shown in Figure 2(b).

Start up and first operational condition
The reactor was operated over 149 days with a hydraulic detention time (TDH) of 72 h and an organic loading rate (OLR) of 1 g.L À1 ·d À1 , comprising the reactor startup and OC1. During the first 28 days, the organic matter removal efficiency went from 51% to 90%. The production of bicarbonate alkalinity was verified since the beginning of the operation; however, the startup period was considered con-

Second operational condition
The HABBR was monitored during 105 days with a TDH of 24 h and OLR of 2.96 g.L À1 ·d À1 during OC2. The average COD removal efficiency was 91 ± 1.8% for unfiltered and filtered samples of 91 ± 2.0%. Biogas presented a methane fraction of 53 ± 27%. The reactor was operated with stability, with an average alkalinity production of 380 ± 32 mg CaCO 3 ·L À1 . After 12 days at this operational condition, a biomass flotation in the first chamber due to an oil and grease load of 0.31 g COD·L À1 ·d À1 was observed.

Third operational condition
During the third experimental condition, the system was operated for approximately 71 days, with a hydraulic detention time of 12 h and an OLR of 5.92 g.L À1 ·d À1 . During this period, the average methane fraction in biogas was 62 ± 12%. The reactor showed consistent production of alkalinity, which was, on average, 355 ± 55 mg CaCO 3 ·L À1 , with IA/PA of 0.2.
Unfortunately, during this condition the reactor constantly presented problems, such as liquid leakage and a partial biogas outlet clogging due to the obstruction caused by the biomass. There was a biomass migration to the fourth compartment, and the treatment using minimum operational maintenance was not viable. For the HABBR operation, with part of its immobilized biomass, the system's floated sludge management must be constant.
Thus, an increase in the values of such biometric parameters was observed when T2 and T3 were applied. T2 showed a 37% and 6% increase for fresh and dry leaf mass, respectively, and a 37% increase in leaf areas com-

Microbial analyses
Microbial analyses were performed to verify the contamination potential of the reactors for food security of the produced vegetables, because the inoculum from the anaerobic reactors can often come from domestic wastewater treatment plants, which also present potential contamination by pathogenic microorganisms. Although the HABBR was fed with simulated dairy effluent, thus theoretically free of microorganisms, it may not be the case for industrial full-scale situations, where the level of asepsis in the dairy products can be very high and where such production units do not mix their domestic effluents with their industrial effluents.

Effluents
The results for counting heterotrophs and total coliforms in the effluent over a 4-week period are shown in Table 3. During the 4 weeks of lettuce cultivation, the effluent produced by the HABBR showed similar concentrations, without statistical differences, for total coliforms and for heterotrophic bacteria, as shown in Table 3. Heterotrophic bacteria, as well as total coliforms, are microorganisms naturally present in water samples and, by analogy, in effluents.
Their presence does not create immediate health risks; however, values above 6.0 log CFU·100 mL À1 (WHO ) may represent contamination risks to the culture that has been irrigated with such source. For the present analysis, the results of counting these microorganisms remained within acceptable limits.   the null hypothesis that the five treatments did not have significant difference in relation to the total counts of mesophilic, psychrotrophic, S. aureus aerobes, total coliforms, molds, and yeasts was accepted at a 5% level of significance, inferring that irrigating lettuce crops with 25%, 50%, 75%, or 100% effluent yields leads to a microbiological contamination statistically equal to irrigation with only water. It is important to note that, in addition, the current results remained within the accepted limit considered safe for human consumption in Brazil (BRASIL ).

Plants
Thus, it is possible to state that even irrigation with a 100% effluent did not represent significant increase in microbiological load compared to irrigation with drinking water alone. This is in accordance with the data obtained in the effluent analyzes previously presented (Table 3), demonstrating that it is safe to use the effluent from the dairy treated in ABBR.

CONCLUSIONS
The HABBR has been proven to be a reliable option for fatty-rich salty wastewater treatment, as demonstrated by the consistent alkalinity production, high organic matter removal (above 90%), and rich methane biogas production.
HRT of 24 h provided the optimal operational conditions among those tested, although hydraulic shocks using 12 h of HRT were well tolerated.
When carefully conducted, the agricultural reuse of the anaerobic-treated effluent could guarantee yields of the lettuce crops. The 50% dose of dairy anaerobically treated effluent resulted in the best conditions for residue utilization in agriculture, presenting an equal lettuce production without nutritional disorders and a reduction in 50% of necessary nitrogenous mineral fertilizer.
Throughout the irrigation time, the treated effluent did not show potential for microbiological contamination, proven by the results of the analysis carried out on the plants, where the count of ended mesophilic aerobes, psychrotrophic, S. aureus, molds and yeasts, and total coliforms were below levels allowed by legislation, thus guaranteeing food safety.