Chemical and biological combined treatment for sugarcane vinasse: selection of parameters and performance studies

Sugarcane vinasse has been reported as a high strength industrial wastewater that could cause severe environmental pollution due to its complex and bio-refractory compounds. Thus, the combined coagulation and sequencing batch biofilm reactor (SBBR) system was employed for the sugarcane vinasse treatment. This study aims to determine the recommended conditions of various parameters under coagulation and SBBR and investigate the effectiveness of combined processes. First, the approach of the coagulation process could achieve the maximum COD reduction and decolorization efficiencies of 79.0 ± 3.4% and 94.1 ± 1.9%, respectively, under the recommended conditions. Next, SBBR as an integrated biofilm reactor showed excellent synergistic biodegradability, removing 86.6 ± 4.3% COD concentration and 94.6 ± 3.8% color concentration at 3.0 g·COD/L of substrate loading concentration. The kinetic studies of SBBR revealed that the first-order kinetic model was the best fit for COD reduction efficiency. In contrast, the second-order kinetic model was the best fit for decolorization efficiency. The SBBR reaction was further investigated by ultraviolet–visible spectrophotometry (UV–Vis). In the combined processes, SBBR followed by the coagulation process (SBBR–CP) showed greater COD reduction and decolorization efficiencies (97.5 ± 0.3 and 99.4 ± 0.1%) when compared to the coagulation process followed by SBBR (CP–SBBR). This study demonstrated the removal performance and potential application of the combined sequential process to produce effluent that can be reused for bioethanol production and fertigation. This finding provides additional insight for developing effective vinasse treatment using combined chemical and biological processes.


Introduction
Sugarcane vinasse is one of the most frequently generated wastewaters in the bioethanol distillery, and 1 L of bioethanol production could generate around 9 to 14 L of sugarcane vinasse (España-Gamboa et al. 2011). Vinasse contains a lot of harmful substances, such as phenols, polyphenols, and heavy metals, as well as significant levels of organic content and dissolved solids. Its characteristics of being acidic (pH of 4.30-4.55), being dark brown, and having a high chemical oxygen demand (COD) concentration (28.5-85.0 g·COD/L) are necessary to be a concern since the phytotoxic and recalcitrant compounds in the sugarcane vinasse could negatively affect the organisms at disposal sites Kiani Deh Kiani et al. 2021;Takeda et al. 2022). Disposing of untreated/ partially treated vinasse on land or in groundwater could Responsible Editor: Ta Yeong Wu have a profound environmental impact. Thus, the bioethanol industry has adopted some technological applications such as fertigation (Carpanez et al. 2022), physico-chemical (Hoarau et al. 2018), and biological treatments (Silva et al. 2021). Fertigation using sugarcane vinasse is commonly applied due to increased crop productivity without complex technologies for its management; however, its uncontrolled practice can cause soil salinization, water sources contamination, and the release of bad odors (Fuess and Garcia 2014).
The coagulation/flocculation method is the most common chemical treatment technology, especially as a pre-treatment process for the vinasse. During the coagulation process, colloidal and finely divided suspended matters are facilitated in the aggregation to generate larger flocs that can be separated through sedimentation or filtration to clarify water from impurities . The coagulation-flocculation process using poly-γ-glutamic acid combined with sodium hypochlorite and sand filtration could reduce turbidity and COD concentration by 70 and 79.5%, respectively, in the tequila vinasse treatment (Carvajal-Zarrabal et al. 2012). Besides, FeCl 3 -involved coagulation post-treatment for biologically treated distillery wastewater could achieve high decolorization efficiency (Zhang et al. 2017). It was discovered that the ferric coagulant preferentially interacts with the aromatic compounds and melanoidins through surface complexation, charge neutralization, or both. Additionally, to reduce the coagulant dosage and increase the degradation potential, Moringa oleifera seed extract (MOSE) was used in the coagulation process with ferric sulfate and aluminum sulfate (alum) (David et al. 2016). Due to its low cost, availability, and simplicity of handling, alum has been widely used in water and wastewater treatment (Hussaini Jagaba 2018). This study selected alum, ferric sulfate, and copper sulfate for the coagulation process to determine the COD reduction and decolorization efficiencies of vinasse treatment.
Biological methods are applied to utilize pollutants for microorganisms' growth and convert the organic compounds into simpler substances through anaerobic and aerobic processes (Fito et al. 2019). Trickling filters, lagoons, and activated sludge are the most common practical conventional biological methods used in industrial wastewater treatment. A biofilm-based technique called sequencing batch biofilm reactor/submerged bed biofilm reactor (SBBR) could efficiently eliminate pollutants from wastewater (Ismail et al. 2018). A submerged fixed plastic carrier serves as the foundation of the SBBR system and supports associated growing aerobic bacteria (El-Shafai and Zahid 2013). The aerobic bacteria are active at eliminating organic carbon and nitrogen. SBBR is a basic system that quickly promotes the microorganism's growth. As a result, SBBRs could remove pollutants, enhance biomass content, and prevent sludge generation while taking up minimal area, being cost-effective, simple to operate and maintain, and decreasing smell and noise (Gómez-Villalba et al. 2006).
Sometimes, a single method is not sufficient for complete distillery wastewater treatment. When the distillery wastewater is partially treated due to the bio-recalcitrant compounds, the dark-brown effluent leaves a foul smell with a high COD concentration. Researchers are thus paying close attention to investigate the combined processes of physico-chemical and biological treatments (Oller et al. 2011). The combination of physico-chemical and biological methods included the combination of chemical coagulation, biodegradation, and photo-Fenton oxidation (Rodrigues et al. 2017), the combined biological-electrochemical oxidation treatment (Vilar et al. 2018), and the aerobic fungal growth followed by ozonation (Reis et al. 2019). The combined treatment showed their capability to simultaneously degrade/reduce the pollutants in COD and color concentrations. The management of distillery effluent using physico-chemical and biological methods together is sustainable and beneficial to the environment by adding value-added products (Ratna et al. 2021).
The focus of this study is to compare the efficiencies of three different treatment strategies: coagulation, SBBR, and a combined process of coagulation and SBBR to produce effluent that complies with discharge standards and allows for water recycling. First, the coagulation process was carried out to examine the effects of various coagulants, initial pH, coagulant dosages, and initial COD concentration in the treatment of vinasse. Next, the effect of the substrate loading concentration was evaluated using SBBR in COD reduction and decolorization efficiencies. The effect of the substrate loading concentration on the SBBR treatment performance was further analyzed using the kinetic models, ultraviolet-visible spectrophotometry (UV-Vis), and analysis of variance (ANOVA). Then, the effluents of the coagulation process and SBBR were subjected to combined sequential treatment process to determine the effectiveness of the combined technologies.

Sampling of wastewater
The wastewater sampling was carried out at the final pond of the sugarcane ethanol industry, Fermpro Sdn. Bhd., Perlis, Malaysia. The sample was kept in the fridge at 4 ± 1 °C to lower the biodegradation of the vinasse. The vinasse is alkaline (pH 8.6 ± 0.1), with a COD concentration and color concentration of 8280 ± 180 mg/L and 46,900 ± 600 Pt/C o , respectively.

Chemicals and materials
The coagulants used are from HmbG Chemicals, which are alum (Al 2 (SO 4 ) 3 ), ferric sulfate (Fe 2 (SO 4 ) 3 ), and copper sulfate (CuSO 4 ). The d-glucose anhydrous, with the molecular formula C 6 H 12 O 6 (Fisher Scientific), was added as the carbon source in the submerged bed biofilm reactor. For the pH adjustment of vinasse, sodium hydroxide (NaOH) and sulfuric acid (H 2 SO 4 ) from Merck and Fisher Scientific, respectively, were chosen in this study. All chemicals were employed without further purification.

Experimental procedures
The experiments were divided into three different sections. First, the COD reduction and decolorization efficiencies were used as indicators to investigate the effects of pH, catalyst dosage, and initial COD concentration in the coagulation process. After that, SBBR was used to study the influence of substrate loading concentration on COD reduction and decolorization efficiencies. The combined sequential treatment methods were then carried out in two steps: coagulation followed by SBBR (CP-SBBR) and SBBR followed by coagulation (SBBR-CP). Both combined processes were conducted under recommended conditions.

Chemical coagulation
As shown in Fig. 1(a), the coagulation operations were conducted at room temperature (25 ± 3 °C) in a Velp Scientifica Jar Test equipment (model JLT6). Each beaker was filled with 250 mL of vinasse, and the pH was adjusted to the desired pH (pH 3, 6, 9, 12) using a dilute H 2 SO 4 or NaOH solution with a concentration of 0.1-1.0 M. After adding the coagulant, the mixture was stirred rapidly (200 rpm) for 2 min and then stirred slowly (100 rpm) for 15 min, as illustrated in the literature (Lau et al. 2014). After the coagulation process, the effluent was settled for 1 h. The supernatant was collected and filtered through Whatman No. 4 filter papers (12.5 cm diameter; 20-25 μm pore size). The filtrates were collected to analyze COD (mg/L) and color concentration (PtC o ). The effects of various operational parameters such as types of coagulant, initial pH, catalyst dosage, and initial COD concentration on the COD reduction and decolorization efficiencies were determined in this study.

Sequencing batch biofilm reactor (SBBR)
The SBBR has a dimension of 30 × 20 × 20 cm 3 with 12 L of total capacity and 4 L of working volume, as shown in Fig. 1(b). The vinasse (influent) was diluted to a 3000 mg/L COD concentration before the feeding stage and added with a 0.5 g/L glucose supplement. The daily operation of SBBR includes fill + react (20 h), settle (3 h), decant (0.5 h), and idle (0.5 h), and one batch cycle is in 7 days. The inoculation of the bio ball, activated carbon, and bio ring was performed with activated sludge for 1 month before being transferred into the SBBR. The inoculation process was conducted under aerobic conditions. The reactor was then operated for 28 days to allow for microbial acclimatization and reactor stability. The diluted sugarcane vinasse was constantly supplied during the acclimatization period. Then, the SBBR was carried out for 35 operational days for five cycle batches. The supernatant was collected daily after the sedimentation phase and analyzed for COD and color concentrations. After each 7-day cycle, 50% of the effluent in the SBBR was evacuated and then fed with 2.0 L of diluted vinasse with a 0.5 g/L glucose supplement. Air was delivered during the fill + react phase at a rate of 200 mL/min using an Atman air pump (HP-4000, China) to maintain aerobic conditions in the SBBR system. The reactor was operated at room temperature (25 ± 2 °C), and the timers (Eurosafe ES-24HT) were used to control the system. The effect of substrate loading concentration on COD reduction and decolorization efficiencies was investigated in this study, with substrate loading concentrations ranging from 1.0 to 5.0 g·COD/L. The effect of the substrate loading concentration was further investigated through zero-order, firstorder, and second-order kinetic studies.

Combined process
The following combined coagulation and SBBR processes were designed and used in the experiments: (Approach 1) Coagulation process followed by SBBR (CP-SBBR) as a combined sequential treatment. First, the sugarcane vinasse was treated by coagulation under recommended conditions. Then, the wastewater that had been treated by coagulation settled for an hour, and the supernatant was filtered. Before going to SBBR, the pH of the supernatant was adjusted to 8.6, which was its original pH. The treatment processes are illustrated in Fig. 1(a) and (b). (Approach 2) SBBR followed by the coagulation process (SBBR-CP) as a combined sequential treatment. The sugarcane vinasse was subjected to SBBR at the recommended substrate loading concentration, and then, the supernatant was collected. The pH of the collected supernatant was adjusted before continuing with the coagulation process. The treatment process is depicted in Fig. 1(b), followed by Fig. 1(a).

Analytical methods
The vinasse treatment performances along the coagulation process, SBBR, and combined process were evaluated by collecting the influent and effluent samples. The samples collected were analyzed for COD and color concentration. Each sample was centrifuged at 4200 rpm for 10 min in a benchtop centrifuge (CENCE L500) before being subjected to color and COD analysis. Using a spectrophotometer (HACH, DR2800, USA), the COD (mg/L) was obtained using the Dichromate Reactor Digestion Method of Wastewater Analysis. A Hach DR2010 spectrophotometer was utilized to measure the color concentration (PtC o ) at 455 nm. UV-Vis Spectrophotometry (UV Professional, China) was used to examine the absorption spectra between 190 and 500 nm. A Methrom 826 portable pH meter was used to measure the pH and temperature during the experiments.

Statistical analysis
In the current study, all values were representative of triplicate experiments, and the data were reported as mean ± standard deviation (SD). Analysis of variance (ANOVA) was also applied to investigate the impact of substrate loading concentration on COD reduction and decolorization efficiencies in SBBR. The Tukey-Kramer post hoc test was employed to examine the significance of the specific substrate loading concentration on COD reduction and decolorization efficiencies. Statistics were considered significant when p-values less than 0.05.

Effect of pH on the coagulation process
In the coagulation-flocculation process, pH is undeniably one of the most important aspects since pH significantly impacts floc structure, size, and the liquid/solid separation effect. The COD reduction and decolorization efficiencies in the coagulation process using different pH values are demonstrated in Fig. 2. It was obvious that the highest COD reduction and decolorization efficiencies achieved by alum and Fe 2 (SO 4 ) 3 were at pH 10. On the other hand, the maximum COD reduction and decolorization efficiencies of the coagulation process by CuSO 4 were at pH 8. The coagulation process using alum achieved 24.5 ± 1.2% and 59.9 ± 3.0% COD reduction and decolorization efficiencies at pH 3, which reflected that the coagulation process using alum was not suitable to carry out in the acidic condition. The high concentration of H + existing under acidic conditions makes it more challenging to hydrolyze the carboxyl groups of organic molecules (Cao et al. 2010). The result was similar to the condition at pH 5 with alum. Low pH may cause the solubility depletion of organic matter and coagulation performance reduction if the primary process relies on charge neutralization (Dayarathne et al. 2021). Alum was more effective at reducing color and COD concentrations at pH 10 (68.1 ± 3.4% and 96.1 ± 1.5%) when compared to pH 5 (23.2 ± 1.4% and 75.5 ± 3.8%), but it became less effective after reaching pH 12 (7.1 ± 0.5% and 20.6 ± 1.2%).
The COD reduction and decolorization efficiencies of alum in the coagulation process were similar to those of Fe 2 (SO 4 ) 3 , which increased from pH 3 (45.6 ± 2.2% and 50.3 ± 2.5%) to pH 10 (73.7 ± 3.6% and 96.3 ± 1.3%). Metal ions tend to precipitate as amorphous hydroxides as pH increases. These colloidal hydroxides can adsorb other soluble species, leading to charge neutralization and destabilization in suspended colloidal systems (Dayarathne et al. 2021). Based on the results, it has been revealed that pH 10 is recommended for the alum and Fe 2 (SO 4 ) 3 in the coagulation process. The formation of the bonding flock particles using alum and Fe 2 (SO 4 ) 3 involves the presence of alkalinity in the water, which is aluminum hydroxide [Al(OH) 3 ] and ferric hydroxide [Fe(OH) 3 ], as shown in Eq. (1) and Eq. (2). Zayas et al. (2007) obtained the highest removal percentages of COD, color, and turbidity for anaerobically treated vinasse by FeCl 3 under slightly alkaline conditions. The COD reduction and decolorization efficiencies of the coagulation process by CuSO 4 increased from pH 3 (48.7 ± 2.4% and 53.5 ± 2.7%) to pH 8 (67.5 ± 2.9% and 95.7 ± 1.7%). The result was lower when the pH decreased to pH 10 and pH 12. Salts of divalent and trivalent metals can be very acidic due to the release of protons during the formation of hydroxy complexes, which lowers the pH (Matilainen et al. 2010). The minimum COD reduction and decolorization efficiencies were achieved for pH 12 by alum, Fe 2 (SO 4 ) 3 , and CuSO 4 . Under the highly alkaline condition, the surface electrical property turned negative, lowering the capability of charge neutralization (Cao et al. 2010). The coagulation process has been extensively studied in vinasse treatment using FeCl 3 , and this study provides an additional option for vinasse treatment in coagulation/flocculation processes.

Effect of coagulant dosage on the coagulation process
The effect of coagulant dosage was assessed for three types of coagulants (alum, Fe 2 (SO 4 ) 3 , and CuSO 4 ) at the recommended pH condition, where this parameter was varied in the range of 0.5-4.0 g/L. Figure 3 displays the sugarcane vinasse's COD reduction and decolorization efficiencies through the coagulation process at different coagulant dosages. The COD reduction and decolorization efficiencies at 0.5 g/L were the lowest compared to higher catalyst dosages for all three coagulants. The result indicated the coagulant is inadequate for complete reaction under sub-optimal doses, then attributed to low charge neutralization and bridging (Wei et al. 2018). With the increase in coagulant dosage to 1.0 g/L, the treatment efficiencies of the three types of coagulant improved. Alum, Fe 2 (SO 4 ) 3 , and CuSO 4 reached the highest COD reduction and decolorization efficiencies when the coagulant dosage was 1.0 g/L. Fe 2 (SO 4 ) 3 achieved the highest COD reduction and decolorization efficiencies among the three coagulants, which are 73.7 ± 3.6% and 96.3 ± 1.3%. The result could be explained by the flock particles (Fe(OH) 3 ) having a significantly higher density than the alum flocks, which could be removed easily through sedimentation. Fagier et al. (2016) reported that increasing the coagulant dosage from 5 to 15 g/L in treating high strength raw vinasse can increase total organic carbon (TOC) removal. The more coagulant is added, the better the adsorption effect is. However, excessive coagulant dosage can increase water turbidity and cause application problems. The statement reflected the lower COD reduction and decolorization efficiencies when catalyst dosage was increased to 2.0 g/L. When simple charge neutralization is the main mechanism, the removal efficiency increases first and then decreases with increasing dosage (Tang et al. 2022). Fig. 2 The study of coagulation process under effect of initial pH in terms of COD reduction and decolorization efficiencies using various types of coagulants (1.0 g/L coagulant dosage; 1.0 g/L initial COD concentration) The effect of coagulant dosage on the coagulation process depends on the destabilization mechanism. Although increasing coagulant dosage may favor the formation of metal hydroxide precipitates and enhance destabilization due to the colloid entrapment mechanism, it can also reverse the charge neutralization effect and promote the re-stabilization of the suspension (Bratby 2016). Underdosing promotes the formation of colloidal particles while overdosing pollutes wastewater by increasing turbidity, organic load, and slurry volume, expanding treatment costs . Sludge volume was also affected by coagulant dosage; the higher the amount of iron added, the greater the volume of sludge formed. However, when the coagulant dosage was increased to 4.0 g/L, the COD reduction and decolorization efficiencies decreased to 58.0 ± 2.0% and 70.8 ± 2.6%, respectively. The excessive coagulants can lead to the re-stabilization of the suspended particles, and thus, the treatment efficiencies were reduced . The increase in the coagulant dosage also resulted in a pH reduction after the coagulation process. Taking Fe 2 (SO 4 ) 3 as an example, the successive increase of iron in solution could contribute to the formation of H + (Eq. (3), Eq. (4), Eq. (5)) (Gao et al. 2020).

Effect of initial COD concentration on the coagulation process
Practical application with the coagulation process shows that the initial COD concentration severely influences the reduction's effectiveness. Therefore, determining the range for the initial concentration is crucial before starting the The increase in initial COD concentration is simultaneous with the rise in color concentration. Figure 4 shows the effect of the initial COD concentration using Fe 2 (SO 4 ) 3 since it achieved the highest COD reduction and decolorization efficiencies (73.7 ± 3.6% and 96.3 ± 1.3%) compared to alum and CuSO 4 . The COD reduction and decolorization efficiencies were 41.5 ± 2.2% and 81.5 ± 2.6% at the initial COD concentration of 500 mg/L, which is lower than the initial COD concentration of 1000 mg/L. The result indicated that the excessive quantity of coagulant causes the colloid to re-stabilize since the recommended dosage was determined according to the initial COD concentration of 1000 mg/L (Sun et al. 2019). Overdosing could lead to nutrient leaching in the coagulated wastewater (Okoro et al. 2021). Furthermore, the highest COD reduction and decolorization efficiencies were obtained at 1000 mg/L under the recommended conditions. However, the COD reduction and decolorization efficiencies decreased at 3000 mg/L (12.2 ± 0.7% and 31.6 ± 1.3%). This result could be related to insufficient doses that reduce the reactive surface of organic matter, thus leading to low adsorption by the coagulant Fig. 3 The study of coagulation process under effect of coagulant dosage in terms of COD reduction and decolorization efficiencies using various types of coagulant (optimum pH for each coagulant; 1.0 g/L initial COD concentration) Fig. 4 The study of coagulation process under effect of initial COD concentration in terms of COD reduction and decolorization efficiencies using Fe 2 (SO 4 ) 3 (initial pH 10; 1.0 g/L coagulant dosage) (Camacho et al. 2017). Increasing the initial COD concentration provides more available pollutant molecules, producing more collisions between the coagulant and pollutants (Wang and Chen 2020). Thus, adequate coagulant dosages are required at high initial COD concentration conditions to achieve outstanding treatment effects.
Meanwhile, the coagulation process was carried out based on a 1:1 ratio of coagulant dosage (g/L) and initial COD concentration (g/L). Figure 5 shows the COD reduction and decolorization efficiencies based on a 1:1 ratio of coagulant dosage and initial COD concentration using Fe 2 (SO 4 ) 3 . The highest COD reduction efficiency (82.8 ± 3.3%) was achieved at an influent COD concentration of 2000 mg/L, while the highest decolorization efficiency (96.3 ± 1.4%) was obtained at an influent COD concentration of 1000 mg/L. The results from influent COD concentrations of 1000 to 5000 mg/L were similar, and the average COD reduction and decolorization efficiencies were 79.0 ± 3.4% and 94.1 ± 1.9%, respectively. This result revealed that the coagulant dosage of Fe 2 (SO 4 ) 3 in the coagulation process was directly proportional to the initial COD concentration. According to Zhao et al. (2021), the dosages of coagulants will be added proportionally by increasing the initial concentration of the wastewater until complete charge neutralization has occurred at a given dose of coagulants. When charge neutralization is the primary mechanism, the efficiency decreases as the dosage increases. Optimizing dosing rates can save additional costs while reducing sludge volume and treatment costs. The decolorization efficiencies between the influent COD concentrations of 1000 and 2000 mg/L were close, but the COD reduction efficiency of the influent COD concentration of 2000 mg was 9.0% greater than that of the influent COD concentration of 1000 mg/L. Thus, the influent COD concentration of 2000 mg/L was selected as the recommended condition based on a 1:1 ratio of coagulant dosage (g/L) and initial COD concentration (g/L).

Sequencing batch biofilm reactor (SBBR)
The SBBR was monitored for 28 operational days for the acclimatization phase, followed by 35 operational days for five cycle batches. SBBR was used to determine how substrate loading concentration affected COD reduction and decolorization efficiencies. The COD reduction and decolorization efficiencies are demonstrated in Fig. 6(a) and (b). The COD reduction and decolorization efficiencies were achieved up to 80.4 ± 4.0% and 92.2 ± 3.7% after 7 days of reaction time under 1.0 g·COD/L of substrate loading concentration. The COD reduction and decolorization efficiencies increased to 84.0 ± 4.2% and 94.8 ± 3.8%, respectively, when the substrate loading concentration was changed to 2.0 g·COD/L. The highest treatment performances of SBBR were achieved at a substrate loading concentration of 3.0 g·COD/L. The SBBR could provide high COD reduction efficiency (86.6 ± 4.3%) and decolorize the wastewater up to 94.6 ± 3.8%. However, only 75.8 ± 3.8% and 84.0 ± 3.4% of the COD reduction and decolorization efficiencies could be obtained using 4.0 g·COD/L of substrate loading concentration. The results indicated the process instability of the reactor occurred when the substrate Fig. 5 The study of relationship between the effects of coagulant dosage and initial COD concentration in terms of COD reduction and decolorization efficiencies using Fe 2 (SO 4 ) 3 during coagulation process (initial pH 10) Fig. 6 Reduction efficiencies of sugarcane vinasse using SBBR under the effect of substrate loading concentration in terms of (a) COD and (b) color loading concentration was too high (Wang et al. 2019). The SBBR needed a longer duration to achieve complete substrate degradation when the substrate loading concentration of the reactor was beyond its limit (Yap et al. 2022). Furthermore, the COD reduction and decolorization efficiencies were 66.8 ± 3.3% and 74.2 ± 3.0% under 5.0 g·COD/L of substrate loading concentration, respectively. High substrate loading concentration could promote high treatment capacity; however, the overly high substrate loading concentration resulted in excess substrates and a saturated state, which affected the microbes' growth (Ni et al. 2020). Due to the over-high substrate concentration, microbes could not completely break down, and the COD reduction and decolorization were less effective. Juang et al. (2011) reported that when the organic loading rate (OLR) increased from 1.96 to 4.46 kg·COD/m 3 ·d, the microbes experienced the inhibitory effect, which caused a depletion in power density.
where S t is the COD concentration at time t (mg/L), S 0 is the initial COD concentration (mg/L), t is the operational day, and k 0 , k 1 , and k 2 are the zero (mg//L•day), first (day −1 ), and second kinetic rate constants (L/mg•day), respectively. Table 1 demonstrates regression coefficients (R 2 ) and reaction rate constants (k) for zero-order, first-order, and secondorder kinetic models in the COD reduction efficiency. The best fit was with the first-order kinetic model, which had the highest average R 2 value (0.91) compared to the zero-order (0.88) and second-order kinetic models (0.85). A similar result in the recirculated sequencing batch reactor showed that the pseudo-first-order kinetic model was the best fit for the COD reduction efficiency (Kee et al. 2022b). The highest COD reduction rate constant (k 1 COD ) was 0.1411 day −1 under a substrate loading concentration of 3.0 g·COD/L. The k 1 COD was 1.79 times higher than when it was 1.0 g·COD/L (0.0786 day −1 ) and 2.66 times higher than 5.0 g·COD/L (0.0531 day −1 ). The variation in k 1 COD associated with various substrate concentrations was attributed to the influence of microbial activity on the reactor's performance. Besides, Table 2 demonstrates R 2 and k for the decolorization efficiencies of zero-order, first-order, and second-order kinetic models. The second-order kinetic model is the best fit model in the decolorization efficiency and had the highest average R 2 value (0.98) compared to zero-order (0.86) and firstorder kinetic models (0.95). The decolorization rate constant (k 2 Color ) was achieved at its highest (4 × 10 −4 L/mg•day) under a substrate loading concentration of 1.0 g·COD/L and decreased to 9 × 10 −6 L/mg•day at the substrate loading concentration of 5.0 g·COD/L. The result revealed that the decolorization rate was high under the low substrate loading concentrations. However, the inhibition of decolorization occurred when the SBBR was added with a higher substrate concentration (Tsafrakidou et al. 2022).
ANOVA provided statistical evidence that demonstrated the effect of substrate loading concentration on the COD reduction and decolorization efficiencies. The ANOVA results showed a significant difference between the five stages of substrate loading concentration (p < 0.05). In contrast, the Tukey post hoc test revealed no statistically significant difference in COD reduction efficiency between the 1.0 and 2.0 g·COD/L, 1.0 and 3.0 g·COD/L, 1.0 and 4.0 g·COD/L, and 2.0 and 3.0 g·COD/L groups. The results indicated that the slight increase in substrate loading concentration had little influence on the COD reduction efficiency (p > 0.05). The Tukey post hoc test yielded a similar result in determining the significant difference in specific substrate loading concentration in decolorization efficiency. However, the 1.0 and 4.0 g·COD/L groups revealed a significant difference. The result could refer to a drastic decrease in decolorization efficiency from 1.0 to 4.0 g·COD/L of substrate loading concentration.  Figure 7 displays the impact of substrate loading concentration on UV-vis absorption spectra (190 to 500 nm) from day 1 to day 7. These results demonstrated that the absorbance bands had decreased after 7 days of reaction time. The results showed that the absorbance peak intensity at 270 nm decreased rapidly after 7 days of reaction time, representing the degradation of organic compounds (Arreola et al. 2020). The absorbance reduction reached 100% when considering 270 nm as the reference wavelength at the substrate loading concentration of 1.0 g·COD/L after 7 days of reaction time. However, the absorbance reduction decreased gradually to 78.2%, 62.7%, 48.5%, and 27.1% when increased the substrate loading concentration to 2.0 g·COD/L, 3.0 g·COD/L, 4.0 g·COD/L, and 5.0 g·COD/L of substrate loading concentration, respectively. Moreover, the absorbance peak intensity of 192-198 nm gradually decreased, especially in Fig. 7(a), (b), and (d). The peak (λ max ) observed at 203 nm might refer to the ethanol in the vinasse (Kee et al. 2022a). After 7 days of reaction time, the absorbance at 194 nm decreased from 1.451 to 0.626 (56.9%) when the substrate loading concentration was 1.0 g·COD/L. The absorbance at 194 nm decreased from 1.451 to 0.626 after 7 days of reaction time under 1.0 g·COD/L of substrate loading concentration, which achieved a 56.9% of absorbance reduction. Furthermore, the absorbance reduction was reduced to 40.6% and 25.3% when the substrate loading concentration was increased to 2.0 g·COD/L and 4.0 g·COD/L, respectively. Under substrate loading concentrations of 3.0 g·COD/L and 5.0 g·COD/L, the λ max was not observed in the UV-vis spectra of 192-198 nm, as shown in Fig. 7(c) and (e). It could be related to the maximum absorbance limit (≤ 3.0) and applying a high dilution factor.

Combined sequential process of coagulation and SBBR
The results of COD reduction and decolorization efficiencies, as presented in Table 3, were related to the treatment performances of the combined processes of coagulation and SBBR under recommended conditions. The SBBR-CP outperformed the CP-SBBR in terms of COD reduction and decolorization efficiencies. For the approach of SBBR-CP, the COD reduction (86.5 ± 1.4%) and decolorization (93.0 ± 0.4%) were achieved in the first approach using SBBR. After the coagulation process, the results improved to 97.5 ± 0.3% COD reduction and 99.4 ± 0.1% decolorization. The result showed that the SBBR could remove most organic compounds and decolorize the vinasse through biodegradation. The growth of microbial attachment in the biofilm can enhance the degradation of COD concentration through the aeration process (Guo et al. 2019). The coagulation process after the SBBR could secure the effluent to reach the discharge standard. For the approach of CP-SBBR, the coagulation process destabilizes the colloids of the sugarcane vinasse in the aggregation during the first approach and achieved a COD reduction efficiency of 81.2 ± 0.2% and decolorization efficiency of 91.1 ± 0.5%. The outstanding performance of the coagulation could be related to the rapid reaction of charge neutralization between the coagulants and vinasse (Crini and Lichtfouse 2018). It provides sufficient coagulant for the pollutant molecules under alkaline conditions and produces more collisions between the coagulant and pollutants. Further degradation by using the SBBR was achieved in the second approach of the CP-SBBR. The SBBR could produce qualified effluent as the COD reduction and decolorization efficiencies achieved 96.3 ± 0.2% and 99.3 ± 0.2%, respectively. Thus, it could be said that both combinations of the coagulation process and SBBR could maximize the reduction efficiencies, where the approach of SBBR-CP achieved a slightly better result when compared to CP-SBBR. Some previous studies focused on combining chemical treatment processes (Zayas et al. 2007;Guerreiro et al. 2016;Poblete et al. 2020). Meanwhile, similar studies have been reported with the combination of chemical and biological treatment (anaerobic and aerobic digestion) (Cabrera-Díaz et al. 2016;Vilar et al. 2018;Reis et al. 2019). The combined processes were recommended to be applied in the treatment of sugarcane vinasse because single technology has difficulty managing vinasse due to its high organic content and complex wastewater containing recalcitrant organics and persistent color (Gebreeyessus et al. 2019). The chemical treatment in the combination process could remove   melanoidin, a complex compound that contributes to excessive coloring in sugarcane vinasse. Melanoidin is difficult to separate and purify through biological treatment. Hence, the biological treatment could degrade the organic matter in the sugarcane vinasse. Thus, the combined chemical and biological treatment process could reduce the toxicity in sugarcane vinasse so the treated effluent could be reused in bioethanol production.

Comparison of the effectiveness of the combined processes with previous studies
The past and current studies on the combined process of vinasse treatment are summarized in Table 4. Most previous studies using combined methods demonstrated excellent treatment efficiencies, which could provide an overall COD treatment of 82.8 ± 14.4%. The integrated processes showed the potential application of processing the sugarcane vinasse into qualified effluent for discharge. 97.5% of the maximum COD reduction was reported in the current study, which was relatively high compared with other combined studies, as shown in Table 4. High COD reduction and decolorization were obtained through either coagulation or SBBR as the first approach in the treatment of sugarcane vinasse. Notably, the combination of chemical coagulation, biodegradation, and photo-Fenton oxidation by Rodrigues et al. (2017) showed high efficiency in reducing COD (91.0%). Three approaches were employed to treat sugarcane vinasse, individually or combined. The combination of chemical and biological treatment systems accomplished harmless effluent and led to similar high COD removal efficiencies compared to the current study. The treated wastewater could be reused in the anaerobic reactor to minimize water usage. Combining chemical coagulation and photo-Fenton oxidation to treat vinasses resulted in a 69.2% reduction in COD, as reported by Guerreiro et al. (2016). Compared to Fenton's oxidation, the operating cost of coagulation is much lower. Besides, the coagulation process could enhance the effluent's biodegradability and eliminate its toxicity to Vibrio fischeri. Thus, the selection of the coagulation process in this study seems to be a promising and economically attractive chemical treatment process compared to others. In addition, Zayas et al. (2007) obtained the highest COD reduction efficiency (99.5%) through coagulation/flocculation and electrochemical processes. This finding indicated the high applicability of coagulation/flocculation in combined treatment, which achieved the purification of biologically treated vinasse effluent in terms of COD, color, and turbidity. The combined process of ultrafiltration and nanofiltration with pre-coagulation was a promising technology for treating sugarcane vinasse, according to Silva et al (2020). Implementing the combined process could provide a 94.0% COD reduction, but the treatment cost was relatively high due to membrane fouling. Lebron et al. (2020) used coagulation, microfiltration, and nanofiltration processes to reduce COD concentration by 99.5%. It was reported that the possibility of membrane fouling could be minimized by increasing floc sizes with the coagulant addition and improving the backward transport velocity of particles. The microfiltration and nanofiltration permeate quality ensure its ability to produce satisfying effluent for water reuse. However, the costs related to membrane replacement and operational expenditures are required to be carefully considered.
The application of Pleurotus sajor-caju, followed by electrochemical oxidation for vinasse treatment, achieved a 71% COD reduction (Vilar et al. 2018). The biological treatment followed by electrochemical oxidation showed high applicability in sugarcane vinasse degradation, similar to the current study. Poblete et al. (2020) elucidated that combining ultrasound and heterogeneous photocatalysis achieved the best COD, color, and polyphenol removal ratios among five individual and combined treatment approaches. Thus, the advanced oxidation process (AOP) could be recommended as an integrated process for treating pisco vinasse with other treatment approaches because of its energy efficiency and relatively high pollutant-removal rates. Furthermore, Reis et al. (2019) reported that the sequence of anaerobic digestion, aerobic fungal growth, and ozone treatment could reach high COD reduction efficiency. The treated vinasse is safe for fertilization and irrigation since most COD, phenolic compounds, and Kjeldahl nitrogen have been removed after the combined processes. The result indicated the outstanding potential of biological treatment for sugarcane vinasse. Lastly, Cabrera-Díaz et al. (2016) reported that the upflow anaerobic filter reactor achieved high COD reduction with methane production, while the ozonation process played an essential role in the complete decolorization of anaerobically digested vinasse. The integrated biological and chemical treatment process was required for the sugarcane vinasse before disposal because of the complex compounds in the sugarcane vinasse. Based on other studies, most combined processes still focus on combining chemical and physical processes. The high removal efficiency and fast reaction in the physio-chemical techniques could explain these phenomena. Typically, biological treatment requires a more prolonged start-up phase due to the slow growth of bacteria and a long hydraulic retention time (HRT). The combined treatment systems are not functional in every case. Thus, this combination of chemical and biological methods provided additional insight for future treatment of sugarcane vinasse. As a result, the effluent quality was enhanced, allowing for subsequent reuse in bioethanol production or fertigation.

Conclusion
In this study, coagulation and SBBR showed their potential application in treating sugarcane vinasse, either in a single or combination process. First, the coagulation process obtained the best treatment performances using Fe 2 (SO 4 ) 3 at an initial pH of 10, resulting in COD reduction and decolorization efficiencies of 79.0 ± 3.4% and 94.1 ± 1.9%, respectively. The recommended proportion between the initial COD concentration (g/L) and coagulant dosage (g/L) was a 1:1 ratio. Then, the maximum COD reduction (86.6 ± 4.3%) and decolorization (94.6 ± 3.8%) were achieved using SBBR at 3.0 g·COD/L of substrate loading concentration. Furthermore, kinetic studies of SBBR were evaluated, and the firstorder kinetic model was best fitted among zero-, first-, and second-order kinetic models. From the UV-Vis analysis, the absorbance bands at 270 nm and 192-198 nm were diminished over time, which indicated the degradation of organic and alcohol compounds in SBBR. Next, two conditions of combined processes, CP-SBBR and SBBR-CP, were assessed. These two combined processes carried out at the recommended operating parameters showed 97.5 ± 0.3 and 96.3 ± 0.2% COD reduction efficiencies, with 99.4 ± 0.1 and 99.3 ± 0.2% decolorization efficiencies for SBBR-CP and CP-SBBR, respectively. Thus, combined sequential processes of coagulation and SBBR were recommended for implementation in sugarcane vinasse treatment, where the approach of SBBR-CP achieved a slightly better result when compared to CP-SBR. The combined chemical and biological process could reduce toxicity and provide value-added products to reuse in bioethanol production and fertigation.