Removal of water-based printing inks in a biofilm sequential batch reactor: start-up and kinetic study

doi:10.21203/rs.3.rs-2993098/v1

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Removal of water-based printing inks in a biofilm sequential batch reactor: start-up and kinetic study

https://doi.org/10.21203/rs.3.rs-2993098/v1

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Water-based inks face new challenges owing to the generation of polluting effluents throughout their manufacturing process as well as during the cleaning of equipment and printing areas. Appropriate treatments are essential to avoid associated environmental problems resulting from toxicity, impeding the passage of light and diffusion of oxygen in water bodies. This study aims to evaluate the ability of the microbial consortium MJ1, isolated from Mexican soil, to remove four commercial water-based inks, namely magenta (MI), yellow (YI), black (BI), and cyan (CI), in batches and in a 6 L sequential batch reactor. FIA-ESI-FTICR-MS analysis identified pigments Violet 19 and Red 122 in the MI ink, Pigment Yellow 74 in the YI ink, and Phthalocyanine and Pigment Blue 15 in the CI ink. The discoloration of the inks depended on the nutrient concentration in the medium. The performance and kinetic parameters of a sequential batch reactor packed with sterilized volcanic rocks, inoculated with the MJ1 consortium, and supplemented separately with MI, YI, BI, or CI inks at concentrations of 3000, 3000, 3000, and 1500 mg L^-1, respectively, were evaluated. The hydraulic retention time showed that most discoloration occurred in the first 3.5 d of treatment. Furthermore, elimination rates of 796.8, 791.1, 601.7, and 223.8 mg L^-1d^-1 were determined for the MI, YI, BI, and CI inks, respectively. A preliminary structure of the generated metabolites was proposed by comparing the results of biodegradation pathway prediction by the EAWAG-BBD database for Violet 19, Red 122, Yellow 74, Phthalocyanine, and Blue 15 pigments to the FIA-ESI-FTICR-MS analysis from biodegradation experiments. In conclusion, the use of a packed sequencing batch reactor has been shown to be an effective method for the discoloration of various commercial water-based print inks. These findings provide important insights into the bioremediation of inks and the associated environmental problems, highlighting the potential for further research and development in this area.

Printing inks

Red 122

Violet 19

Yellow 74

Blue 15

discoloration

sequential batch reactor

wastewater treatment

Printing inks are essential raw materials used in the graphics industry. The composition of printing inks is complex and variable depending on the intended use and patented formulation of the manufacturer. Typically, printing inks comprise a matrix of colorants (pigments and dyes), additives (defoamers, driers, waxes, and dispersing aids), auxiliary solvents (glycols, alcohols, and glycol ethers), and supports or vehicles (emulsions and alkali-soluble or colloidal dispersion chemistries) (Noonpui et al. 2010; Pérez-Ramos et al. 2019).

The socio-economic impact of the graphics industry has stimulated research on improving the performance of printing inks, such as the development of brighter colors, better fixing capacity, and greater durability of the printing medium. One of the notable advances has been the formulation of biodegradable inks, such as water-based inks. These inks have gained popularity in the flexographic sector because of their environmental friendliness compared with inks based on organic solvents (Balea et al. 2017). In 2000, the consumption of water-based inks used in flexography in the United States, Europe, Latin America, and Japan was approximately 133,333, 72,000, 11,333, and 9000 t, respectively (Ma and Xia 2009).

However, the use of water-based inks presents new challenges due to the generation of polluted effluents during the manufacturing and cleaning of equipment and printing areas (Balea et al. 2017; Papadopoulos et al. 2019). Printing ink effluents are characterized by high pigment and dye contents, non-biodegradable compounds, and traces of toxic metals such as cadmium and mercury (Zampeta et al. 2022). Thus, it is crucial to apply appropriate treatments to mitigate environmental problems. Recalcitrant compounds in untreated effluents disrupt the ecological balance of water bodies by hindering sunlight penetration and oxygen diffusion (Benkhaya et al. 2020). Furthermore, the intrinsic toxicity of polluting compounds poses a risk to living organisms in contaminated soils and bodies of water (Kishor et al. 2021).

Because of the complex composition of printing ink effluents, an effective treatment is required to remove the most recalcitrant contaminants. Commonly applied technologies include chemical coagulation, microfiltration (Wang et al. 2015), adsorption (Maknakorn et al. 2019), enzymatic degradation (Indumathi et al. 2022), and electrocoagulation (Papadopoulos et al. 2019). Although biological processes have not been adequately evaluated, some microorganisms can develop at contaminated sites and use contaminants as growth substrates, making them useful in bioremediation processes (Viesser et al. 2020). However, the biodegradation of printing ink wastewater is challenging, and studies on the biodegradation of commercial inks are scarce. For instance, Zhang et al. (2003) demonstrated that N and P sources influenced the treatment of printing ink wastewater using Bacillus sp. immobilized onto a honeycomb. Montañez-Barragán et al. (2020) showed that color removal for azo dyes depends on the nutrient and dye concentrations and hydraulic retention time using Halomonas sp.

To evaluate the efficiency of the biotechnological solution, consortium MJ1 was isolated from soil and tested for its ability to decolorize synthetic water-based inks. Color removal was optimized by varying the nutrient and ink concentrations, and the decolorization process was carried out in a packed sequential batch reactor under optimal nutrient conditions. Kinetic studies were performed, and preliminary identification of metabolites was proposed.

2.1 Water-based inks characterization and metabolite analysis

Commercial liquid water-based inks comprising magenta (MI), yellow (YI), black (BI), and cyan (CI) colors were procured from a local market. The densities and UV-vis spectra of the inks were determined using a HACH DR 5000 spectrometer. Calibration curves were constructed using appropriate water dilutions at λ_max for each ink at pH values of 4, 5, 6, 7, and 8.

To identify the compounds, present in the MI, YI, BI, and CI inks, 10 µL aliquots of each sample were individually injected into a Solarix 7T Bruker ultra-high-resolution cyclotron resonance mass spectrometer (FIA-ESI-FTICR-MS). The treated samples were centrifuged at 10,000 rpm for 15 min, and 10 µL of the supernatant was injected into the FIA-ESI-FTICR-MS system for metabolite elucidation and profile comparison before and after treatment.

An Agilent 6000 series HPLC system was employed, equipped with a Zorbax Eclipse Plus C18 column (4.6 mm x 150 mm, 5 µm particle size) operating at a flow rate of 1 mL min^− 1, and the DAD detector was set at 260 and 530 nm. Mobile phase A comprised a 2.5% acetic acid solution, and mobile phase B contained acetonitrile. The elution program consisted of an increase to 24% B in 15 min, followed by a 12-min hold, an increase to 100% B in 10 min, a 4-min hold, and finally return to 100% phase A.

Mass spectra were analyzed using the data analysis software for Solarix 7T Bruker. The results obtained from the FIA-ESI-FTICR-MS analysis of the biodegradation experiments were compared with the metabolites proposed by biodegradation pathway prediction using the EAWAG-BBD database (http://eawag-bbd.ethz.ch/) for Violet 19, Red 122, Yellow 74, Phthalocyanine, and Blue 15 pigments.

2. 2 Microorganisms

The consortium was isolated from polluted soil in Mexico State, Mexico, by serial dilution in basal medium (BM). BM was composed of the following (g L^-1): MgSO₄·7H₂O, 10; KCl, 10; KH₂PO₄, 0.028; K₂HPO₄, 0.028; meat extract, 0.11; and glucose, 0.5, as described by Montañez-Barragán et al. (2020). Individual experiments were performed by incorporating each ink into the medium.

2.3 Inoculum preparation

A 2 mL aliquot of biomass suspension, with Abs_600nm adjusted to 1.0, was supplemented with 20 mL of nutritive medium (NM) composed of casein peptone (5 g L^− 1) and meat extract (3 g L^− 1). The samples were then incubated at 35°C and 120 rpm for 48 h. After incubation, the culture broth was centrifuged, and the biomass pellet was washed with distilled water to eliminate residual nutrients. The washed pellet was then re-suspended in BM and used for microcosms experiments.

2.4 Evaluation of water-based inks discoloration in batch experiments

3 mL of inoculum was added to 50 mL vials containing 30 mL of BM supplemented with each printing ink separately. The vials were incubated for 24 h at 35°C and 120 rpm, followed by another 24-h incubation at 35°C under static conditions (Montañez-Barragán et al. 2020). Subsequently, a 2 mL aliquot was withdrawn and centrifuged at 10,000 rpm for 10 min. The absorbance of the supernatant was measured at the appropriate λ_max and pH. The cells were then re-suspended in distilled water, and the absorbance at 600 nm was measured. Color ink removal (CIR) was calculated using Eq. 1.

$$\%CIR=\frac{Ci-Cf}{Ci}x100$$

where %CIR is the color ink removal percentage and C_i and C_f represent the initial and final ink concentrations (mg L^− 1), respectively. All experiments were carried out in duplicate, and results were reported as mean ± standard deviation.

2.5 Experimental design to evaluate the effect of water-based inks and nutrient concentrations on the discoloration process

As in previous studies (Montañez-Barragán, 2020), it was observed that the efficiency of color ink removal depended on the concentrations of ink and nutrients. To evaluate the individual and interactive effects of these variables on the percentage of CIR for MI, YI, BI, and CI inks, we performed a Central Composite Design (CCD). For the CCD, the minimum and maximum ink concentrations (mg L^− 1) were set as follows: 800 (-1) and 4000 (+ 1) for MI and YI, 600 (-1) and 3000 (-1) for BI, and 500 (-1) and 2500 (+ 1) for CI. The nutrient concentration levels were varied by mixing BM with 15% (-1) to 85% (+ 1) of NM. The CCD model was designed using the statistical software Design Expert V 6.0®. The %CIR was measured in nine experiments (in duplicate), and response surfaces were generated for each ink. Furthermore, the equations of the models that describe the discoloration and conditions for the optimal discoloration of the inks were determined.

2.6 Biofilm sequential batch reactor: start-up and kinetic study

A 6 L Plexiglas sequential batch reactor (SBR) was filled with sterilized volcanic rocks (diameter of 1 cm and porosity of 0.47) supported by a perforated resting plate perched 5 cm above the bottom of the reactor with the purpose of discoloring printing inks. The SBR was then filled with reactor medium (RM) composed of the following (g L^-1): MgSO₄.7H₂O, 8.3; KCl, 8.3; KH₂PO₄, 0.023; K₂HPO₄, 0.023; meat extract, 0.591; casein peptone 0.833; and glucose, 0.41. The pre-grown inoculum was added and allowed to grow and adhere to the packing material for 5 d. This process was repeated thrice by emptying the SBR and introducing fresh RM and biomass.

The SBR was then filled with sterile RM supplemented with separate MI, YI, BI, or CI inks (3000, 3000, 3000, and 1500 mg L^-1, respectively) and operated without aeration in the typical sequence of the fill, react, and draw phases. The filling and drawing phases occurred almost instantaneously. Discoloration was performed for each printing ink, and the reaction time for each ink was measured when its concentration reached a constant value. The samples (25 mL) were withdrawn from the bottom of the reactor every 12 h. A 2 mL aliquot was centrifuged for 10 min at 10,000 rpm, and the ink concentration was measured in the supernatant. The kinetic parameters obtained by fitting a suitable kinetic equation to the variation in substrate concentration with respect to time are important indicators of the efficacy of the consortium and the reacting system. Equations 2 and 3 were used to determine the rate constant and kinetic model of ink decolorization.

First-order kinetics:

$$ln\frac{Ct}{Ci}= -{k}_{1} t+b$$

Second-order kinetics:

$$\frac{1}{Ct}={k}_{2}t+b$$

where C_t is the ink concentration at each time of the experiment (mg L^-1), t is the reaction time (day), k is the first-order rate constant (day^-1), and k₂ is the second-order rate constant (L mg^-1 day^-1).

2.7 Analytical methods

The total organic carbon (TOC) and inorganic carbon (IC) were measured using a Sievers Innovox instrument. 40 mL of the treated ink sample, medium without ink, and water containing 3000 mg L^− 1 of MI, YI, and BI inks and 1500 mg L^− 1 of CI ink were used for analysis. The Persulfate Thermic Method was used for ink dissolution. The pH was measured using a UB-10 pH/mV Meter UltraBasic potentiometer.

3.1 Water-based inks characterization

One of the greatest challenges in the treatment of contaminated effluents is the significant complexity and diversity of the chemical compounds present. The performance of biological treatments is affected by fluctuations in the composition of the contaminants, including their physical, chemical, and physicochemical characteristics. Consequently, there is a need to develop more robust bioremediation methods to treat a wide spectrum of contaminants. In this study, we investigate the biological treatment of synthetic effluents contaminated with water-based printing inks. However, it is necessary to conduct a general analysis of water-based inks to identify the pigments used in commercial formulations. Although the biodegradation mechanisms of azo dyes have been widely studied and documented, information on other pigment classes is limited.

The densities for MI, YI, BI, and CI in g mL^-1 were 1.0155, 0.9488, 0.662, and 0.997, respectively. The maximum wavelengths for MI, YI, BI, and CI were 544, 423, 551, and 626 nm, respectively. The absorption coefficients of MI (0.012 L mg^-1 cm^-1), YI (0.014 L mg^-1 cm^-1), and BI (0.015 L mg^-1 cm^-1) did not change in the pH intervals analyzed. The absorptivity of CI changed with pH; it decreased at pH 4 (0.014 L mg^-1 cm^-1) but remained constant from pH 5 to 8 (0.027 L mg^-1 cm^-1).

Two main pigments contributing to the coloration were identified in the MI ink. One of these compounds was Pigment Violet 19 (C₂₀H₁₂N₂O₂, Fig. 1a). The other was Pigment Red 122 (C₂₂H₁₆N₂O₂, Fig. 1b). Both structures are derivatives of quinacridone and β-quinacridone. These pigments are in high demand for the production of water-based printing inks and solvent-based paints owing to their high resistance and thermal stability (Lomax, 2005). These dyes possess a heterocyclic structure that is believed to contribute to their high chemical stability and recalcitrance.

Pigment Yellow 74 (C₁₈H₁₈N₄O₆, Fig. 1c) is a monoazo dye found in YI ink formulations. It is highly soluble in water (UL Prospector 2022) and used in the production of water-based inks, plastics, paints, and other products.

Phthalocyanine (C₃₂H₁₈N₈, Fig. 1d) was detected in the CI ink. This bluish-green compound has a central structure that can coordinate with a metal atom; if copper is the metal atom, it is Pigment Blue 15 (C₃₂H₁₆CuN₈, Fig. 1e), which is a blue color. In addition, it can also coordinate with non-metallic elements, such as chlorine and sulfonic groups, which can produce various shades of green and blue (Garza and Kharisov 2008).

Figure 1

3.3 Nutrient’s effect on the discoloration of water-based inks

Bioremediation is a promising biotechnological strategy for addressing the severe water crisis currently afflicting the world, which is largely caused by the presence of recalcitrant contaminants, such as synthetic dyes, from residual discharges in industries such as textiles and graphics (Ihsanullah et al. 2020). Microbial cells, like any cell, require adequate amounts of carbon, nitrogen, phosphorus, and sulfur to perform their vital functions. During bioremediation, the proper supply of nutrients and other chemical components, as well as the favorable manipulation of environmental conditions that directly affect microbial growth, are essential for promoting the metabolic degradation of pollutants (Ihsanullah et al. 2020; Mehrotra et al. 2021).

This study demonstrates that the extent of discoloration of water-based printing inks depends on the type and concentration of the printing inks and the nutrient concentration (Table 1). A second-order polynomial equation was obtained through the CCD design, which described the discoloration process for all the inks. The quadratic model equations for the discoloration of each ink, expressed as significant (p < 0.05) coded factors, are listed in Table 1. In all cases, a significant positive coefficient was obtained for factor A (%NM), indicating that degradation was favored with a greater amount of nutrients. The interaction between the nutrient and ink concentrations is illustrated on the response surfaces of the CI in Fig. 2. Similar patterns were observed for all the inks.

Table 1

Coded polynomial equations for each response in the CCD experimental design.
Type and concentration of ink [mg L^− 1]	Equation for color ink removal	R²
MI [800–4000]	%CIR = + 93.92 + 12.57A-8.16B-11.11A²-6.64B² + 31.06AB	0.78	2.9–97.6
YI [800–4000]	%CIR = + 0.002 + 14.66A-6.90B + 28.43A² + 35.14B² + 25.87AB	0.93	27.9–88.6
BI [600–3000]	%CIR = + 98.47 + 31.06A + 1.45B-20.98A²-6.67B²-6.78 AB	0.86	17.4–99.8
CI [500–2500]	%CIR = + 50.98 + 20.71A-7.5B-13.32A²-2.97B² + 2.82 AB	0.62	0.0-67.4
Notes. %CIR: Color ink removal (%); A: Nutritive medium concentration (%); B: ink concentration (mg L^− 1). Bold letters indicate significant terms (p < 0.05).

Table 1

Figure 2

The synthetic dye concentration in effluents significantly affects their biodegradation processes (Ikram et al. 2022). The response surface shown in Fig. 2 indicates that increasing the concentration of NM (i.e., high nutrient concentration) increased ink discoloration in all cases. Regarding BI and CI inks (Table 1), it can be noted that their discoloration process was more dependent on the concentration of NM (higher coefficient value of A).

High removal percentages were achieved for MI, YI, and BI, indicating a wide tolerance range of the consortium to high concentrations of the synthetic dyes. However, the low degradation of the CI ink may be attributed to the presence of Cu, which is a part of Pigment Blue 15 and may be toxic to microbial cells, thus affecting their degradation capacity.

In bioremediation processes, added nutrients generally serve as electron sources. External organic nitrogen sources, such as the casein peptone and meat extract found in NM, are particularly important (Seyedi et al. 2020). Shah and Bera (2021) reported discolorations of Reactive Red 3 under microaerophilic conditions of up to 96% using peptone as a nitrogen source, whereas discoloration with meat extract was only 56%. In another study, it was observed that a decrease in nitrogen sources (peptone/meat extract, P/E) did not substantially affect the capacity of Halomonas to decolorize Reactive Black 5, going from P/E:5–1.5 to P/E:E:1.66–0.5, but a decrease to P/E:0‒0.05 resulted in a drop in discoloration ability to 46% (Montañez-Barragán et al. 2020). This is due to the participation of exogenous sources of organic nitrogen in the regeneration of NADH, which acts as an electron donor (Garg et al. 2020). It has been shown that the decolorization performance of azo dyes is lower than that of reactors free of nitrogen sources, owing to the availability of O₂ from inorganic nitrogen sources such as NO₃ or NO₂, which are preferred as final electron acceptors (Tizazu et al. 2022). This reveals that treatments aimed at decolorizing dyes present in inks require organic nitrogen sources, making it essential to continue further studies to evaluate economical sources of nitrogen that can be utilized on an industrial scale.

3.4 Degradation of water-based inks in SBR-packed bioreactor

3.4.1 Kinetics of discoloration of water-based inks

The correlation of the experimental data showed first-order kinetics to explain the behavior of the discoloration of MI, YI, and BI inks during treatment. Conversely, the discoloration of CI ink was better fitted to a second-order model (Table 2).

Table 2

Mathematical representation of the discoloration kinetics of water-based inks
Colour ink	Kinetic model	Model equations	R²
Magenta	First order (day^− 1)	S=-0.0585x + 0.0464	0.9908
Yellow	First order (day^− 1)	S=-0.0335x + 0.0591	0.9641
Black	First order (day^− 1)	S=-0.0178x + 0.21	0.9661
Cyan	Second order (L mg^− 1 day^− 1)	S = 1x10^− 5x + 0.0004	0.8745

Chen et al. (2011) investigated the discoloration of Reactive Black 5 (1000 mg L^-1) by Enterobacter sp. GY-1 and reported that a first-order kinetic model was appropriate for reduction kinetics using biological elements. The dependence of this model on the dye concentration suggests that an external carbon source is required to provide an excess of reducing equivalents, as observed in the treatment of MI, YI, and BI inks. However, it is important to consider the potentially toxic effects of inks and their degradation metabolites on microbial growth and discoloration.

Table 2

3.4.2 Performance of the SBR bioreactor on the discoloration of water-based inks

One of the most significant advantages of packed-bed bioreactors is their ability to immobilize microbial cells on the support surface, which prevents biomass elimination during treatment and protects the cells from mechanical damage (Reddy and Osborne 2020). In addition, some studies have reported the formation of microniches within the pores of packing materials, resulting in varying concentrations of oxygen, which may lead to simultaneous oxidative and reducing conditions (Barragán-Huerta et al. 2007; Montañez-Barragán et al. 2020).

In this study, the discoloration efficiencies of water-based printing inks MI, YI, BI, and CI were evaluated using an MJ1 consortium in a sequential batch reactor (SBR) with a packed bed. Table 3 presents the performance parameters, including conductivity (µS cm^-1), total organic carbon (TOC, mg L^-1), inorganic carbon (IC, mg L^-1), pH, hydraulic retention time (HRT, d), and color elimination rates (mg L^-1d^-1).

Table 3

Performance parameters of the SBR bioreactor during discoloration treatments of water-based printing inks
Water-based ink color	Conducivity (𝜇S cm⁻¹)		pH		Biomass		TOC (mg L^− 1)		IC (mg L^− 1)		*CIR (%)	HRT (d)	*CER (mg L^− 1d^− 1)
	Initial	Final	Initial	Final	Initial	Final	Initial	Final	Initial	Final	Final	Final	Final
MI	15.85	18.76	6	7	0.103	0.375 ± 0.10	513.6 ± 12.6	630.0 ± 68.0	0.08 ± 0.1	67.2 ± 8.1	92.9 ± 3.3	3.5	796.8 ± 28.4
YI	16.19	18.91	5.81	7.34	0.147	0.342 ± 0.04	638 ± 17.6	620.0 ± 11.5	0	56 ± 6.6	92.3 ± 1.3	3.5	791.1 ± 10.9
BI	16.02	18.6	5.39	7.31	0.121	0.337 ± 0.03	501 ± 7.2	665.3 ± 8.6	0	65.9 ± 4.4	70.2 ± 0.1	3.5	601.7 ± 1.12
CI	16.13	16.02	5.42	7.57	0.102	0.328 ± 0.07	637 ± 46.4	549.7 ± 4.7	0	91.8 ± 4.6	52.2 ± 0.8	3.5	223.8 ± 6.4
Notes: * Initial concentration = 3000 mg L^− 1 for MI, YI, and BI, 1500 mg L^− 1 for CI; Color Ink Removal, %CIR; Color Elimination Rate, CER.

Conductivity is an indirect measure of the salinity of a medium and indicates the amount of total dissolved solids (Corwin and Yemoto 2020). Although there is no established range for conductivity readings in wastewater, it can be observed from the results presented in Table 3 that the conductivity increased during the discoloration treatments, possibly due to the ions released during the biodegradation process. The final electrical conductivity values for the discoloration treatments ranged from 16.02 to 18.91 𝜇S cm^-1. Electrical conductivity is a parameter that indirectly estimates the level of contamination in an effluent. However, high values may be associated with the presence of ions with high conductivity potential, such as chloride ions (de Sousa et al. 2014).

The SBR bioreactor utilized in this study demonstrated the ability to decolorize up to 92.9% of MI ink and 92.3% of YI ink, as shown in Table 3. However, the CI ink exhibited the lowest %CIR at 52.2%, indicating its high recalcitrance. Notably, the initial ink loads were high at 3000 mg L^-1 for MI, YI, and BI and 1500 mg L^-1 for CI. In comparison to other studies, Kumar et al. (2021) achieved similar results in an SBR bioreactor with Acid Red 3BN dye but with a workload ten times less (339 mg L^-1) than that evaluated in this study. Franca et al. (2020) also implemented an SBR system and achieved the total discoloration of effluents contaminated with Acid Red 14 dye but with initial loads of up to 100 mg L^-1. Both studies attributed the decrease in the discoloration capacity to the toxicity of the dye and/or its metabolites.

The phthalocyanines found in CI inks have been poorly evaluated in terms of their biodegradation. Fu et al. (2002) evaluated the discoloration of an initial load of 20 mg L^-1 of Reactive Blue 21, a copper phthalocyanine, using sludge acclimatized under aerobic conditions. The authors determined a dye removal of 29.5% in 12 h, which later reached approximately 80% after the tenth day, which they attributed mainly to the structural complexity of the pigment that prevented its rapid metabolism by microorganisms.

Table 3 lists the TOC values obtained at the end of the discoloration treatments for the water-based inks. TOC is a water quality parameter that estimates the efficiency of a biological process in removing contaminants from effluents (de Sousa et al. 2014). Although some decolorization was observed, the TOC values showed only slight changes with the treatment, suggesting that the biodegradation process was incomplete. Increased TOC values from the discoloration treatments suggest an increase in the metabolic rate of bacteria that involves the generation of biomass and metabolites related to microbial growth and development, which exceed the degradation of the organic matter of the dyes present in the printing inks (Ajaz et al. 2020).

Water-based printing inks are composed of a complex variety of additives such as emulsifiers, defoamers, waxes, and alcohols (Hou et al. 2019), which can interfere with the mechanisms involved in the total degradation of organic matter. Therefore, once decolorization is carried out in the SBR bioreactor under microaerophilic conditions, additional treatment with aeration is required to increase the TOC removal percentage. A higher value of IC is observed for the CI ink treatment (91.8 ± 4.6 mg L^-1) than for MI, YI, and BI, even though the %CIR is lower for the CI ink (52.2%), which could be due to differences in the CI ink composition.

One of the main challenges in the bioremediation of industrial effluents contaminated with synthetic dyes is the HRT. As shown in Table 3, the HRT values of 3.5 d were determined for the discoloration of water-based inks. These results highlight the efficiency of the process carried out in the SBR-packed bed bioreactor, especially considering the high loads of the treated water-based ink. Moreover, the porosity characteristics of bioreactor packaging could enhance the HRT by promoting better adherence of biomass and contributing to its discoloration capacity. Porous materials allow for the appropriate fixation, growth, and development of the metabolic activity of microbial cells (Montañez-Barragán et al. 2020).

The HRT values determined in this research are similar to those reported by Sathian et al. (2014), who achieved a maximum discoloration of 71.3% in textile wastewater with an HRT of 3 to 5 d. However, the authors complemented this treatment with subsequent aeration. Ma et al. (2011) achieved a 93% removal of Methylene Blue using an SBR bioreactor with an HRT of 173 d, starting from an initial concentration of 66.67 mg L^-1 of dye. Another study (Qu et al. 2012) showed a 95% removal of Acid Red B dye (100 mg L^-1) at an HRT of 12 h.

Table 3

3.4.3. Metabolite’s analysis of ink biodegradation in an SBR bioreactor

The only azo dye identified in the water-based inks was the pigment Yellow 74, which was also present in the YI ink, as determined by spectrometric analysis. The mechanism of azo dye biodegradation has been the subject of numerous investigations but has not yet been fully elucidated. However, under reducing conditions, it occurs via a co-metabolic and non-specific mechanism that involves the transfer of four electrons to the azo dye group, resulting in the formation of colorless aromatic amines (Franca et al. 2020). In this study, the SBR was operated under aerobic conditions; nevertheless, the porous packing material used as the bed allowed for the establishment of microniches. In these areas, the dissolved oxygen tension decreases until it reaches a microaerophilic environment that favors the prevalence of reducing conditions necessary for azo bond cleavage (Montañez-Barragán et al. 2020).

To the best of our knowledge, no studies have been conducted on the biodegradation of quinacridone pigments such as Red 122 and Violet 19, and studies on phthalocyanine-derivative pigments are limited. Analysis of the molecular mass abundances of biodegradation intermediates using principal component analysis (PCA) demonstrated that biological ink treatment with the MJ1 consortium produced mixtures with molecular masses different from those of the parent compounds (see Fig. 3).

Figure 3

Comparison of the molecular weights and structures of the biodegradation intermediates predicted for Red 122, Violet 19, Yellow 74, Blue 15, and Phthalocyanine pigments using the EAWAG database with those obtained by FIA-ESI-FTICR-MS analysis showed the presence of several predicted intermediates. Information regarding the preliminary identification of metabolites is provided in the Supplementary Material. These results support the IC, CIR, and TOC results, indicating that pigment transformation occurs, but longer or combined treatments are necessary for complete mineralization.

The EAWAG-Pathway Prediction system has been previously used to study the biodegradation of xenobiotics (Sivakumar et al. 2017; Tam et al. 2021; Arora et al. 2022). This tool is useful for predicting the degradation of xenobiotics, which has not yet been reported. In this study, the EAWAG-Pathway Prediction system was used to facilitate the identification of the metabolite in complex samples, such as printing inks. Table 4 shows the metabolites identified in treated samples.

Table 4

Identified metabolites in the biodegradation process of water-based inks
Pigment	Metabolites	Molecular weight (g mol^-1)
Red 122	a) 2-(hydroxymethyl)-9-methyl-5,7,12,14-tetrahydro-5,12-diazapentacene-7,14-dione	355.108
	b) 9-methyl-7,14-dioxo-5,7,12,14-tetrahydro-5,12-diazapentacene-2-carbaldehyde	353.092
	c) 2-amino-3-(2,3-dihydroxy-5-methylbenzoyl)-7-(hydroxymethyl)-9,10-dihydroacridin-9-one, and three isomers	391.129
	d) 2-(2-amino-7-methyl-9-oxo-9,10-dihydroacridin-3-yl)-2-oxoacetate	296.079
Violet 19	a) 2-amino-3-(2,3-dihydroxybenzoyl)-9,10-dihydroacridin-9-one, and one isomer	347.103
	b) 3-(2-amino-9-oxo-9,10-dihydroacridine-3-carbonyl)-2-oxopent-4-enoate	350.09
	c) 2-oxopent-4-enoate	114.031
	d) 2-(2-amino-9-oxo-9,10-dihydroacridin-3-yl)-2-oxoacetate	282.064
	e) 2-[2-(2-aminobenzoyl)-2-carboxylatoeth-1-en-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylate	377.077
	f) 2-amino-9-oxo-9,10-dihydroacridine-3-carboxylate	254.069
	g) 3-[6-amino-4-(2-aminobenzoyl)-2,3-dihydroxybenzoyl]-2- oxopent-4-enoate, and one isomer	384.095
	h) 2-[2-(2-aminobenzoyl)-2-carboxylato-1-hydroxyethyl]-4-oxo-1,4-dihydroquinoline-3-carboxylate	395.087
Yellow 74	a) N-(2-hydroxyphenyl)-2-[(1Z)-2-(3-methoxy-4-nitrophenyl) diazen-1-yl]-3-oxobutanamide and one isomer b) 2-[(1Z)-2-(3-methoxy-4-nitrophenyl) diazen-1-yl]-3-oxobutanoic acid c) 2-methoxyaniline. d) 2-[(1Z)-2-[4-(hydroxyamino)-3-methoxyphenyl]diazen-1-yl]-N-(2-methoxyphenyl)-3-oxobutanamide	373.114 281.640 124.076 373.151
Phthalocyanine	a) (20Z,26Z)-6H,13H,30H,32H-phthalocyanine-22,23-diol, and various oxidized intermediates with molecular weights ranging from 577.137 to 609.127	547.163
Note: For more information on the FIA-ESI-FTICR-MS analysis, please refer to the "Supplementary Material."

Table 4

Thus, one of the most important contributions of this work is that the isolated microorganisms are not only capable of degrading azo dyes, such as pigment Yellow 74 identified in YI ink, but also synthetic dyes derived from quinacridone, Red 122, and Violet 19 present in MI ink, which are synthetic dyes, and their applications are growing because they are stable and attractive compounds for the industry. However, there are no studies on their treatment of remediation, particularly biodegradation, as well as the pigments derived from phthalocyanine present in CI ink, which are characterized by the presence of copper, a heavy metal that can significantly affect the efficiency of conventional biodegradation processes. With this information, the present investigation establishes the basis for further explorations aimed at elucidating the biodegradation mechanisms of these chemical classes of dyes, which will improve the operating conditions of the implemented bioreactor and subsequently scale bioremediation processes.

In this study, a biological treatment was implemented for the bleaching of four water-based inks (Magenta, Yellow, Black, and Cyan) using a sequential batch reactor (SBR) with a packed bed. The decolorization capability of the MJ1 consortium was strongly influenced by the presence of meat extract as an external source of organic nitrogen; however, minimal amounts were required. Similarly, the initial concentration and type of contaminant significantly affected the degree of discoloration. It was discovered that it was more difficult to remove inks which contained copper, which could be toxic to microorganisms. This study pioneered the biological treatment of water-based printing inks. This demonstrates the high potential and broad spectrum of the microbial consortium for the synthetic dyes’ biodegradation of three different chemical classes: azo, quinacridone, and phthalocyanine derivatives. The improvement in the performance parameters of the sequential batch reactor, degradation mechanisms of the other classes of dyes identified, and toxicity of the effluents obtained should be studied in greater detail in future research.

Statements & Declarations

Ethical approval

Not applicable.

Consent to participate

Not applicable.

Consent for publication

Not applicable.

Author contributions

All authors contributed to the conception of this study. This study was performed based on the planning of Barragán-Huerta and done under her guidance. Material preparation and data collection were performed by Maryjose Gallegos-Araujo and Yair Cruz-Narvaez. Metabolite analysis was performed using the Yair-Cruz-Narvaez method. All authors analyzed the results and wrote the first draft of the manuscript. All authors commented on the previous versions of the manuscript. All the authors have read and approved the final manuscript.

Funding

This research was performed with funding granted by the Secretaría de Investigación y Posgrado del Instituto Politécnico Nacional (IPN) through the project SIP 20230639 and Project CONACYT: 302670 in the 2019 call for support for the acquisition and maintenance of infrastructure in specialized research institutions and laboratories, CF-2019/6669 in the frontier science call; through projects SIP 20220296 and 1533-2021, and through the innovation and development project of the call for technological development or innovation projects in the IPN 2022.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

Availability of data and materials

Data supporting the findings of this study are available in the article and its supplementary material. Raw data is available from the corresponding author, (BEBH), upon reasonable request.

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GallegosAraujoSupplementaryMaterialSENT.docx

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Removal of water-based printing inks in a biofilm sequential batch reactor: start-up and kinetic study

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Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Water-based inks characterization and metabolite analysis

2. 2 Microorganisms

2.3 Inoculum preparation

2.4 Evaluation of water-based inks discoloration in batch experiments

2.6 Biofilm sequential batch reactor: start-up and kinetic study

2.7 Analytical methods

3. Results and discussion

3.1 Water-based inks characterization

3.3 Nutrient’s effect on the discoloration of water-based inks

3.4 Degradation of water-based inks in SBR-packed bioreactor

3.4.1 Kinetics of discoloration of water-based inks

3.4.2 Performance of the SBR bioreactor on the discoloration of water-based inks

3.4.3. Metabolite’s analysis of ink biodegradation in an SBR bioreactor

4. Conclusion

Declarations

References

Supplementary Files

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