Brazilian Journal of Chemical Engineering APPLICATION OF RESIDUAL YEAST AS A SOURCE OF REDOX MEDIATORS FOR THE ANAEROBIC DECOLORIZATION OF A MODEL AZO DYE

This work investigated the anaerobic degradation of the model azo dye Remazol Yellow Gold RNL in batch reactors using discharged residual yeast as the source of redox mediators (RM). Two yeast lysis methods (mechanical lysis and sonication) were tested and optimized to produce a riboflavin-rich yeast lysate. The reactors were operated at 25 C for 48 hours, evaluating the effect of external carbon source (glucose) and RM (from residual yeast lysate and commercial yeast extract) addition. The results showed that color removal efficiencies for the batch reactors fed with commercial yeast extract reached 90%, whereas those fed with discharged yeast lysate reached 80% (sonication) and 73% (mechanical lysis). These values were statistically higher when compared to reactors operating without RM (48 to 66%), demonstrating that yeast extract enhances azo dye degradation in anaerobic conditions and that the residual yeast is a cheap and alternative source of carbon and of the RM riboflavin.


INTRODUCTION
The textile industry is an important economic sector for the Brazilian economy.The textile sector represented in 2013 about US$58.2 billion, which is equivalent to 5.7% of the total production value of Brazilian manufacturing industry -excluding the activities of mining and construction (IEMI, 2014).Unfortunately, the textile production increase con-tributes to the escalation of severe pollution problems.Textile wastewater contains a variety of chemicals such as dyes and starch, which confer color and organic matter to the effluent.Removal of dyes is a major concern when treating textile wastewater due to carcinogenic and mutagenic properties of some dyes and their degradation byproducts (Baêta et al., 2012).
The main technologies used nowadays for the treatment of textile wastewater are physicochemical Brazilian Journal of Chemical Engineering and biological (mainly aerobic) processes.Although the physicochemical techniques are effective in removing particulate organic matter, they are inefficient in degrading azo dyes and dissolved organic compounds (Kunz et al., 2011).As a result, destructive techniques, such as biological treatment, are normally preferred.Although azo dyes are good electron-accepting compounds in reducing environment (anaerobic), in aerobic systems oxygen is the preferential electron acceptor when compared with azo dyes, due to the presence of electrophilic functional groups in their structure, making them more resilient to conventional biological aerobic treatment (Alvarez et al., 2010).
Anaerobic digestion has been considered as one of the best technologies for color removal from textile effluents (Georgiou et al., 2004).Removal efficiencies of an anaerobic reactor can reach up to 80% during the treatment of azo dye solutions (Méndez-Paz et al., 2005;Dos Santos et al., 2006).However, the electron transfer between different species of the anaerobic consortia and the final acceptor (azo dyes) can be limited and be a major drawback for the treatment of textile effluents in anaerobic reactors.
According to Dos Santos (2005a), anaerobic treatment efficiency can be enhanced by using redox mediators, which act by improving electron transfer between the donor (source of carbon) and acceptor (azo dye) (Dos Santos, 2005b), thereby increasing the decolorization rates.Several studies (Rau et al., 2002;Cervantes et al., 2001;Brady, 2003) have shown the ability of vitamins such as riboflavin and other purified substances such as quinones (e.g., sulfonated antraquinone -AQS) to act as redox mediators.Field et al. (2002) has shown that reduced flavins, such as riboflavin, were responsible for the direct chemical reduction of azo dyes (non-enzymatic), instead of acting as a vitamin.
Saccharomyces cerevisiae and Ashbya gossypii are normally employed for industrial riboflavin production, since riboflavin extraction is more costeffective than its chemical synthesis (Santos et al., 1995).Côrrea et al. (2009) evaluated the use of yeast extract as a source of riboflavin to improve the color removal in anaerobic digestion, and found that the efficiencies increased by 30%.Baêta et al. (2012) also performed studies that showed increased color removal efficiencies in the presence of commercial yeast extract as a source of riboflavin, but in a largescale treatment plant, its use would raise the costs of treatment.
Residual yeast, regarded a solid waste from the fermentation process in brewery and other fermenta-tion industries, could be a viable source of redox mediator if cheaper lysing methods are employed for extract production.Therefore, the main objective of this paper is to present results on the application of a fermentation industry residue as the source of redox mediators for the anaerobic decolorization of a model azo dye (Yellow Gold Remazol) commonly used in the textile industry.Two yeast lysis methods (mechanical lysis and sonication) were tested and optimized to produce a riboflavin-rich yeast extract.

Pretreatment of the Yeast Biomass
The yeast biomass was collected from a brewery fermentation vat where the production process was already over.The first step to produce yeast lysate from the residual yeast biomass was clarification, consisting of a sequence of centrifugations and ethanol solution (10% M/M) addition.The final solution of this process, hereby called whole cells clarified solution (WCCS), was further submitted to distinct cell lysis processes aiming at the release of riboflavin.

Experimental Design
A screening procedure was initially performed to determine which variables and their levels resulted in the best experimental condition for residual yeast lysis.For this, two factorial designs at two levels (2 3 and 2 2 ), both with triplicates at the central point, for sonication and blender, respectively, were carried out to optimize the best experimental conditions for riboflavin release in the screening step.The effects of variables were calculated at the level of significance of 0.05 by using the software PASW Statistics 18®, and a response surface methodology (MATLAB R2011a®) was used to explore the relationship between the variables.

Cell Lysis Methods
Sonicator: The WCCS, as described above, was prepared according to each experimental condition described by the factorial experimental design, and the variables studied during sonic disruption were the cell density in g L -1 (x 1 ), duty cycle in % (x 2 ) and contact time in minutes (x 3 ).A Branson 250 sonicator was used with a titanium¾" probe; the solutions were prepared at 4 ºC and maintained in ice bath to prevent over heating.
Brazilian Journal of Chemical Engineering Vol. 33, No. 04, pp. 705 -711, October -December, 2016 Mechanical Lysis: The mechanical lysis was performed using the blender method (rotating blades).A bench scale industrial blender (448 W) was employed to grind and disperse the WCCS.The solutions were prepared according to each experimental condition described by the factorial experimental design, and the variables studied during the mechanical disruption were the cell density in g L -1 (x 1 ) and the contact time in minutes (x 2 ).

Batch Experiments
The batch experiments were carried out in triplicate with solutions of the model azo dye Remazol Yellow RNL Gold (50 mg.L -1 ) (C 16 H 18 N 4 O 10 S 3 ; CAS 12226-47-0; molecular weight of 522,54 g mol -1 ) using amber glass flasks (20 mL) duly sealed with rubber stoppers and aluminum caps.This work evaluated the azo dye without hydrolysis, as normally happens for this type of dye in the industry surveyed, since the dye is readily soluble in water.Anaerobic conditions were ensured by purging nitrogen gas into each flask for about 2 min.The flasks were then incubated in a shaker at 25 °C for 48 hours using anaerobic sludge (~1,000 mg.L -1 of volatile suspended solids -VSS) as inoculum.The sludge was collected from a demo scale UASB reactor installed at the Centre for Research and Training in Sanitation (CePTS) UFMG/COPASA, located at the Arrudas WWTP, in Belo Horizonte -Brazil.The incubation conditions are detailed in Table 1.

Analytical Procedures
Riboflavin Quantification: Riboflavin quantification analyses were carried out in a high performance liquid chromatography (HPLC) system (Shimadzu -LC-20A) equipped with a fluorescence detector (SPD -20A) setting the wavelengths at λ ex = 450 nm and λ em = 525 nm, with an analysis time of 20 min per sample, according to the validated method of Bueno Solano et al. (2010).The chromatographic separation was performed on a Phenomenex Luna 5mM C18 (150 mm × 4.6 mm and 5 mM) column at 25 ºC, using 0.005 M ammonium acetate and methanol (72:28 v / v) as mobile phase under isocratic flow (1.0 mL min -1 ) with an injection volume of 30 µl.A calibration curve was generated in the range of 0.005 to 0.2 mg.L -1 and resulted in very good linearity (r 2 = 0.9996).
Color Analyses: For color analyses an HP 8453 UV-Visible (California, U.S.A) spectrophotometer was used previously set at the wavelength where the azo dye exhibited the maximum absorption (λ max = 410 nm).All samples were centrifuged at 2,500 rpm for 20 minutes before the analyses, which were carried out with samples collected at 0h, 12h and 48h.A calibration curve was generated in the range of 1 to 100 mg.L -1 (r ² = 0.9977) to allow the calculation of azo dye removal efficiency.
Fig tw lys

Table 1 : Operational conditions for the batch experiments that evaluated the anaerobic decolorization of azo dye solutions.
**** Azo dye provided by a local textile industry and used without purification or pre-treatment, as per its industrial use.