A novel textile wastewater treatment using ligninolytic co-culture and photocatalysis with TiO2

Textile industries produce effluent waste water that, if discharged, exerts a negative impact on the environment. Thus, it is necessary to design and implement novel waste water treatment solutions. A sequential treatment consisting of ligninolytic co-culture with the fungi Pleurotus ostreatus and Phanerochaete crhysosporium (secondary treatment) coupled to TiO2/UV photocatalysis (tertiary treatment) was evaluated in the laboratory in order to discolor, detoxify, and reuse textile effluent waste water in subsequent textile dyeing cycles. After 48 h of secondary treatment, upto 80 % of the color in the waste water was removed and its chemical and biochemical oxygen demands (COD, and BOD5) were abated in 92 % and 76 %, respectively. Laccase and MnP activities were central to color removal and COD and BOD5 abatement, exhibiting activity values of 410 U.L-1 and 1 428 U.L-1, respectively. Subjecting waste water samples to 12h of tertiary treatment led to an 86 % color removal and 73 % and 86 % COD and BOD5 abatement, respectively. The application of  a sequential treatment for 18 h improved the effectiveness of the waste water treatment, resultingin 89 % of color removal, along with 81 % and 89 % COD and BOD5 abatement, respectively. With this sequential treatment a bacterial inactivation of 55 % was observed. TiO2 films were reused continuously during two consecutive treatment cycles without thermic reactivation. Removal percentages greater than 50 % were attained. Acute toxicity tests performed with untreated waste water led to a lethality level of 100 % at 50 % in Hydra attenuata and to a growth inhibition of 54 % at 50 % in Lactuca sativa. Whereas sequentially treated waste water excreted a 13 % lethality at 6.25 % and aninhibition of 12 % at 75 % for H. attenuata and L. sativa, respectively. Finally, sequentially treated waste water was reused on dyeing experiments in which 0.86 mg.g-1 adsorbed dye per g of fabric, that is equivalent to 80 % of dye adsorption.


Introduction
Textile industries release considerable volumes of water-soluble waste substances because of fabric dyeing processes.This waste consists of dyes (azoic, disperse, anthraquinone, etc.) and dye-related compounds (surfactants, A novel textile wastewater treatment using ligninolytic co-culture and photocatalysis with TiO 2 salts, polymers and solubilizing agents).Usually, these chemicals are discarded in the sewage, ending up in water bodies where they become a serious threat to the environment due to their chemical complexity and low biodegradability indexes (BOD 5 /COD < 0.5).This BOD 5 /COD is the ratio between the biochemical oxygen demand (BOD 5 ) and the chemical oxygen demand (COD) of a given waste product.Biodegradability indexes, such as BOD 5 /COD, depend on dye type, concentration and its mixture with other compounds.
Azoic dyes comprise an important type of industrial dyes extensively used for dyeing cotton-made fabrics.Structurally, azoic dyes consist of three main components, (i) a chromophore formed by three aromatic rings linked together by an azo bond, (ii) an auxochrome which modifies the chromophore's light absorption, and (iii) a solubilizer.Azoic dyes bind covalently to cellulosic fibers and can thus withstand temperature, friction, and pH changes (Kongliang et al., 2014;Satapanajaru et al., 2010).During fabric dyeing processes, a fraction (20 % -30 %) of the dye is not attached to the fiber and it is therefore released, producing color effluents with high COD and BOD 5 , entailing considerable amounts of solids, and having alkaline pH values and high conductivities (Qingxiang et al., 2009;Waghmode et al., 2011).In complying with environmental acts and regulations, textile industries have adopted different strategies to minimize the impact of dye residues.These strategies consist of implementing physical, chemical, and biological wastewater treatment protocols, based on sedimentation, coagulation, activated sludge, anaerobic reactors, and bio-filtration.However, these protocols have proved to be effective only to certain extent (Waghmode et al., 2011), and they are not sufficient to reduce color and toxicity, deriving in documented adverse effects on aquatic ecosystems (Jadhav et al., 2010;Punzi et al., 2015).
Complementary or sequential technologies can be used to improve conventional textile effluent wastewater treatments.Ligninolytic fungi and photocatalysis with TiO 2 make suitable secondary and tertiary treatment options, respectively (Deveci et al., 2016).Ligninolytic fungi are more efficient than aerobic and anaerobic bacteria at removing and bio-transforming dye waste due to their oxidative enzymes with low substrate specificity (i.e.peroxidases and polyphenol-oxidases) (Morales et al., 2016;Morales et al., 2017;Rivera-Hoyos et al., 2013).This group of fungi is also capable of removing heavy metals and reducing COD and BOD 5 , thus increasing waste biodegradability ratios to values higher than 0.5 (Maalej et al., 2009;Puentes et al., 2012a;Sathian et al., 2014).However, they have certain limiting factors such as long processing periods, secondary sludge (biomass) production, and progressive fungal biomass inactivation as the system becomes saturated (Castillo et al., 2012).
Heterogeneous photocatalysis with TiO 2 represents an attractive tertiary treatment application that can enhance the effectivity of ligninolytic-based protocols (Deveci et al., 2016).This technology uses natural or artificial light to produce two main photo-chemical reactions that take place at the TiO 2 surface trough UV-light irradiation: (i) photo-induced redox reactions of adsorbed compounds and (ii) hydrophilic photo-induced conversion of TiO 2 .Complete or incomplete dye mineralization via electromagnetic radiation (≥ 3.2 eV) is based on electron hole pair production followed by recombination due to a charge transference (oxidation or reduction) process (Claudinei et al., 2015;Fernandez et al., 2015;Fernandez et al., 2016).Thus, a desirable sequential textile effluent wastewater treatment would use ligninolytic fungi to reduce high organic material and color, suspended solid concentrations, and facilitate photocatalyst action, coupled to a photocatalysis course that abates colored byproducts and decreases wastewater toxicity (Nogueira et al., 2015;Punzi et al., 2015).
The present study aimed at assessing a non-conventional biologic secondary textile wastewater treatment with an active ligninolytic fungal biomass coupled to a TiO 2 physical-chemical tertiary treatment.To this end, ad hoc prepared TiO 2 films were reused trough four consecutive cycles to study progressive loss of photocatalytic activity.Additionally, acute toxicity tests were performed before and after sequential treatment using the fresh-water polyp, Hydra attenuata, and Lactuca sativa, seeds as toxicity indicators.Finally, sequentially treated wastewater was utilized to carry out dyeing cotton fabric batch experiments in the laboratory.

Characterization of wastewater
A set of physical, chemical and microbiological textile effluent wastewater parameters were evaluated before and after treatments (Table 1).All fungal biomass was removed from wastewater samples using a Thermo Scientific R Sorvall RC6 Plus centrifuge (10 800 g, 10 min, 4 • C), and the following parameters were measured in the supernatant: discoloration percentage, calculated on color units according to Pallerla & Chambers (1997); COD, calculated with the 5222D method (APHA, 2012); and BOD 5 , calculated with the 523D method (APHA, 2012).Further, conductivity and pH were determined using a digital sensor, and plate count agars were used to culture heterotrophic bacteria (CFU mL −1 ) (Fernandez et al., 2015).(2012a).TiO 2 films were prepared placing each Soda-lime glass substrate on a 9-cm diameter petri dish.A total of 20 mL of a TiO 2 /TIP (TiO 2 USP/ titanium (IV) tetraisopropoxide solution was poured over each soda-lime glass substrate and allowed to dry during 5 h at 50 • C to form the film.Later, TiO 2 films w ere t hermally t reated a t 4 50 • C f or 1 h .T iO 2 fi lm surface characteristics were observed using Scanning Electron Microscopy (SEM) (JEOL JSM 6490-LV electron microscope).Crystallographic characteristics of TiO 2 films (Anatase and Rutile phases) were determined by X-ray diffraction as in Fernández et al. (2016).

Parameter
Two cubic reactors of 20 x 40 x 5 cm (width, length, and depth), were utilized.Each one was divided into two equally-sized sections (20 cm wide, 20 cm long, and 5 cm deep) to perform 2 experiments simultaneously under the same conditions (photoreactor 1 and 2).Air glass diffusers of 19 cm 2 , with 2-mm pores, were set inside each section.Then, UV radiation was administered using two mercury UV lamps (Philips TUV 18, 15 W, wavelength 254 nm) located parallel to each reactor, 10 cm away from the TiO 2 films.Both reactors were placed inside a box made of galvanized metal sheets (60 x 50 x 30 cm) with an internal division to separate each reactor (Suppl.1).Each section contained 8 TiO 2 films (1 000 mg TiO 2 ) placed in contact with 400 mL of wastewater treated for 6 h with VB LCC of P. ostreaus and P. chrysosporium.
UV lamps were turned on following adsorption/desorption time under dark conditions during 20 min.A preliminary photocatalysis test was carried on samples that were previously subjected to VB LCC secondary treatment for 0 h, 6 h, 24 h, and 48 h.These experiments were performed in triplicate with 4 h UV irradiation.Downstream color removal percentage and COD abatement levels served to establish level analysis results on samples from this photocatalysis test, the conditions for the subsequent photocatalytic experiments.
Photocatalysis (TiO 2 /UV), Photolysis (UV) and adsorption removal (TiO 2 without UV light) experiments were performed in triplicate for 12 h.Color removal, COD and BOD 5 abatement, and microbial inactivation assays were performed on samples taken every hour along the process.
TiO 2 film recycling The number of operational cycles of each TiO 2 film w as empirically determined.To this aim, a batch of eight TiO 2 (1 000 mg TiO 2 ) thin films was prepared per section, as previously described, and used in the treatment of wastewater previously subjected to secondary treatment for 6 hours (1 700 ± 34 CU and 2 345 ± 123 mg L −1 COD).The same TiO 2 thin films were then reused for up to four tertiary treatment cycles.At the end of each cycle, wastewater was replaced.Wastewater from each tertiary treatment cycle was tested for discoloration and COD abatement.In addition, SEM was used to document differences between films at the beginning and end of four-cycle runs.Particular attention was payed to looking for microorganisms attached to the film's surface.

Toxicity tests
Acute toxicity tests with (L.sativa) seeds, variety Batavia Gran Lago, were carried out contrasting the toxicity of untreated wastewater, wastewater subjected to secondary treatment with ligninolytic co-culture, and wastewater subjected to secondary treatment and tertiary photocatalysis for 18 h (VB LCC /TiO 2 UV Sequential treatment).Additionally, TiO 2 toxicity was also evaluated.Static acute toxicity tests, exposing L. sativa seeds for 120 h to the three types of wastewater were performed according to the methodology described by McInnis (1989).Toxic effects were recorded as percentage of germination inhibition.Furthermore, an acute toxicity assay was also performed with fresh-water polyps H. attenuata for 96 h following the methodology described by Trottier et al. (1997).Wastewater toxicity in H. attenuata was expressed as polyp mortality percentage at the highest wastewater dilution capable of driving the expected effect.C and 700 mg of sodium chloride (additive) was added.Dye and additive were mixed during 10 min at 100 rpm, and then a piece (0.7 g) of fabric was immersed in the solution.Each cotton fabric piece (7.0 x 7.0 cm) was degreased and subjected to detergent and impurity elimination prior to immersion in the dyeing solution.Each tested dyeing solution, with one immersed cotton fabric, was then heated up to 90 • C for 45 min and regularly stirred (each 15 min) with a 30-cm glass rod to guarantee uniform staining.Subsequently, all pieces of fabric were cooled down for 1 h at room temperature.The dye concentration (mg L −1 ) in the remaining solution was used to calculate the q of adsorbed dye by cotton fabric in mg g −1 , see Eq. 1.
In Eq. 1, V is the volume of the dyeing solution, C is dye concentration (mg L −1 ), C o is the initial dye concentration (mg L −1 ), and x is the cotton piece weight in g.The reported q is the average of the five replicates per treatment.

Statistical Analysis
Normality and variance homogeneity of untreated and treated (secondary, tertiary, and sequentially treated) data were determined with Shapiro-Wilk and Levene tests.Kruskal-Wallis nonparametric tests were performed to establish whether there were differences between treatments.Statistical significance was determined at the 95 % (α = 0.05) level.All analyses were conducted with the Statistical Analysis System (SAS 9.0).Data from each independent experiment were presented as the means ± standard deviation of the mean.

Characterization of textile wastewater
All variables analyzed in textile effluent wastewater before and after treatments are shown in Table 1.Remarkably, untreated textile effluent wastewater variables such as color units, COD, BOD 5 , and pH showed values above levels permitted by local environmental authorities (i.e.Resolución 3957 of Secretaría Distrital de Ambiente, Bogotá -Colombia).Moreover, the effluents had heterotrophic bacteria with morphology associated with Gram-negative and Gram-positive bacilli.Thus, tested effluents were not considered apt for discharging.
Secondary treatment with ligninolytic co-culture Ligninolytic co-culture experiments were undertaken to assess how effectively VB LCC , NVB LCC and WLCC bacteria inoculates in textile effluent wastewater reduced its color and its COD and BOD 5 .Experiments were carried without pH control under non-sterile conditions.In terms of discoloration efficiency at 48 h, treatments VB LCC , NVB LCC and WLCC bacteria resulted in color removal percentages of 80 %, 35 %, and 11 %, respectively (Fig. 1A).Significant abatement of COD, by 92 %, and BOD 5 , by 76 %, were observed in the treatment with VB LCC at 48 h, whereas percentages of COD and BOD 5 abatement obtained after treatment with NVB LCC at 48 h were moderate, 41 % and 25 % respectively.The treatment with WLCC bacteria removed as low as 30 % and 10 % of COD and BOD 5 under the same operational conditions (Fig. 1B and C).Statistically significant differences between treatments were observed for color removal, COD and BOD 5 abetment, and it was established that treatment VB LCC was more efficient (p < 0.0001) than NVB LCC and WLCC bacteria .
Ligninolytic enzymes played an important role in color removal and COD and BOD 5 abatement.Laccase activity increased progressively reaching values of 452 U L −1 at 18 h of treatment.Later, laccase activity decreased to 410 U L −1 after 48 h of treatment.MnP activity reached a maximum activity value of 1 428 U L −1 at 48 h (Fig. 2A).In the WLCC bacteria treatment, Laccase and MnP activities were not detected.Wastewater alkaline values of 8.0 were observed at the beginning of the experiments; pH gradually decreased to 5.5 ± 0.5, probably due to consumption of simple forms of organic carbon (residual glucose: 0.1 g L −1 at 48 h) and production of citric and oxalic acids by fungi and bacteria (Fig. 2C).Bacterial growth was higher during treatment with VBLCC (10x10 13 CFU mL −1 ) in comparison with the other two treatments, in which bacteria concentrations attained values of 10x10 8 and 10x10 9 CFU mL −1 for NVB LCC and WLCC bacteria , respectively (Fig. 2B).higher resolutions (25000x and 50000x) a rough surface, presence of different aggregate sizes, various morphologies (spherical and ovoid forms), and small cracks were revealed.These cracks are probably caused by heat treatment at 450 • C. The TiO 2 films had signals matched with Anatase crystalline planes (101, 103, 004, 112, 200, 105, and 211) (Fig. 3C).

Photocatalytic discoloration and COD and BOD 5 abatement
Wastewater treated after 6 h, 24 h, and 48 h by VB LCC was used for preliminary photocatalysis tests.Photocatalytic treatment for 4 h resulted in a 25 % discoloration and in COD and DOD 5 abatement in wastewater (Fig. 4).Based on these results, photocatalytic experiments were performed for 6 hours on wastewater previously treated by VB LCC .
A maximum discoloration of 86 % was obtained after 12 h of photocatalytic treatment of wastewater treated with VB LCC .The percentages of discoloration due to photolysis and adsorption were 45 % and 33 %, respectively (Fig. 5A).
The efficiency of the VB LCC -TiO 2 UV sequential treatment (secondary treatment with VB LCC for 6 h and tertiary treatment with TiO 2 UV for 12 h) was established based on the initial values of color, COD, and BOD 5 levels of untreated textile effluent wastewater.Accordingly, total removal/abatement percentages were as high as 89 %, 81 % and 89 % for color, COD and BOD, respectively.Additionally, a microbial inactivation of 53 % was attained (Table 1).

Recycling of TiO 2 films without thermal reactivation
After 12 h of tertiary treatment during test cycle 1 (C 1 ) of the TiO 2 films, a discoloration value of 81 %. was observed.Reusing the same films in subsequent cycles led to decreeing discoloration percentage of 79 %, 33 %, and 32 % for C2, C3 and C4, respectively (Fig. 6).Under the evaluated conditions, TiO 2 films can be reused without thermal reactivation during two consecutive cycles.Concerning COD abatement and heterotroph bacteria inactivation, no statistical differences were determined between C1 (57 % COD and 54 % heterotroph inactivation) and C2 (56 % COD and 51 % heterotroph inactivation).In C3 and C4, COD abatement and heterotroph bacteria inactivation were less than 40 %.Moreover, SEM images showed that bacterial biofilm formation increased from new (Fig. 6A) to C4 (Fig. 6B and Fig. 6C) TiO 2 films.The bacterial morphology corresponds to Bacillus with terminal spores.Biofilm formation can be associated to gradual loss of photocatalytic activity, especially after C3 and C4.

Acute toxicity test
Wastewater without any treatment was toxic to both fresh-water polyps and letucce seeds.In the polyp, 100 % at 50 % lethality was observed.Moreover, acute toxicity tests with L. sativa showed 54 % at 50 % of inhibition.After comparing the toxicity of untreated wastewater with samples of those treated by sequential treatment (VB LCC /TiO 2 UV) during 18 h, toxicity on H. attenuata increase to (13 % at 6.25 %) and decreased on L. sativa (12 % at 75 %).Additional tests were performed in order to verify TiO 2 toxicity at 1 % (w/v).According to data, no adverse effects of TiO 2 were noticed on L. sativa (0 % at 100 % by triplicated).On the other hand, acute toxicity tests on H. attenuata revealed that TiO 2 lethality (56 % at 12.5 %).Most of the toxicity found on H. attenuata may be related to by-products formed during sequential treatment.Minor toxicity effects could be associated to TiO 2 .

Dyeing tests using treated effluent water
Regarding the amount (mg g −1 ) of adsorbed dye per gram of cotton fabric, no statistical differences were observed between wastewater treated with VB LCC /TiO 2 UV at 18 h, drinking water and distilled water (0.86 ± 0.02, 0.85 ± 0.04, and 0.88 ± 0.03 mg g −1 ; Fig. 7A).According to the visual assessment, the brightness and final aspect of the fabrics after dyeing were similar in all treatments.By the end of the process, pH values ranged between 8.0-8.5 and average conductivity was of 5.27 mS cm −1 (Fig. 7B and Fig. 7C).This can be related to the presence of sodium chloride acting as dyeing additive (Fig. 7B and Fig 7C).Moreover, the liquid residue obtained after dyeing tests may be treated by the sequential system and re-used in a new dyeing cycle.This will represent a zero-discharge system at the laboratory level.

Discussion
Secondary treatment with ligninolytic co-culture Treating wastewater material with microorganisms can increase the efectivity of sequential or integrated oxidation (i.e.photocatalysis) procedures.This is possible because biological treatments can remove different charges of organic matter (Henao et al., 2011 andPunzi et al., 2015).
The fact that NVB LCC showed some adsorption capacity can be a consequence of the heat treatment (autoclaving) itself.The heat treatment to which NVB LCC inoculates were subjected can generate a considerable number of functional groups that enhance color removal and led to the observed moderate COD and BOD 5 abatement levels (Russo et al., 2010).Further, in the experiments without ligninolytic co-culture (WLCC bacteria ), the observed levels of COD, BOD 5 abatement and color removal were related to bacterial activity, probably as a result of enzyme activities such as peroxidases, azoreductases, riboflavin r eductases, NADH-DCIP reductase and aminopyrine N-demethylase (Singh et al., 2015).
Positive interactions between fungi and bacteria have been previously described by Pedroza et al. (2018).These authors found that gram-positive and gram-negative bacteria, along with two white rot fungi (Pleurotus ostreatus and Trametes versicolor), can efficiently remove dyes from wastewater.Similar results were found in the present study, indicating that ligninolytic co-cultures can be used in bio reactors without specific s terile c ontrol c onditions and that fungi are not inhibited by bacteria.Moreover, the presence of fungi may favor bacterial growth as evidenced by a bacterial biomass increase from 6.0 x 10 6 to 10 x 10 10 CFU mL −1 .Fungal ligninolytic enzymes are likely to attack complex molecules producing simpler ones that can be transformed by bacteria (Pedroza et al., 2018;Singh et al., 2015;Fig. 2A and 2B).
Concerning enzymatic activities, Laccase and MnP activity increased gradually.This may be related to an inducer effect caused by the dyes.In fact, the chemical structure of azoic dyes, which is made of aromatic rings, resembles that of phenyl propane units present in lignin.This has an enzyme enhancement effect.A possible scenario can be proposed in which the chromophore group is modified by the action of Laccase and then aromatic rings are transformed by MnP.This is drawn from findings b y M orales e t a l. ( 2016) i n which P. ostreatus laccase modified chromophore groups of Malachite Green, and then peroxidases were able attack aromatic rings.
Tertiary treatment with TiO 2 /UV In the present study, preliminary tests showed that high concentrations of color and elevated COD values (as seen in wastewater without treatment) affected photocatalytic degradation.This can be explained by color and organic matter loads acting as a screen blocking the passage of UV light; thus affecting electron hole pair formation and leading to deficient rates of reactive oxygen species production.The effect of pollutant concentration on photocatalytic degradation has been previously reported by Puentes et al. (2012a).These authors evidenced that at high concentrations (80 mg L −1 and 100 mg L −1 ) of the azoic dye Reactive Black 5 (RB5), discoloration and COD abatement percents were as low as 15 % and 10 % , respectively, at 10 h of treatment.Conversely, at low pollutant concentrations (10 mg L −1 , 50 mg L −1 and 70 mg L −1 ), discoloration was as high as 80 % during the sames period of time.
After analyzing tertiary treatment results, it was noted that photocatalysis (TiO 2 /UV) was more efficient than ultraviolet light and adsorption.The process was initiated by adsorption of residual dyes thanks to the pH conditions of the wastewater that facilitated residual dye transformation.This transformation is likey due to UV light interactions with the catalyst that lead to reactive oxygen species pruduction ending in dye structural modifications, namely at N=N and C-N b onds.Aditionally, this process would produce by-products like aromatic (phenols, amines, quinones etc), aliphatic compounds, and dissolved ions, such as NO − 3 , N 2 and SO 4 2 (Henao et al., 2011;Puentes et al., 2012a).
The observed limited re-usability of TiO 2 films c ould b e r elated to progressive inactivation of TiO 2 excreted by adsorbed by-products onto the semiconductor's oxide surface.These by-products could block available active sites and prevent photonic excitation.Another aspect that could affect TiO 2 performance at each usage cycle can be associated to bacterial population size (9.0 x 10 9 CFU mL −1 ).Bacteria can progressively cover TiO 2 film surface creating biofilms.In addition, bacteria could behave like suspended solids at high concentrations blocking UV light.This assumption was confirmed by SEM of TiO 2 films obtained prior and after TiO 2 film usage cycle C4 (Fig. 7), in which a bacterial biofilm was observed.This may be associated with the presence of azoic dyes, additive salts, and solubilizers which have been previously reported as toxic and potentially carcinogenic (Almeida & Corso 2014).
Wastewater toxicity on H. attenuata increased by the end of the sequential treatment, probably due to the presence of by-products formed during the process and the TiO 2 washed off from the films.This compounds and particulate material would be absorbed by the target organisms, causing alterations in their digestive tract and producing morphological changes (Castillo et al., 2013, Allouni 2009, Rizzo 2011).The current results agree with the observation by Henao-Jaramillo et al. (2011) that the toxicity of an RB5 (300 mg L −1 ) solution increased after being treated with Trametes versicolor and photocatalysis with TiO 2 .
Lettuce (L.sativa) was less sensitive than H. attenuara, and responded more favorably to sequentially treated wastewater.L. sativa seeds were likely to be less sensitive to toxic effects due to a strong external layer made of hemicellulose and cellulose.These natural polymers tend to be hydrophobic and protect the seeds.Similar results are recorded by Puentes et al., 2012a who evaluated RB5 toxicity pre and post photocatalytic treatment.They showed that a solution of RB5 without treatment causes an inhibition percentage of 10 % at 100 %.This result was slightly different in comparison to the one obtained in the present study (56 % at 50 %).A possible reason for this difference could be the synergic toxic effect of the mixture of dyes and additives present in textile wastewater as opposed to that of a single dye.
Adsorption of RB5 takes place in 5 stages.First, the dye is adsorbed by cellulose fibers and then it diffuses in the matrix.Second, the dye is fixed to the fabric under favorable conditions of alkaline pH, additives and temperature.
Next, the dye attaches to the active sites of the fiber through covalent bonds (nucleophilic substitutions).The last stage is the final wash and drying; in which unabsorbed dye is washed out from the fabric, and it acquires brightness and a uniform color (Sufian et al.,2 016).In the present work, just 20 % of the dye was lost in the final wash out.On average, 30 % dye losses could be expected in textile dye procedures, thus the results are promising and show that textile effluent wastewater recycling after treatment is possible.However, large scale studies should be performed to determine the optimal number of treated wastewater re-usage cycles.The process can help to reduce the volume of wastewater produced and is a good approach to zero discharge in the textile industry.

Conclusions
A laboratory-scale sequential system to treat textile wastewater was developed and evaluated.The present work showed that final textile effluent wastewater can be treated by subjecting it to a secondary treatment with a fungal co-culture for 6 h, coupled to a tertiary treatment consisting of photocatalysis with TiO 2 for 12 h.
Thanks to this proposed sequential treatment, wastewater color was removed and COD and BOD 5 abated.However, treated wastewater remained ostensibly toxic to fresh-water polyp, H. attenuata, and lettuce, L. sativa, seeds.
The reutilization of TiO 2 films and treated wastewater was evaluated.TiO 2 films c an b e r eused f or t wo c ontinuous o perational c ycles a nd treated wastewater can be recycled and used in cotton fabric dyeing.

Figure 1 .
Fig. 3A and fig.B show SEM images of TiO 2 films prepared at 50 • C and heat treated at 450 • C.Under low magnification (5000x), a full oxide semiconductor coating on the substrate and a film's uniform appearance was revealed.At

Figure 4 .
Figure 4. Effect of four secondary ligninolytic treatments on photocatalytic activity at 4 h, as evidenced on A) COD abatement percentage and B) discoloration percentage.Ligninolytic treatments: 1. Wastewater without any secondary treatment; 2. Wastewater at 6 h of secondary treatment or VB LCC ; 3. water at 24 h of secondary treatment; and 4. water at 48 h of secondary treatment or VB LCC .

Figure 6 .
Figure 6.Discoloration, COD abatement, and microbial inactivation throughout TiO 2 film recycling e xperiments.Letters represent Tukey homogeneous subsets.a corresponds to the cycles with the best removal, followed in order by b and c.

Figure 7 .
Figure 7. Reuse of sequentially treated wastewater in cotton fabric dyeing test.(ICF) Initial cotton fabric color.1. Dyeing solution with treated wastewater with ligninolytic co-culture/TiO 2 /UV at 18 h.2. Dyeing solution with drinking water.3. Dyeing solution with distilled water.Letters represent Tukey homogeneous subsets.a corresponds to best dyeing treatments.

Table 1 .
Chemical, physical and microbiological variable values of textile effluent wastewater before and after sequential treatment with ligninolytic co-culture/TiO 2 /UV.
Distilled water, drinking water, and sequentially VB LCC /TiO 2 UV treated wastewater were used in dyeing fabric experiments.This dyeing process was performed in the laboratory in quintuplicate.In each dyeing experiment, 35.8 mg of azoic dye (Reactive Black 5) were mixed with 28 mL of the test waters (distilled, drinking, and VB LCC /TiO 2 UV treated wastewater) in 250 mL flasks at 19 • C. Each solution was heated up to 90 Universitas Scientiarum Vol.23 (3): 437-464 http://ciencias.javeriana.edu.co/investigacion/universitas-scientiarumCotton fabric dyeing tests with treated wastewater (Morales et al., 2017;Puentes et al., 2012b) values were associated to the presence of five different azoic dye types in textile effluent wastewater.Hence mechanisms of textile effluent w a s tewater d i s coloration a l s o led to abatement of COD and BOD 5 values.Two mechanisms can relate to discoloration via VB LCC .First, dyes can be absorbed into fungal walls, which possess functional groups acting as ligands, (i.e.carboxyl, amine, hydroxyl, phosphate, thiol, etc.)(Morales et al., 2017;Puentes et al., 2012b).Second, following dye adsorption, ligninolytic enzymes can generate modifications in the chemical structure of the dyes.These enzymes can cleavage azo bonds (N=N) modifying chromophore groups, and therefore changing visible spectrum adsorption.Later, byproducts may be oxidized, reduced, demethylated, and hydroxylated producing benzene or substituted quinones Previous studies have tested film reusability.Yue e et al. (2010) created a novel composite film made of Cds/TiO 2 /NTFs for methyl orange discoloration.This novel material removed 90 % of color and endured five continuous usage cycles of 10 h each.However, in Yue e et al. (2010), a single dye at low concentration was evaluated, in contrast with the dye mixture used in our study.Some toxic by-products could be produced after industrial wastewater treatment by combined technologies.These can sometimes be even more toxic to different organisms than the original untransformed compounds.Hence, it is important to verify water quality in terms of toxicity.Based on the current results, wastewater without any treatment was slightly toxic to H. attenuata and L. sativa, being H. attenuata more sensitive than L. sativa.