Various forms of industrial waste are major sources of environmental pollution due to the release of several unidentified toxic pollutants. The majority of agro-based industries, i.e., distilleries, tanneries, and pulp paper are major sources of aquatic and soil pollution. Due to the release of huge amounts of waste water and sludge, sugarcane-molasses-based distilleries are among the most polluting industries in India. Distilleries release 12 to 15 l of spent wash per liter of alcohol produced. Currently, there are more than 319 distilleries in India, reflecting the magnitude of the problem due to the presence of various complex pollutants in post-methanated distillery effluent (PMDE) and post-methanated distillery sludge (PMDS). The detection and detoxification of distillery waste is a challenge for the safe disposal of effluent and sludge. Safe disposal of PMDS in the environment is of paramount importance currently due to the presence of various unidentified complex organic and inorganic pollutants (melanoidins, phenolics, and sulfur compounds as well as heavy metals) (Chandra and Kumar, 2017a). These complex pollutants in PMDS are generated during the process of the distillation of fermented molasses slurry and the subsequent methanogenesis of the spent wash. The toxicity of PMDS and PMDE to the terrestrial and aquatic environment are well documented due to the presence of heavy metals and organic compounds (Bharagava and Chandra, 2010). The seed germination (SG) test has indicated stunted stem growth and reduced root systems in Phaseolus mungo at higher concentrations of sludge-amended soil (Chandra et al., 2008).

Microbial communities are fundamental components of any ecosystem, playing a primary and critical role in the metabolism of organic matter to maintain biogeochemical cycles in various critical environments (Fuhrman, 2009). They are predominantly involved in the bioremediation of contaminated sites, and several microorganisms, which degrade a wide range of pollutants have been described (Loviso et al., 2015). A detailed knowledge of microbial communities at any polluted site not only reflects their relationships with pollutants, but also reflects information with respect to the bioremediation potential of microbes on specific pollutants. This may indicate a direction of the bio-stimulation or bio-augmentation for the restoration of any polluted ecosystem.

However, recent progress in molecular microbial ecology has shown that traditional culturing methods are insufficient to allow detailed analysis of the microbial diversity at any contaminated site. This is because only a small proportion of viable microorganisms from a sample are recovered by culturing techniques. So, to show the full extent of microbial diversity various molecular techniques have been applied (Zhongtang and Mohn, 2001). The study of pollutants and their influence on the microbial communities of WWTPs can provide useful information for solving the problem of fluctuation in WWTPs. The study has also demonstrated that different configurations of treatment plants influence the structures of microbial communities (Gich et al., 2000).

Some significant research has been carried out on the analysis of bacterial communities derived from the activated sludge of wastewater treatment processes and bioreactors, which was designed to evaluate the process performance of treatment plants for the biodegradation of hazardous chemicals (Whiteley and Bailey, 2000; Forney et al., 2001). For the assessment of the biodegradability of toxic compounds and the measurement of whole bacterial communities, substrate utilization has been reported using phospholipid fatty acid (PLFA) analysis and fluorescence in situ hybridization (FISH) techniques (Maharana and Patel, 2014). Rapid community fingerprinting by the polymerase chain reaction (PCR)-based denaturing gradient electrophoresis (DGGE) of 16S rDNA also indicated highly structured bacterial communities growing in treatment plants and different classes of bacteria were detected (γ-Proteobacteria, Firmicutes, etc.; Kapley et al., 2015). Some gene probes have also been used as a tool to detect the degradation of phenolic compounds in wastewater treatment systems (Purohit et al., 2003). A few studies have been published in the literature on microbial community indices using fatty acid or phospholipid analysis as a tool for the assessment of bioremediation of pollutants and eco-restoration of contaminated sites (Maharana and Patel, 2014). Detailed knowledge regarding organic pollutants and the growth of autochthonous bacterial communities at several complex polluted industrial waste sites is lacking. These recalcitrant pollutants persist in the environment and cause adverse effects on aquatic and soil ecosystems, and on flora and fauna, as well as on humans due to the presence of some unidentified mutagenic and androgenic compounds, which have, so far, not been isolated. So, the extraction of organic pollutants from PMDS using appropriate solvents would allow the extraction of the maximum number of toxic organic pollutants present in sludge. Their further detection by gas chromatography-mass spectrometry (GC-MS) will show the properties of the pollutants that challenge safe disposal, while simultaneously investigating the autochthonous bacterial communities present in disposed PMDS that mediate the bioremediation of toxic compounds in situ. Hence, an analysis of the growing autochthonous bacterial communities present in PMDS after 90 days of in situ bioremediation would not only provide information on the bacterial communities, it will also highlight a prerequisite step for the monitoring of polluting discharges from sugarcane-molasses-based distillery waste. Furthermore, the characterization of metabolic products will also provide important information about the environmental fate of these pollutants. The in situ bioremediation of organochlorine-pesticides-contaminated soil by using gene probes has been reported, but there is no knowledge of the chemical properties of pollutants present in complex industrial sludge discharge from sugarcane-molasses-based distillery waste after bio-methanogenesis. Furthermore, the autochthonous bacterial communities growing in sludge, which have potential capabilities for in situ and ex situ bioremediation remain unidentified.

In the present study, we have detected organic pollutants by GC-MS extracted with organic solvents to ensure the extraction of the majority of pollutants from disposed sludge. Energy dispersed spectroscopy (EDS) of sludge was also carried out for qualitative and quantitative analysis of heavy metals and of various salts. Potential autochthonous bacterial communities have also been detected using culture-independent methods, i.e., restriction fragment length polymorphism (RFLP) during in situ bioremediation to show the relationship between pollutants and the potential autochthonous bacterial communities responsible for biodegradation of PMDS. These findings will allow exploration of the potential autochthonous bacterial communities existing in the extreme environment of PMDS, which are capable of the bioremediation of inorganic and organic pollutants that are rich with phenolic and melanoidin compounds. Such bioremediation currently poses a challenge for the safe disposal of PMDS in the environment due to its complex structural and recalcitrant properties and its toxic effects on ecosystems.

Physicochemical analysis showed that sludge and leachate both have a high pH. This may be due to the presence of large amounts of salt, but the slight reduction in various physicochemical properties, as measured for leachate, was due to bioremediation by autochthonous bacterial communities; this alters the original chemical structure and properties of various organic constituents and salts in the sludge (Chandra and Kumar, 2017b). The alkalinity of the waste arises from the combined residual effect of carbonates, bicarbonates, and hydroxides, which are used in a distillery for pH adjustment during the fermentation process and the washing of fermentation products (Tiwari et al., 2013). While the EC of sludge and leachate was found to be 4.1 and 3.9, respectively, the high values of EC indicated the role of various cations and anions, such as sodium, chloride, nitrate, and ammonium ions, which are present in both fresh distillery sludge and leachate. The high pH and EC of sludge could be due to the presence of high concentrations of soluble salts. Several authors have also reported higher values of these cations and anions in distillery waste (Chandra et al., 2008; Chandra and Kumar, 2017b). Furthermore, in the present study the presence of various heavy metals along with melanoidins has also been shown. Heavy metals have a strong tendency to bind to melanoidins (Migo et al., 1997). The high content of heavy metals in distillery sludge could be due to the corrosive effect of sugarcane juice during the sugar manufacturing process. Metals may be added further during the fermentation and distillation processes of sugarcane molasses in distilleries, as it is finally discharged as a spent wash under highly acidic conditions and again, undergoes treatment in an anaerobic reactor and all of these processes potentially induce metal corrosion of metal pipes. This may be the main source of the heavy metal content of distillery sludge. The high concentrations of heavy metals and salts in PMDS are due to the condensation process that takes place during sugar manufacturing and alcohol production. Our findings corroborated well with earlier data (Chandra and Kumar, 2017b). The high levels of organic and inorganic parameters in PMDS are also concordant with previous reports (Chandra et al., 2008). The high content of several heavy metals is an environmental health hazard because they are known to be damaging to humans and animals due to their persistence and accumulation in the environment from contaminated sites. They are known to cause neural toxicity, renal disorders, asthma, and carcinogenic effects in humans and animals (Barakat, 2011). Therefore, the sludge has toxic properties and apparently does not degrade. There was a subsequent reduction in various physicochemical parameters of the distillery leachate after 90 days of in situ bioremediation; possibly due to the action of the microbial community resulting in the mineralization of complex compounds. The majority of bacteria, actinomycetes and fungi are saprophytes and so they decompose organic matter. They hydrolyze and oxidize various complex organic and inorganic compounds through enzymic processes, resulting in the reduction in some physicochemical parameters following in situ bioremediation.

Fourier transform-infrared spectrophotometry spectroscopy is an extensively used method for the determination of the functional groups present in organic compounds. The observed change in the peaks in FTIR spectra analysis of control and degraded sludge leachate suggests degradation of organic compounds has occurred after 90 days of bioremediation in situ. This indicated the conversion of complex toxic compounds into simpler, non-toxic molecules. The autochthonous bacterial communities employed during in situ remediation during the investigation potentially resulted in the cleavage of various chemical linkages.

Gas chromatography-mass spectrometry is a good technique for determining the organic pollutants in the environment. In our study, the majority of the organic compounds detected in distillery sludge and leachate posing a threat to the environment and human health are substances derived from biochemical processes at the various stages of sugar and alcohol production, and of effluent treatment. GC-MS analysis of organic extracts in the sample identified more than 35 organic compounds some of which were mutagens, carcinogens, and environmental endocrine disrupters. Octadecanoic acid and hexadecanoic were detected in our study, which have been reported to be anti-quorum sensing molecules in bacterial products (Singh et al., 2013). However, in some studies octadecanoic acid has also been reported to be a toxic compound in aquatic systems (Kamaya et al., 2003), while hexadecanoic acid has been reported to be a DNA fragmentation inducer in a human melanoma cell line (de Sousa Andrade et al., 2005). Similar compounds have been reported by various researchers, and so our data support results reported previously (González et al., 2000; Quinn et al., 2007). This finding also corroborated previous studies (Kaushik et al., 2010). These organic compounds constitute the main components of wastewater and discharge in sludge from yeast or original sugarcane molasses, which remains after the secondary treatment of distillery effluent. Apart from this, other compounds were also identified as stigmasta-5, 22-dien-3-ol(3 β,22E), stigmasterol and β-sitosterol. These are major phytosterols (plant sterol) with a chemical structure similar to cholesterol, soluble in water at all pH values and previously reported to be present in sugarcane (Saccharum officinarum L.) and the wastewater from sugarcane molasses. These compounds are screened under the list of environmental endocrine-disrupting chemicals (EDCs; as in USEPA, 2012). In a previous study, it was reported that in nature, aerobic, and/or anaerobic microorganisms may transform β-sitosterol and other sterols into androgenic hormones such as 5-β-androstan 3, 17-dione and androstan 4-en-3, 17-dion (Taylor et al., 1981). Such androstan derivatives of sterols may ultimately interfere with endocrine systems and produce hermaphroditism or other morphological defects. A role of these compounds in masculinizing the fish population and reducing fish numbers has been suggested (Jenkins et al., 2003). However, tetradecane and other similar phyo hydrocarbons (heptacosane dotriacontane, lanosta-8, 24 dien-3-one) have also been identified, and reported as key constituents of environmental pollutants responsible for dermal irritation (Muhammad et al., 2005). We have also detected several other organic compounds in control and degraded sludge leachates. This might occur as a residual fraction of the distillation process during ethanol production from fermented molasses slurry by yeast cells. However, the role of several other organic compounds detected in the environment is still of interest and requires detailed investigation for environmental safety.

Sequence analysis of the 16S rRNA genes of bacteria and archaea has been frequently used to characterize the taxonomic composition and phylogenetic diversity of environmental samples (Langille et al., 2013). In the present work, the RFLP technique was used to divulge phylogenetic relatedness among uncultured bacterial clones. RFLP analysis disclosed the presence of the major phyla Bacillus, followed by Enterococcus. The genus Bacillus is also characterized by its cellular properties, which are Gram-positive, have a low G+C content, are mostly straight rod shaped, and occur singly, in pairs, or chains. They also form endospores and grow aerobically. So, in the presence of organic compounds under anaerobic conditions they could grow slowly or are in the inactive phase and become viable after the discharge of sludge (Whitman, 2009). The heat resistant nature of the endospores accounts for the presence of Bacillus sp. in cooling tower water, as the heat-exchange process selects for heat resistant microorganisms that become even more dominant as water is recycled (Sharmin et al., 2013). Also, Park et al. (2007) reported the dominance of Bacillus sp. growing on a Rotating Activated Bacillus Contactor Biofilm (RABCS) as used for advanced wastewater treatment by culture-dependent methods. The second member of the phylum Firmicutes noted in this study was Enterococcus sp. These bacteria are characterized as being Gram-positive, cocci-shaped, often occurring in pairs (diplococci) or short chains, non-endospore forming, aerobic or facultative anaerobes, capable of the fermentation of sugars and of producing lactic acid or other acidic products. One study has also revealed the presence of Bacillus and Enterococcus sp. (España-Gamboa et al., 2012) in the anaerobic sludge of an upflow anaerobic sludge blanket (UASB) reactor for the treatment of distillery spent wash. This indicated that bacteria are able to exploit carbon compounds from spent wash as a necessary source of energy, and can adapt to this environment by using their endogenous enzymatic systems. This corroborates our results.

Based on sequence analyses recovered from the clone libraries, the phyla Firmicutes represented the dominant group of bacteria. PhyloChip analysis has also been used to assess the dominance of Firmicutes growing within sugarcane processing plants (Sharmin et al., 2013). Acharya et al. (2011) also studied the dynamics of the microbial communities within anaerobic biphasic fixed film bioreactor treatment distillery spent wash, showing the clear dominance by Firmicutes in a methanogenic bioreactor, which indicates the high degree of diversity of this phylum. The presence of Firmicutes as a dominant group is quite rare for natural samples. However, lignocellulosic material, such as sugarcane molasses/sugarcane bagasse represents an abundant, inexpensive source of organic material, which can be a carbon source for a growing bacterial population. Hence, the results of the current study agree with previously reported data from autochthonous bacterial communities in similar environments (Ch]andra et al., 2012). PMDS contains much more various organic and inorganic complex pollutants at much greater concentrations, but the autochthonous bacterial communities identified were specific and capable of growing in this environment.

The SG test is a common and basic tool used for toxicity evaluations of the environmental safety of industrial waste (OECD, 2003). A SG rate of 100% occurred with a 1% (v/v) concentration of fresh leachate and as the concentration increased, so the percent germination decreased. The promotion of seedling growth by lower concentrations of leachate might be due to the presence of lower concentrations of toxicant in samples tested. While the suppression of germination at high concentrations of leachate might be due to the presence of highly toxic organic compounds and dissolved solid, which were absorbed by the seeds before germination and affected various physiological and biochemical processes of SG (Bharagava and Chandra, 2010). Increases in the percentage germination in degraded sludge leachate may be due to the presence of less organic compounds, so creating a favorable environment for germination and utilization of nutrients present in the leachate prepared from the degraded sludge. Different concentrations of leachate and the different degrees of seed coat permeability led to different degrees of inhibition of germination. The inhibitory effect of the fresh sludge leachate on SG and seedling growth might be attributed to the high salt load and metal content, which induces both a high osmotic pressure and anaerobic conditions. It has also been reported that a high salt load and metal content acts as an inhibitor of plant hormones (amylases, auxins, gibberellins, and cytokinins), which are required mainly for SG, seedling growth and plant development, respectively (Ahsan et al., 2007). One study showed that distillery-sludge-amended soil delayed flowering and reduced pod formation in P. mungo L., which apparently provides evidence for the suppression of reproductive hormones by the toxicants present in distillery sludge (Chandra et al., 2008). These findings support the presence of high levels of EDCs plus other toxic compounds in distillery sludge.

Many national and international studies have focused on genotoxicity evaluation of industrial wastewater in different test models (Hemavanthi et al., 2015; Kumari et al., 2016). We selected the A. cepa assay due to its sensitivity and effectiveness in assessing effluent pollutants. The assay demonstrated that untreated samples were more toxic than the respective treated samples. The results of genotoxicity tests showed that MI decreased with increasing concentrations of fresh sludge leachate as compared to degraded sludge leachate. Trace metals and other organic pollutants have been considered responsible for diminishing the MI of A. cepa exposed to industrial wastewater (Chandra et al., 2005; Carita and Marin-Morales, 2008). A cytogenic effect was also observed that might be due to the presence of genotoxic compounds within the PMDS. We observed various chromosomal aberrations including morphologically altered cells with loss of genetic material, disturbed metaphase, c-mitosis, chromosome bridges, laggard chromosomes, sticky chromosomes, polyploidy cells, and apoptotic bodies. The reason for such effects could be due to the presence of toxic substances in the liquid medium, which may disturb division, causing a relatively high number of aberrations. c-Metaphase may result from the action of aneurogenic agents on the cell; compounds that promote complete inactivation of the mitotic spindle (Fiskesjo, 1993; Fernandes et al., 2007). Such alterations may generate other types of cell abnormalities, such as polyploid cells (Odeigah et al., 1997). The presence of chromosomal adherence reinforces the evidence of the aneugenic action of organic pollutants present in distillery leachate. However, organic pollutants disturb the balance in the quantity of histones or other proteins responsible for controlling the proper structure of nuclear chromatin. Chromosome stickiness reflects a highly toxic effect, which probably leads to cell death (Fiskesjo, 1997). Similar observations have been reported in A. cepa root after treatment with distillery effluent (Hemavanthi et al., 2015). This increased stickiness also leads to the formation of chromosome bridges. However, the frequency of cells with chromosome bridges significantly increased with increases in the concentration of distillery sludge leachate, indicating a clastogenic effect of leachate (Lerne and Marin-Morales, 2009) involving one or more chromosomes. Chromosome bridges were observed here, and may have occurred due to the misrepair of DNA, telomere end fusions or even from chromosome adherence. Our findings are well corroborated with earlier studies (Hemavanthi et al., 2015).

This study has revealed that PMDS and its leachate contain several residual recalcitrant organic pollutants plus heavy metals, the majority of which are environmental toxicants (i.e., octadecanoic acid), endocrine disrupting chemicals (i.e., β-sitosterol) and also DNA fragmentation inducers (i.e., tetradecanoic acid), which are still fairly unknown. During bioremediation of PMDS in situ, Bacillus sp., and Enterococcus sp., were noted growing dominantly as autochthonous bacterial communities of the phylum Firmicutes. This showed a capability to degrade the toxic pollutants present in PMDS. This bacterial community also demonstrated a special niche in high concentrations of Fe, Zn, Cu, Mn, and Pb, and an environment rich in complex organic compounds containing mutagenic molecules and EDCs. The findings of the present study will be useful for monitoring and managing distillery waste environmentally and for the eco-restoration of polluted sites.

RC did leading role for designing of experiments, analysis of pollutants, and bacterial community. While, VK played role as team member and supported in experimental work and data arrangement. Further, the co-author also supported to corresponding author, i.e., RC for manuscript formatting and phytotoxicity and genotoxicity assay and graph preparation. Thus, both authors justified their role for manuscript preparation and communication.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.