Bioremediation of lignin derivatives and phenolics in wastewater with lignin modifying enzymes: Status, opportunities and challenges

. The presence of thesecompounds inwastewaterisa criticalissue from environmental and toxicolog-icalperspectives.Somechloro-phenolsareharmfultotheenvironmentandhumanhealth,as theyexertcarcino-genic,mutagenic,cytotoxic,andendocrine-disruptingeffects.Inordertoaddressthesemosturgentconcerns,the useofoxidativeligninmodifyingenzymesforbioremediationhascomeintofocus.Theseenzymescatalyzemod-i ﬁ cation ofphenolicandnon-phenolic lignin-derivedsubstances, andinclude laccaseanda rangeofperoxidases, speci ﬁ cally lignin peroxidase (LiP), manganese peroxidase (MnP), versatile peroxidase (VP), and dye-decolorizing peroxidase (DyP). In this review, we explore the key pollutant-generating steps in paper and pulp processing,summarizethemostrecentlyreportedtoxicologicaleffectsofindustriallignin-derivedphenoliccom-pounds, especially chlorinated phenolic pollutants, and outline bioremediation approaches for pollutant mitiga-tioninwastewaterfromthisindustry,emphasizingtheoxidativecatalyticpotentialofoxidativeligninmodifying enzymes in this regard. We highlight other emerging biotechnical approaches, including phytobioremediation, bioaugmentation, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based technology, protein engineering, and degradation pathways prediction, that are currently gathering momentum for the mitiga-tionofwastewaterpollutants.Finally,weaddresscurrentresearchneedsandoptionsformaximizingsustainable biobased and biocatalytic degradation of toxic industrial wastewater pollutants.


H I G H L I G H T S
• Systematic outline of key pollutantgenerating steps in the paper and pulp industry • Toxic pollutants chemistry and their environmental and human health impact Environmental pollutants derived from large scale industrial wood processing represent a critical global challenge for sustainable development.The discharge of processing pollutants from the paper and pulp industry has improved significantly during the last 10-20 years, but this industry still generates wastewater at an immense scale that contains high amounts of phenolic, chlorinated, complex lignin-derived and sulfonated pollutants (Mandeep et al., 2019;Singh and Raj, 2020).In China, for example, the demand for paper products and hence paper and pulp effluent loads increased enormously during the decades of booming economic growth and these effluents now contribute about 10% of China's industrial wastewater emissions, but 25% of the chemical oxygen demand (COD), and the paper industry is thus considered a major source of industrial wastewater emissions in China (Zhang et al., 2020).Likewise, in India, the paper and pulp industry, which supplies about 3% of the world's paper production, is classified as one of the most waterpolluting industries (National Mission for Clean Ganga, 2019).The industry encompasses wood-based paper processing mills and smaller-scale industrial pulp manufacturing from agro-industrial lignocellulosic residues (Kumar et al., 2020a, b, c).
The high amounts of wastewater and the discharge of significant amounts of potentially harmful compounds are produced during several different papermaking process stages, including pulping, bleaching, and washing (Chandra et al., 2018;Mehmood et al., 2019).Per each ton of paper products produced, it is estimated that 70-225 m 3 of wastewater effluent is discharged; approximately 20-25 m 3 is from the pulping step, and up to 80-100 m 3 stems from the bleaching processthe volumes vary depending on the raw material, processing regime, and extent of water recirculation (Hubbe et al., 2016;Wagle et al., 2020).To put these numbers in perspective, a conservative estimate is that about 2.5 million tons of chemical pulp is produced per year in India, of which 60% is bleached using chlorine and chlorine-based chemicals (Malhotra et al., 2013).
The paper and pulp industry strives to transition to become more sustainable, and elemental chlorine-free and total chlorine-free paper processes do exist, which have reduced the presence of toxic substances profoundly in certain countries (Hubbe et al., 2016).Yet, both conventional chemical pulping and classical bleaching steps based on sequential addition of chlorine, hypochlorite, and chlorine dioxide, along with the use of acid sulfur dioxide washing and sodium dithionite addition, are still widely used to obtain bright, white paper products (Kumar et al., 2020a, b, c;Malhotra et al., 2013).These processing steps result in the discharge of numerous free constituent chlorophenols and related compounds, some of which resist spontaneous degradation.The wastewater produced from major current paper and pulp processing steps is thus characterized by containing an array of problematic substances, i.e. chlorophenols, sulfonated fragments of lignin, a range of sulfide byproducts, various resins, along with different high biological oxygen demand products, and high levels of inorganic salts (Choudhary et al., 2015;Kumar et al., 2020a, b, c).The effluents are potentially toxic and hazardous to the environment (Fig. 1).In addition, as discussed later, the toxicity effects of some of these compounds on human health are significant and include mutagenic and possible endocrine-disrupting effects (Chandra et al., 2018).
As hinted above, the pulping and bleaching are the main steps wherein different toxic contaminants are formed, i.e. the chlorophenols, the lignin-derived phenolic compounds, the sulfonated substances, and the volatile organochlorine compounds (Du et al., 2014;Qin et al., 2019;Singh et al., 2019).The effluents from the pulping and bleaching processes are indeed characterized by their high COD (1000 to 7000 mg L −1 ) and their low biodegradability, with biodegradability/ COD ratios ranging from 0.02 to 0.07 even at moderate levels of suspended solids (500 to 2000 mg L −1 ) (Hubbe et al., 2016;Mehmood et al., 2019;Mounteer et al., 2007;Uğurlu and Karaoğlu, 2009).
To our knowledge, a clear overview outlining the effects, advantages, disadvantages, and application records of these enzymes in relation to scavenging of industrial pollutants, including chlorinated phenols in wastewater does not exist.This critical review attempts to outline the significant polluting steps in paper manufacture.It particularly focuses on addressing newer biotechnological options for sustainable, efficient, and eco-friendly mitigation of the most hazardous phenolic and chlorophenolic wastewater compounds.The core of the review focuses on exploiting oxidative microbial lignin modifying enzymes, including laccases and certain peroxidases for conversion and removal of industrially relevant toxic effluent compounds, especially the toxic pollutants resulting from industrial pulping and pulp bleaching in pulp paper manufacturing processes.Moreover, other bioremediation techniques and the use of predictive and advanced molecular tools, including novel molecular and microbial engineering approaches and in silico prediction approaches are considered for enhanced bioremediation.

Structural and chemical aspects of lignin
Lignin consists of three specific phenylpropanyl units that are biopolymerized in the plant cell wall to function as a three-dimensional amorphous polymer: guaiacyl alcohol (G unit), p-coumaryl alcohol (H unit), syringyl alcohol (S unit).In the lignin polymer, the three constructive monolignols are attached and linked by one of the two or both types of linkage forming a C\ \C bond or a C\ \O\ \C bond.In more detail, the units are mainly linked by aryl ether (β-O-4), phenylcoumaran (β-5), resinol (β-β), biphenyl ether (5-O-4), dibenzodioxocin (5-5/β-O-4) type linkages (Munk et al., 2015).As a structural material of wood, lignin is vital and might comprise up to 25% of dry plant biomass.Lignin and its synthesis vary slightly from plant to plant, and differences occur between chemical components, especially in linkages among different plant types, either softwood or hardwood (Mu et al., 2018;Sher et al., 2020).Typically, more than two-thirds of monolignols units are connected by ether linkages (Guadix-Montero and Sankar, 2018; Ralph et al., 2004).Fig. 2  the components of the lignin polymer including lignin monomer units, coupled dimeric moieties as well as dibenzodioxocin and different linkages alongside percentages.

displays
Due to it being a polyaromatic, ether-linked hydrophobic polymer, lignin is "waterproof" and quite resistant to biological degradation.For this reason, harsh chemicals and high temperatures are used in paper and pulp processing, in turn resulting in the generation of harmful paper mill effluents (Kamimura et al., 2019;Pu et al., 2015).The prime purpose of the pulping process is to remove the lignin without damaging the cellulose fiber strength.Hence, a critical point in paper production is the removal of the lignin from the cellulose fibers and the elimination or bleaching of any impurities that cause discoloration and possible disintegration of the paper (Bajpai, 2018).As outlined below, the pulping process relies on chemical additives, followed by bleaching that involves a series of chemical and oxidative treatment steps to obtain white paper (Bajpai, 2018).

Lignin processing and pulp bleaching treatments-based wastewater pollutants
Different types of lignin-related compounds have been identified even in treated wastewater from paper industries (Chung and Washburn, 2016).To produce high-quality paper, lignin and hemicellulose contents must be removed.For this purpose, the pulp undergoes treatment with alkali, and sodium sulfide, chlorine dioxide, or sulfite or bisulfite at elevated temperature, i.e. beyond 150 °C (usually at 170-180 °C).This chemical digestion treatment breaks the bonds that tie the lignin to hemicellulose and cellulose and even hydrolyzes the lignin and turns the lignin into water-soluble substances (Mathew et al., 2018;Shrotri et al., 2017).The combined alkali and sulfide pulping are called the Kraft process or sulfate pulping process.The process was invented about 140 years ago in Germany in 1879, yet, although Kraft pulping has been improved since then, the principles behind this pulping procedure remains the commercially dominant pulp process for converting wood to a pulp for papermaking today.As discussed further below, Kraft lignin, soda lignin, organosolv lignin, and sulfonated lignin are the most common types of technical lignin resulting from the pulping process.The pulping process may also include the reaction of residual lignin with chlorine dioxide, a processing step that results in the formation of a significant amount of adsorbable organic halogen (Shi et al., 2019).Also, the addition of different organic solvents, i.e., alcohols, acids, esters, and ketones may be used during pulping to promote the cleavage of lignin bonds between monomer units (Shrotri et al., 2017).As a result, free lignin units form during the depolymerization of lignin.
After digestion, the raw pulp is screened and is usually cleaned again to remove the water.A noticeable amount (up to 6%) of lignin contents is left and persists in the raw cellulose, which, therefore, cannot be used directly as paper (Julkapli and Bagheri, 2016).For further breakdown and modification of lignin contents, the pulp then undergoes a bleaching process (Fortunati et al., 2016;Hubbe et al., 2016;Kumar et al., 2020a, b, c).Pulp bleaching is usually accomplished using a series of chemical treatments designed to produce white, high-quality paper products.The most common bleaching agents include H 2 O 2 , sodium sulfite, sodium hydroxide, chlorine, chlorine dioxide, and also enzymes, notably endo-xylanases, which have been reported useful in the bleaching process of woody pulp (Bajpai, 2018;Kumar et al., 2020a, b, c).In this series of mechanical pulp bleaching processes, that are designed to remove chromogens and brighten the pulp with minimum loss of mass, H 2 O 2 is currently the most widely used chemical (Bajpai, 2018).Chelating agents may be added to control metal ions that otherwise may impart the bleaching via decomposing the H 2 O 2 (Hintz, 2001).
The initiation of the bleaching process starts with oxygen and ozone, followed by the treatment with bleaching agents.Treated or bleached pulp is washed with sodium hydroxide, then treated with chemicals in a sequence.During these treatments lignin becomes modified, and as a result, sulfonated lignin or lignosulfonates are generated in significant amounts as byproducts (Hintz, 2001).Due to the profound number of lignin functional groups as well as the large number of carbohydrate degradation products, including oligophenols that can form during heat treatment by self-condensation and other reactions (Rasmussen et al., 2017), many different chemical compound products may be generated during industrial pulp and paper processes.

Technical lignin
The composition of technical lignin varies considerably with the raw material, pulp conditions, and processing chemicals used.The Kraft lignin process remains the most dominant industrial chemical pulping procedure, which is why Kraft lignin (extracted from pulp mill black liquor) is the most common type of technical lignin product from the paper and pulp industry (Zakzeski et al., 2010), yet obviously, the composition of Kraft lignin varies depending on the raw materials and processing conditions (Ház et al., 2019).
Lignin derivatives from the Kraft process constitute toxic and recalcitrant substances that accounts for the high BOD and COD and cause the dark brown appearance of effluents generated during the pulping process in the paper mill (Roppola et al., 2009).During bleaching of pulp in the papermaking process, chlorine is reacted with lignin and lignin-derived compounds, chlorinated compounds formed by chemical alteration, which are present in the pulp (Sudarshan et al., 2017).In particular, oxidation, hydrolysis, and pyrolysis (methoxy-substituted phenols and cresols) generate lignin-derived compounds in high yield (Alekhina et al., 2015).The recalcitrant and toxic compounds generated from pulping and bleaching operations include chlorinated lignosulfonic acid, chlorinated resin acids, chlorinated phenols, vanillins, catechols, benzaldehyde, guaiacols, and syringe-vanillins along with chloropropioguaiacols (Choudhary et al., 2015;Thakur, 2004).Some well-known lignin-derived, phenolic, and complex pollutants, including EDCs generated from industrial pulp and paper processing operations and their presence in wastewater are listed in Table 1.
For the sake of completion, it should be noted that other commercially available lignin forms are soda lignin, hydrolyzed lignin, organosolv lignin, and lignosulfonates (Zhong and Nie, 2017).Ligninderived value-added products for lignin valorization via biorefinery processes are increasingly being investigated for the production of different bio-based chemicals (Becker and Wittmann, 2019;Gillet et al., 2017).Lignin usage is sometimes considered in a lignin first concept in bioeconomy research.The extraction of lignin as a primary product from lignocellulosic materials processing is predicted to expand in the future (Radotić and Micic, 2016).

Health hazards associated with wastewater from the paper industry
As already indicated above, in the paper finishing steps, additives and chlorine for bleaching are added to achieve the desired paper quality, resulting in the generation of various toxic polychlorinated compounds (Table 1).Fortunately, regulatory restrictions enforced since the 1990s to control the use of dioxin, polychlorinated phenols, and dibenzofuran in paper manufacturing in North America/Canada and Europe, have limited the levels of dioxins in pulp and paper mill effluents in these countries (Hubbe et al., 2016).However, in other places, it is mentioned that dioxins and furans release into the wastewater may result from malpractices such as when plastics are used as boiler fuel (National Mission for Clean Ganga, 2019).Dioxins and dioxin-like polychlorinated biphenyls are notoriously recognized as hazardous chemicals and known to elicit developmental toxicity, carcinogenicity, and endocrine-disrupting properties in humans (Sappington et al., 2015;Urbaniak et al., 2017).The organochlorine compound, 2,3,7,8tetrachlorodibenzo-p-dioxin has for instance long been known as a multisite carcinogen associated with specific tumors (Torén et al., 1996), and polychlorobiphenyls have been classified as probable carcinogens in occupational workers related to pulp and paper industries (Monge-Corella et al., 2008;Soskolne and Sieswerd, 2010;Torén et al., 1996).
Pollutant-rich effluents are generally discharged into water bodies directly or indirectly and thus pose a risk of inducing environmental toxicity (Singh et al., 2019).The presence of various carcinogenic and androgenic components and chloro-lignin in the effluent can trigger the toxicological impact on human health (Monge-Corella et al., 2008).Also, poly-aromatic hydrocarbons can negatively impact human health.These pollutants have often been reported to exert negative effects on the reproductive system of both males and females; moreover, adverse effects on infant development have been detected (Jan et al., 2007;Jan and Vrbic, 2000;Joffe, 2003;Vecoli et al., 2016).
Organosulfur compounds (methyl mercaptan), a specific chemical also generated from the pulp and paper industries during chemical processing, can be released in the water bodies through the generated wastewater; as a result, damaging of the electron transport system in Table 1 Major environmental contaminants emitted from industrial processes, incl.pollutants from paper and pulp processes (reported in effluents).The living organisms by the mechanism of oxidase inhibition may result (Adeel et al., 2017;Lee et al., 2002;Torén et al., 1996).The health hazard or risk surrounding the workplace of paper industries has previously been reported to include risk of malignant disease, lung cancer, skin problems, respiratory disease, autoimmune and cardiac conditions (Torén et al., 1996).However, during the last 15-25 years, the pulp and paper industry has made huge efforts to avoid occupational health hazards by reiterating the processes and decrease their possible negative impact (Hubbe et al., 2016).Nevertheless, chlorine, sodium hypochlorite, and chlorine oxide are indeed used in the paper and pulp processes in certain countries (Kumar et al., 2020a, b, c).The exploitation of chlorine-based chemicals in the bleaching process leads to organochlorine compounds being produced from this step, and many chlorinated phenols indeed have adverse effects on human health (Table 1).
EDCs are a significant class of known chemicals that can interfere with the natural hormonal system of humans and animals.These effects on the hormonal system are either hyper or hypo, and as a result, various medical conditions and deformities may occur in the fetus development (Vilela et al., 2018).Some environmental chemicals or pollutants that act as EDC include phthalates, bisphenol, and alkylphenols, which are all substantial wastewater components that people come into contact with daily (Jambor et al., 2018).The EDCs may emerge from various agricultural, industrial, and household sources and, as a result, reach our drinking water and, ultimately, humans (Gelbke et al., 2004).It is uncertain if effluents of paper mills may contain steroidal estrogens e.g.17β-E2,17β-E2, which may cause serious human reproductive disorders, but steroidal estrogens have been detected at sites close to large wastewater treatment facilities (Adeel et al., 2017).Beyond reproductive toxicity, the development of breast cancer, prostate cancer, potentially negative neuroendocrine effects, harmful metabolic and cardiovascular effects are key problematic features of EDCs in human physiology (Sigman et al., 2012).Data from animal models, human clinical studies, and epidemiological studies indicate EDCs as an emerging environmental health hazard.Beyond steroidal estrogens, including the synthetic estrogen ethinyl estradiol (Adeel et al., 2017), the problematic chemical compounds suspected to be EDCs and/ or which may have cancerogenic effects include alkylphenols (4octylphenol; 4-nonylphenol), nonylphenol, octylphenol, bisphenol along with its alternatives and phthalates (Calafat et al., 2008;Kasahara et al., 2002;Nimrod and Benson, 1996).Some known pollutants, including chlorophenols that may be present in industrial paper and pulp wastewater and their associated human disease/medical conditions are summarized in Table 1.
The human homeostasis system might be altered by environmental exposure to environmental endocrine disruptors (EEDs).Animal model-based studies and other clinical observations and epidemiological studies have reported and suggested that EEDs are potential endocrine-disrupting compounds that cause severe human medical conditions and affect key functions in the human system, including the nervous system, reproductive system, thyroid, cancer, lungs, and may moreover induce obesity.The male reproductive system and development, female reproduction and development, thyroid function, obesity, and diabetes are the most significant abnormalities caused by EEDs (Ropero et al., 2008;Sigman et al., 2012).A diagrammatic representation of human health hazards from multiple environmental pollutants (Exposure of EEDs and its path to toxic endpoints) is shown in Fig. 3.

The effluents of the paper industry: environmental impact and implications
The adverse effects of a wide variety of toxic compounds (phenolics, lignin-derivatives, guaiacol, and heavy metals) present in effluents generated from the different stages of the papermaking process (Hubbe et al., 2016) also include undesirable environmental effects, which contribute to a critical concern for the toxicity of the water.Aquatic toxicity and impacts on the food chain have been identified (Shi et al., 2016) and several plant model systems have been evaluated for possible hazards of wastewater-mediated toxicity in different endpoints (Yu et al., 2019).Haq et al. (2016).Such toxic compounds have shown the phytotoxicity and cytotoxicity triggered by paper mill wastewater on plant model systems.For phytotoxicity research, in particular two plant model systems have been examined (Vigna radiata, and Allium cepa) to assess the possible toxicity under specific environmental conditions in laboratory experiments.Papermill consequences in the Allium cepa root tip cells appear extremely problematic involving inducing chromosome aberration (genotoxicity).Nath (2016) reported morphological and hematological effects on fish species Amblyceps mangois.Moreover, contamination from the paper industry was anemic to fishes (hemolytic disease) (Nath, 2016).A new study concludes that the highly toxic effects of paper mill effluents on Cyprinus carpio L. induced developmental and lethal impact on the organs, including the gills and fins (Dey et al., 2018).Abhishek et al. (2017) reported that Kraft lignin as an active ingredient of paper mill effluents causes acute toxicity, as evaluated by reactive oxygen species generation and a cytotoxicity assay on human keratinocyte (HaCaT) cell line (Abhishek et al., 2017).A diagrammatic illustration of wastewater-induced toxicity on the model system is shown in Fig. 4.

Current concerns and need for bioremediation
Environmental pollution is a major cause of significant damage to the environment in today's global context.Paper industries contribute considerably by emitting various hazardous compounds into the environment through effluents.Phenolic and chlorinated contaminants appear to be the most dangerous and complicated, owing to their recalcitrant and slow degradation potential (Azubuike et al., 2016).Enzyme-mediated bioremediation is a promising approach because enzymes in their nature can selectively catalyze the conversion of many of the problematic compounds, even if the compounds are present in the water at modest concentrations for "chemical reaction".As mentioned earlier, the lignin modifying enzymes are promising biocatalysts for sustainable mitigation of various pollutants of concern as these enzymes have the potential to degrade a wide variety of lignin, methoxylated compounds, phenol, polyphenol, EDCs, and non-phenolic compounds in an eco-friendly manner.Enhanced catalytic potential and production of lignin-modifying enzymes may be achieved with enzyme immobilization (Zdarta et al., 2019) and via engineered microbes (Azad et al., 2014;Dvořák et al., 2017).

Conventional bioremediation approaches for pollutants mitigation
Bioremediation is a promising practice wherein natural resources such as bacteria and plants are used in an "eco-friendly" way to eliminate toxic organic pollutants (Tekere, 2019).The current bioremediation strategies are focused mainly on biodegradation and this approach involves the complete elimination of harmless organic toxic substances from a highly contaminated medium or site.Many biological degradation processes and pathways have been reported to work, either in the presence or absence of oxygen (Ghattas et al., 2017;Ronen and Abeliovich, 2000;Wang et al., 2019).Bioremediation research focuses on enhancing the strength of the remediation process by supplying optimum concentrations of biocatalysts, chemicals and nutrients, usually including oxygen, necessary for the degradation and detoxification of toxic components through microbial metabolism and/or enzymatic conversion (Adetutu et al., 2015).
Microbial assisted pollutant remediation involves using microorganisms to either completely degrade toxic compounds into water and carbon dioxide (organic pollutants) or to catalyze their conversion into less toxic forms (Malla et al., 2018).Because of its low cost and biologybased approach, this technology provides an efficient alternative to conventional chemical treatment methods (Kang, 2014).Bioremediation is thus considered an economical, versatile, effective, and environmentally sustainable solution compared to physical and chemical approaches to treat various environmental contaminants (Jeon and Madsen, 2013).Several remediation approaches are based on a bacterial-derived enzymatic system, some are bioreactor based, and a few others include plant-based approaches (see phytoremediation section).Using a bacterial-mediated remediation strategy, the in situ or ex situ mitigation of pollutants is implemented to clean up an affected site from contaminants (Ali et al., 2013;Baric et al., 2014).In situ infers that the bioremediation occurs directly at/within the contaminated site.At the same time, ex situ implies that the microbial clean up may be applied off-site from the contamination location.In situ, remediation is considered slow and frequently difficult in the natural environment to control and optimize various bioremediation parameters.In this context and ex situ remediation, it is advantageous to use specially designed bioreactors to boost remediation.Bioreactors have been designed for use in bioremediation processes to achieve the optimum conditions, including aeration, microbial growth, and biodegradation to meet the various bioremediation goals.The bioreactors designed for bioremediation include packaged, stirred tanks, airlift, slurry phase, partitioning phase reactors are reported to be used in bioremediation of different organic pollutants (Pathak et al., 2020).
Numerous bacterial species producing lignin modifying enzymes have been described over the last decades, and this type of enzymes have been studied to remove lignin, chlorinated lignin, and organic phenol and chemicals that cause endocrine disruption in human (Bilal et al., 2019;Falade et al., 2018;Grelska and Noszczyńska, 2020).A newer peroxidase enzyme has been recognized as a dye decolorizing peroxidase.As will be discussed later, this enzyme shows particular promise to be used as a biocatalyst for the removal of a range of toxic pollutants in wastewater.This enzyme acts on various substances, including; lignin-derivatives, dyes, and EDCs compounds (Brissos et al., 2017).Some important well-known microorganisms for lignin-modifying enzyme production have been described in Table 2 and lignin compounds (lignin model compounds) that have been studied are listed in Table 3. Fig. 5 explains traditional bioremediation techniques in the treatment of lignin derivatives, phenolics, EDCs, and complex pollutants.

Biostimulation
This form of bioremediation strategy is related to promoting indigenous micro-organisms by injecting relevant nutrients on-site (soil and groundwater).It tends to focus on stimulating the natural or indigenous microorganisms, whether bacterial or fungal communities.Firstly, by providing inorganic nutrients, growth supplements, and trace minerals, secondly, through other environmental conditions such as pH, temperature, and oxygen to enhancing their metabolism and metabolic pathway.The inclusion of small amounts of pollutants can also serve as a stimulant by switching on the enzyme's operons for bioremediation.Nitrogen, phosphorus, and carbon are all necessary for this type of bioremediation, that to our knowledge is not currently in use or considered used industrially.

Bioaugmentation
The bioaugmentation approach concerning wastewater bioremediation relies on that a particular microorganism is added to the wastewater, and that the ensuing microbial growth can increase the rate of pollutant degradation.Accelerating population growth and improving natural microbial degradation, using microbes that preferably feed on contaminated sites, is a crucial feature of the bioaugmentation concept.It can be used to eliminate and alter micro-organisms, such as ethylene and chloride, that are not toxic (Cavinato et al., 2017).Natural or indigenous microbial species are usually not feasible to rapidly break down pollutants of concern.Hence, DNA manipulation or genetically modified/engineered microorganisms have been designed to promote more efficient and robust degradation of pollutants (Pieper and Reineke, 2000;Sayler and Ripp, 2000).However, despite this goal having been preceded for decades, there are still no full-scale applications (Janssen and Stucki, 2020), probably due to technical, economical, and ethical obstacles to the release of engineered microorganisms (Deeba et al., 2018).

Microbial bioreactors in bioremediation
Bioreactors are used to treat contaminated soil and water in meticulous and efficient processes, the point being to convert the contaminated media (e.g.wastewater rich in contaminants) into less toxic compounds via promoting a sequence of biological reactions (Tekere, 2019).Because temperature, pH, nutrient levels, and agitation can be measured in the reactors, microbial activity, and thus contaminant degradation, can be augmented (Jesitha and Harikumar, 2018;Pandey et al., 2009;Robles-González et al., 2008).Microbial bioreactors have been implemented in several laboratory and pilot bioremediation studies for different contaminants (Chikere et al., 2012;Pino-Herrera et al., 2017;Tekere et al., 2005).Flexibility in bioreactor design for various processes and remediation applications makes bioreactors preferable for bioremediation (Azubuike et al., 2016).The design should consider high cell biomass growth, nutrient supply, and waste removal from the system.The bioreactor technique has been practiced for effective use for organic pollutant remediation at a few underground leaking storage tanks at industrial sites (Iorhemen et al., 2016;Lone et al., 2008).

Phytoremediation
The use of plants to mitigate or eliminate inorganic and organic contaminants from the environment offers a practical, "clean and green," environmentally friendly, low-cost, and environmentally friendly technology (Pilon-Smits and LeDuc, 2009).Phytoremediation is a strategy of bioremediation employing designated plants (sometimes in combination with microbes) to remove, transfer, stabilize and/or eliminate soil and groundwater contaminants.The phytoremediation mechanism often involves several other mechanisms, i.e. phytodegradation, phytostabilization, phytoaccumulation, rhizofiltration, etc.Generally, phytoremediation removes pollutants or converts pollutants into their simplest or less toxic form (Saxena et al., 2019).Several plants can extract and concentrate on certain toxic elements from the environment, thereby providing a permanent remediation method.Phytoremediation is commonly recognized as a cost-effective technology for environmental restoration (Lone et al., 2008).According to the environment and types of contaminants, different phytoremediation technologies are available to perform bioremediation (Malla et al., 2018).Volatile organic compounds have been reported to be eliminated from the environment and be assimilated with plants (Pariselli et al., 2009).Phytoremediation for wastewater is a plant-mediated emerging bioremediation  technology, where plants utilize contaminated industrial wastewater and groundwater to eliminate toxic pollutants (Stefanakis and Thullner, 2016).Recently many transgenic plants have been reported for different forms of phenolic and chlorophenol pollutants elimination using phytoremediation (Cherian and Oliveira, 2005;Eapen et al., 2007;James and Strand, 2009;Macek et al., 2008;Wang et al., 2004).Plant roots may produce potent redox enzymes (oxidoreductases) such as peroxidases, encompassing non-specific oxidation polymerization reactions as a base for plant cell wall growth.These enzymes may also play a significant role in reactions for promoting detoxification and play a role in phenol and chlorophenol removal.Laboratory studies have been performed to examine pollutants reduction through phytoremediation with different plants, for pulp and paper wastewaters (Kumar and Chopra, 2016).Recently phytoremediation has been implemented and reported for use in removing pollutant load from paper and pulp industries (Kumar et al., 2020a, b, c).However, although phytoremediation technology has shown enormous promise, large scale field studies are scant (Beans, 2017).
Further research is also needed to ascertain the fate of various compounds in the plant metabolic cycle to ensure that no toxic or hazardous chemicals contribute to the food chain.Also, the disposal of harvested plants can be bothersome if they have elevated heavy metal levels.The application of phytoremediation is typically restricted to areas with low contaminant levels and contamination of shallow soils, streams, and groundwater.

The core advantages of bioremediation
▪ It is a natural process, time-saving, as a waste treatment method suitable for harmful content.Microbes are capable of pollutant degradation, and when the microbe has been properly selected, the number of microbes will be rising when there is a pollutant.The treatment residues are usually harmless or less hazardous.The process avoids the transfer of waste off-site and potential hazards to the environment and human health that could arise during the catabolism of pollutants.▪ It may be implemented efficiently and effectively and is more costeffective than other conventional technologies used in hazardous waste mitigation.▪ It often contributes to mitigating the contaminants.Several hazardous compounds may be converted into harmless substances.This characteristic also eliminates potential liability for the treatment and disposal of contaminated materials.▪ No hazardous chemicals are used with this method.Added nutrients, especially fertilizers, activate and boost microbial growth.Bioremediation transforms dangerous substances into harmless substances and gasses.▪ Pollutants are destroyed, not just transferred to various environmental media.▪ Non-intrusive, which may allow continued use of the site.▪ Relatively easy to implement.

The key disadvantages of bioremediation
▪ Not all hazardous substances can be degraded quickly and completely ▪ There are some issues regarding the persistent or toxicity of biodegradation products over the relative compound.▪ Unique biological processes are often crucial site factors.The existence of microbial communities needed for achievement in suitable environmental growth conditions and adequate nutrient and contaminant levels.▪ From bench and pilot studies, it is difficult to extrapolate into fullfield operation.▪ Research is necessary to develop and create technologies for the bioremediation of sites with complicated mixtures of pollutants, not similarly distributed in the environment.Solids, fluids, and gasses can be contaminants.▪ It is often slower than other treatment alternatives.▪ Uncertainty in the regulatory framework persists concerning acceptable bioremediation performance criteria.No accepted definition of "clean" is given.It is challenging to evaluate bioremediation performance.▪ No complete information can be obtained, in terms of toxicity and environmental fate of all transformed compounds, that is a significant flaw in bioremediation.Exploratory and preliminary investigative studies have been been implemented by in silico approaches in bioremediation (described in Section 7.1).

Lignin-modifying enzymes for sustainable mitigation of lignin, phenolics, and a wide variety of pollutants
Microbially derived lignin modifying enzymes have been studied extensively for their catalytic potential concerning the removal of pollutants.As already indicated in the introduction, the enzymes include laccases, LiP, MnP, VP, and dye decolorizing peroxidase.Based on the catalytic mechanism, it is noted that these enzymes can be classified into two categories: 1. laccases that use O 2 as oxidant (electron acceptor) in the enzyme-catalyzed reaction, not requiring H 2 O 2 for reaction and 2. peroxidases, which are H 2 O 2 dependent.
The LiPs are involved in the oxidative degradation of lignin by catalyzing bond breakage, and may also degrade lignin-derived oligomers, including non-phenolic and phenolic compounds (Lundell et al., 2010;Qi-He et al., 2011).
LiP, VP, and DyP act on diols and sulfonic substituents on benzene rings and non-phenolic lignin compounds, whereas laccase and MnP can catalyze the oxidation of available phenolic components of lignin directly, but laccases cannot directly catalyze bond breakage in lignin unless mediator compounds are present (Munk et al., 2015).Bacterial laccases and peroxidases are structurally different from the most common fungal counterparts, and the fungal-derived laccases and peroxidases also appear to be more studied than the bacterial ones.However, a few bacterial species (Firmicutes, Gamma proteobacteria, and Actinobacteria, as well as Rhodococcus and Streptomyces) express peroxide-dependent peroxidases, including DyP enzyme types (de Gonzalo et al., 2016).DyPs have been found to oxidize a range of diverse substrates, including lignin, lignin-derived compounds, phenolic compounds, and various synthetic dyes (Brissos et al., 2017;Colpa et al., 2014); and an engineered variant (with an asparagine-to-alanine substitution (N246A) of the Rhodococcus jostii bacterial dye decolorizing peroxidase is able to efficiently catalyze the transformation of hardwood Kraft lignin substances (Singh et al., 2013;He et al., 2017).Contemplation of the data indicate that among the lignin modifying enzymes, laccase and LiP appear to be the most promising biocatalysts for wastewater remediation, but DyP may also have bioremediation potential due to its broad substrate use but has been less studied.These enzymes act broadly on non-phenolic lignin compounds, various dyes, and several endocrine disputing compounds (Blánquez et al., 2019;Falad et al., 2017;Pramanik and Chaudhuri, 2018;Wang et al., 2018).A comparative catalytic pattern of lignin modifying enzymes is shown in Fig. 6.

Plant based peroxidases for pollutants remediation
Peroxidases from plants have received some interest in the past for their ability to eliminate phenolic pollutants from synthetic and industrial wastewater (González et al., 2008;Kurnik et al., 2015).Plant peroxidases belong class III peroxidases, and their catalytic mechanism is similar to that of standard peroxidases (Chagas et al., 2015).The plant peroxidases include soybean peroxidase, horseradish peroxidase, and turnip peroxidase (Qayyum et al., 2009).Horseradish peroxidase was previously considered a most promising plant peroxidase candidate for wastewater contaminated with phenolic compounds (Ashraf and Husain, 2010), but appears to be overtaken by other peroxidases (and microbially produced laccases).Enzyme immobilization has been explored to improve enzyme robustness and biocatalytic efficiency: Akhtar and Husain used various immobilized plant-derived peroxidases for removal of phenolic pollutants (Akhtar and Husain, 2006).Qayyum et al. (2009) reported enzyme-catalyzed degradation and transformation of numerous PAHs, PCBs, organochlorines, phenolic compounds, and several dyes with immobilized plant peroxidase (Qayyum et al., 2009).Kurnik et al. (2015) performed a similar study and reported a high phenol decontamination potential in industrial and synthetic wastewaters (95% phenol removal efficiency) of a potato pulp-derived peroxidase (Kurnik et al., 2015).Nevertheless, the application of plant derived enzymes appears to have been overtaken by microbially produced enzymes that can be recombinantly produced easier and cheaper than plant peroxidases, although certain plant peroxidase have been reported recently for efficient removal of phenols (see Section 6.3).

Microbial biodegradation of lignin and its derivatives
High molecular weight (HMW) technical lignin is present in the paper industry's wastewater as a contaminant (Baghel et al., 2020).A complex chemical structure, composed of stable biological bonding, is resistant to microbial degradation (Chen et al., 2012).However, several microorganisms with lignin degradation potential, including fungi, actinomycetes, and bacteria, have been found in nature (Janusz et al., 2017;Kirby, 2005).White-rot and brown-rot fungi have been thoroughly studied for lignin degradation over the past few decades (Kirk and Farrell, 1987).
As persistent organic contaminants, both lignin and lignin-derived compounds can persist in the environment and trigger environmental hazards.Lignin-based pollutants in wastewater from the paper & pulp industry are also the primary source of pollution.It should also be minimized to eliminate the risks (Haq et al., 2017;Vashi et al., 2018).Enzymatic lignin degradation has been mostly investigated with different lignin model compounds rather than natural lignin and its components.
Certain lignin modifying peroxidases may act on organic pollutants by cleaving of β-O-4 (ether linkage), generating free radicals, using one-electron oxidation.Generated cation radicals can spontaneously undergo chemical reactions, including hydroxylation or C\ \C cleavage, leading to hydrophilic products (Bosque et al., 2017).

Biocatalytic biodegradation of phenolic compounds
The lignin modifying enzymes or peroxidases have been reported for the bioremediation of phenolic contaminated wastewater (phenols, cresol, and chlorinated phenols), emerged from industrial processing (Ong et al., 2011).Peroxidases, including LiP and horseradish peroxidase, have been reported as efficient tools for transforming pentachlorophenol at different concentrations in the presence of H 2 O 2 (Kim et al., 2006;Zhang et al., 2007).MnP is an extracellular enzyme produced by various microorganisms and its ability to eliminate phenolic compounds by oxidative catalysis relies on a mechanism that involves oxidation of Mn 2+ into Mn 3+ using one electron, and H 2 O 2 as cosubstrate, hence its name (Hakala et al., 2006).In recent years, various plant peroxidase mediated detoxification of phenol from synthetic and industrial wastewater has been reported in laboratory studies.Various plant peroxidases including, turnip, potato, soybean, gourd, rapeseed, etc. have been reported for the elimination of a wide variety of phenols and phenolic compounds, including 2,4-dichlorophenol, guaiacol, mcresol, p-cresol, o-cresol, anisole, resorcinol, catechol, pyrogallol, hydroquinone, and veratryl alcohol (Kurnik et al., 2015;Kurnik et al., 2018).

Biodegradation of endocrine-disrupting chemicals
Discharge of wastewater from the paper sector often contained numerous phenolic derivatives, and endocrine-disrupting chemicals (EDCs) reported as harmful in several respects to human health.EDCs are the chemical compounds that interact with the endocrine system, impacting developmental, reproductive, and neurological by causing negative effects in humans and animals (Cooke et al., 2013).Different techniques are involved and reported to eliminate EDCs from wastewater exploiting enzymatic treatment with microbially derived laccase and peroxidase (Falade et al., 2018;Grelska and Noszczyńska, 2020).However, it has been documented that traditional wastewater treatment is less effective in removing such EDCs; meanwhile, peroxidases have the potential for efficient removal of EDCs (Zheng and Colosi, 2011).Their substrate versatility can catalyze reactions that eliminate or reduce major environmental pollutants, such as chloroanilines and aromatic polycyclic hydrocarbons from various sources (Harayama, 1997).The assessment of the removal of phenolic EDCs has been reported in recently reported studies exploiting plant peroxidase.Among such studies, several phenolic compounds bisphenol-A (BPA), 2,4-dichlorophenol (2,4-DCP), 4-tert-octylphenol (4-t-OP), and pentachlorophenol (PCP) were successfully removed, exploiting plant peroxidase (Reis et al., 2014).The subsequent experiments of the above studies showed that a significant amount of EDCs were removed by using plant-based peroxidase (POs) assisted treatment (Reis et al., 2014).In a similar study, few selected endocrine-disrupting chemicals, including bisphenol-A, along with some estrogenic compounds (estrone (E1), 17β-estradiol, and 17α-ethinylestradiol) from real and synthetic wastewater were removed exploiting VP by use of a two-stage system (Taboada-Puig et al., 2015).The degradation rates of EDCs (estrone, 17β-estradiol, and 17α-ethinylestradiol) were reported to be in the range of 28-58 μg/(L•min) (Taboada-Puig et al., 2015).

Recent advances in bioremediation for pollutant remediation
Bioremediation provides an effective, eco-friendly, and effective way to combat wastewater pollution concerns, including the discharge of phenolics and lignin-derived pollutants to the environment (Hlihor et al., 2017).Several microbial species, including bacteria and fungi, have shown their potential in bioremediation of relevant pollutants by secretion of potential lignin modifying enzymes, and bacterialmediated bioremediation has long proven an effective and low-cost sustainable mitigation strategy for removal of pollutants from wastewaters (Raj et al., 2014).Several bacterial species, including; Pseudomonas sp.Pycnoporus, Agrobacterium, Bacillus, Rhodocococcis erythropolis, Rhizobium sp. have thus been reported useful for biodegradation of pollutants from the environment (Kang, 2014;Kumar et al., 2013;Urgun-Demirtas et al., 2006).However, the field trials have not always been successful, pointing at the need for further development.
In order to maximize the degradability and enhanced degradation rate of bioremediation approaches, there has recently been substantial progress via the use of advanced biotechnology methods, including the development of genetically modified organisms, metabolic engineering, and e.g.use of in silico modeling and prediction approaches and protein engineering of enzymes to target and enhance efficiency and robustness (Aukema et al., 2017;Gauchotte-Lindsay et al., 2019).In the recent few years, genetically modified organisms have been constructed to increase the degradability of pollutants for the bioremediation of numerous carcinogenic, phenolic substances, heavy metals, and so on (Dvořák et al., 2017;Liu et al., 2019;Rucká et al., 2017;Sanghvi et al., 2020;Tay et al., 2017).In addition to genetically modified (or engineered) microorganisms, it deserves to be mentioned that other innovative techniques are also currently being used for the clean-up of pollutants and restoration of ecological niches.These techniques include, for example, photocatalysis and advanced oxidation processes (AOPs) that have been promoted for different organic pollutant removal from wastewater (Carbajo et al., 2017;Poulopoulos et al., 2019).
7.1.In silico approaches, molecular modeling, and degradation pathway prediction In silico bioremediation, the approach integrates various computational techniques, including molecular docking, molecular dynamics simulation, homology modeling, degradation pathways predictions, etc. (Singh et al., 2020a;Singh et al., 2021a, b).Conventional bioremediation techniques are quite a time talking and long-term strategies for remediating various phenolic, lignin-based pollutants.Sometimes traditional remediation techniques based on oxidoreductase may fail to perform mitigation of organic pollutants effectively.Extrinsic factors, i.e., temperature, pH, nutrition, etc. are responsible for failure or slower degradability.Without knowledge of complete bio-transformed compounds from bioremediation, the environmental fate and toxicity profile of each transformed compound cannot always be determined by conventional bioremediation (Sanghvi et al., 2020).In silico bioremediation approaches exploit and rely on various fields, including genomics, computational biology; proteomics; bioinformatics; molecular modeling, molecular dynamics simulation, and a specialized algorithm for pathways prediction (Karp et al., 2011;Kleinman et al., 2014;Parenty et al., 2013).Such integrated techniques provide a quick insight into enzyme and pollutants for their binding, catalysis, and degradation mechanism.Such advances are highly useful to the virtual screening of concern pollutants, especially when conventional bioremediation fails to perform.In silico toxicology, part of predictive bioremediation provides quick toxicity endpoints prediction in a sophisticated and timesaving manner, which cannot be accomplished through wet lab toxicity assays (Raies and Bajic, 2016;Rim, 2020;Singh et al., 2020a, b).In silico bioremediation approach is known for its potential as it is performed on specialized computer systems; however, this approach simplifies the problem to be further conducted in vitro and in vivo assays and makes it easier than traditional bioremediation (Aukema et al., 2017).During catalysis, the actions of catalytic enzymes (oxidoreductases) can be modeled and visualized through molecular dynamics simulation.This technology can help predict the catalytic potential and binding pattern of pollutants to enzyme proteins, and thus predict the potential for enzymatic biodegradation (Chen et al., 2011).Besides, the amino acids involved in the catalytic mechanism in the active site of the enzyme, the molecular basis for activity, the binding mode of diverse substrates, and even quantitative structure-activity relations can be predicted through molecular docking (Liu et al., 2018;Mehra et al., 2018a;Mehra et al., 2018b), and such predictions can therefore serve to target rational engineering of new enzymes for bioremediation of specific substrates and hence pollutants (Liu et al., 2018;Mehra et al., 2018b).Molecular docking has indeed been applied to screen of enzyme catalytic binding modes and enzyme-substrate affinities for laccase to different lignin-derived phenolics (Mehra et al., 2018a;Mehra et al., 2018b) and was recently used in relation to remediation of industrial textile dyes (Srinivasan et al., 2019).
Degradation pathway prediction is another key computational approach that can be exploited for predictive bioremediation or in silico bioremediation approach (Karp et al., 2011;Riaz et al., 2020).Using the PathPred pathway prediction tool/server developed by Kenehisa and coworkers (Moriya et al., 2010), environmental fate and degradation pathways can be predicted for a series of target and enzymatically transformed compounds, and such pathway degradation modeling can help predict the environmental fate and potential toxicity profile of environmental pollutant chemicals (Moriya et al., 2010;Wackett, 2013).The pathways prediction uses the biochemical reactions information already stored in the server, based on pre-existed available knowledge from literature and databases (Moriya et al., 2010).The in silico approaches for bioremediation of organic pollutants are important novel techniques, which can help develop novel bioremediation strategies and even help select the most optimal enzymes for enhanced bioremediation.Fig. 7 outlines the in silico-based bioremediation approaches to sustainable mitigation of environmental contaminants.

Protein engineering approaches
As already mentioned above, innate or natural bacterial-mediated bioremediation processes for organic pollutants are effective strategies for cleaning-up industrial pollutants from the environment (Benjamin et al., 2019).Intrinsic and genetically engineered microorganisms can indeed mitigate such contaminants efficiently and quickly (Sayler and Ripp, 2000).Wild type biocatalysts may require stabilization or, as mentioned above, molecular improvement to target specific problematic compounds to be efficient for practical bioremediation (Lendvay et al., 2003;Liu et al., 2019).Although stabilization may be accomplished by physical immobilization (as e.g.used by Zdarta et al., 2019), molecular evolution or protein engineering provides an innovative opportunity to apply novel and sophisticated biological materials to mitigate pollution (Singh et al., 2008).Until now, significant efforts have been directed at identifying, documenting, and understanding the catalytic capabilities and robustness of laccases and lignin modifying peroxidases in relation to degradation of specific compounds (including pollutants) (Kallio et al., 2011;Kalyani et al., 2016;Karigar and Rao, 2011;Kim et al., 2006;Mehra et al., 2018a, Mehra et al., 2018b;Miki et al., 2011;Min et al., 2015;Sáez-Jiménez et al., 2016;Santos et al., 2014;Singh et al., 2013).Protein engineering of enzymes is widely used in a range of processing industries to enhance e.g. the activity, robustness, or inhibitor resistance of enzymes.At this point, it is uncertain if protein engineered enzymes have been used in practice for large scale pollutant bioremediation, but in agreement with the perspectives outlined in a recent review (Mishra et al., 2020) we foresee an increased emphasis on protein engineering in the near future to develop efficient biocatalysts for pollutant elimination.

Genetically engineered microbes (GMOs)
Environmental management of environmental contamination by microorganisms is a feasible strategy to ensure environmental sustainability (Pieper and Reineke, 2000).Even though the natural or intrinsic bacterial species are best known for performing bioremediation processes to clean up various pollutants (PAHs, PCBs, pesticides, and dyes), either ex situ or in situ (Leigh et al., 2006;Sachan et al., 2019;Wang et al., 2018).However, the restoring rate to balance the perfect environment from natural bacterial species is relatively slow.Nearly every day, the pollutant loads are growing significantly from paper industries and other pollution emitting industries.Thus, GMOs technology currently performed for pollutants of interest has been used to mitigate all these concerns (Singh et al., 2011).Microbial-based bioremediation has received considerable global attention to reducing pollution due to environmental-friendly, global acceptance, and reduced health hazards and genetically modified bacteria are gaining increasing attention for pollutant reduction in the field of environmental restructuring (Saxena et al., 2020).For GMO's development, four basic principle approaches have been proposed concerning the application in bioremediation (Jariyal et al., 2020): 1) modification of enzyme specificity and affinity (see the previous section); 2) pathway construction and regulation (as already outlined above); 3) bioprocess development, monitoring, and control; and 4) bioaffinity bioreporter sensor applications for chemical sensing, toxicity reduction, and end analysis (Jariyal et al., 2020).Nevertheless, there is some concern that certain genetically engineered microorganisms are being introduced into the environment, implying further delaying field-testing of these organisms until safety and environmental issues can be addressed.

Gene editing CRISPR aided approaches in bioremediation
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) is a newly identified gene-editing technique for cells and bacteria.Numerous bacteria have been edited for their gene/genome for diverse applications (Behler et al., 2018;Donohoue et al., 2018;Liu, 2020;Tapscott et al., 2019).Due to its high degree of flexibility and accuracy in DNA cutting and pasting, it is now the globally accepted and widely used technique for editing genes of interest.Gene editing has thus drawn significant attention in the scientific community due to its extraordinary capabilities.This approach (CRISPR-Cas9) is thus being applied in agriculture and human health for a vast number of applications, and genome-edited microorganisms are being introduced in several sectors, even for designing edible probiotic microbes (Yadav et al., 2018) and has recently been considered used in bioremediation (Jaiswal et al., 2019).In addition to applying CRISPR to microorganisms, plants may also be genome edited for improved tolerance of organic and inorganic pollutants in the environment (Saxena et al., 2020).Several plant genomes have already been edited for applications/phytoremediation of possible hazardous contamination mitigation (Basharat et al., 2018;Bortesi and Fischer, 2015;Cherian and Oliveira, 2005;Yin et al., 2017;Zaidi et al., 2017).CRISPR technology could thus strengthen the natural process of bioremediation without high risks and costs (Cheng et al., 2015;Khorsandi et al., 2018;Huang et al., 2019;Yadav et al., 2018).In a recently published review, Basharat et al. (2018) reported an interesting vision of a futuristic phytoremediation scenario based on CRISPR-mediated genome reprogramming of plants.In line with this vision, we foresee that significant breakthroughs will be achieved on the bioremediation front not only using CRISPR-aided engineering but exploiting the selectivity and sustainability of biobased approaches in general for the safe removal of pollutants from industrial wastewater.

Future perspectives
Although paper production relies on the use of renewable feedstock material, the current processing practices create significant amounts of wastewater that contains toxic pollutants that need to be removed for environmental and human health reasons.
The pulping and bleaching processes are the key steps wherein the chemical conversion of the lignocellulosic material process occurs, notably leading to modification and transformation of the chemical composition of the lignin that is removed to manufacture the cellulose-rich paper.During such operations, technical lignin is abundantly produced as a by-product.Such technical lignin and several toxic compounds pose a negative impact on the environment.The paper industry's byproducts have been recognized as; lignin in different forms (Kraft lignin, soda lignin, organosolv lignin, hydrolyzed lignin, sulfonated lignin), phenolics, chlorinated, and several EDCs.Kraft lignin is the most frequent and abundant type of technical lignin resulting from large scale pulp and paper processing.Scientific studies have shown that Kraft lignin is a potentially toxic agent, as confirmed by various toxicology assays, and genotoxicity and cytotoxicity assessments using cell lines.In addition to the toxicity of Kraft lignin, certain toxic compounds like chloro-lignins, sulfonated compounds, phenolics, and EDCs in wastewater inflict harm to the environment.Many scientific studies have reported that hazardous compounds (dioxin, furan, chlorinated, and phenolic compounds) are also prevalent in wastewater from paper and pulp processing.These compounds trigger human endocrine disorders and may induce developmental deformities in the developing embryo.Bioremediation and more recently biocatalytic and enzyme-assisted pollutants removal appear promising.Implementation of newly emerged technologies, integration of omics, and computational biology could be addressed the existing concerns and fill the gap for bioremediation.Suggested implementation further may provide a direct look at the pollutant removal obstacles.The current critical analysis has revealed the current status and practices in bioremediation processes, but there are still many obstacles that could be reduced by taking the following points into consideration: 1. Enhancing the production yields of the relevant bacterial or fungal-derived enzymes, current costs of enzyme production and low availability is still a challenge for large scale implementation of enzyme-based bioremediation.
2. Existing lignin-modifying strains have certain limitations and sometimes fail to eliminate the target pollutant compounds.In the case of chlorinated lignin, endocrine-disrupting chemicals, the wild type lignin-modifying microbial strain may not eliminate the compounds sufficiently fast or completelythe compounds are indeed toxic, so the survival and robustness of the microbes may be a barrier.GMOs for different pollutants degradation pathways associated with gene integrations can be constructed to simplify the current multiple pollutant and enzymatic selectivity, and/or microbial bioremediation may be combined with targeted enzymatic pollutant removal strategies.3. Available conventional bioremediation techniques are cost-effective but time-consuming.Enzymes can be engineered to efficiently convert target compounds and plant and microbes may be designed to integrate different genes that promote certain pathways of degradation and then eliminate different problematic pollutants, including chlorophenols and EDCs.Targeted bioengineering for bioconversions could be accelerated using novel biotechnologies such as predictive molecular modeling, protein engineering for accelerated molecular protein evolution of enzymes, cloning, and genome editing (CRISPR).These types of advanced biotechnology approaches are expected to significantly impact bioremediation and also help understand how the biological systems respond to pollutants. 4. Establishing effective biobased remediation of pollutants may be expedited by using targeted in silico approaches.The key in silico methods include molecular docking, molecular dynamics simulations, degradation pathways predictions, and predictive toxicology.The use of these techniques can also advance the understanding of the environmental fate and potential toxicology of the converted degradation compounds.Therefore, integration of predictive toxicology, predictive bioremediation tools/servers is recommended for achieving targeted bioengineering and maximum degradation.Consequently, sustainable, eco-friendly mitigation strategies, including targeted enzyme-based clean-up approaches could be introduced faster to improve environmental protection and avoid healthhazardous compounds to be spread.

Concluding remarks and research trends
Lignin derivatives and chlorinated phenolic compounds are the most prevalent contaminants that end up in wastewater from paper production.The problematic compounds are mainly produced at the pulping and bleaching stages.Several of these compounds are acutely toxic having adverse effects on plants (being phytotoxic), the aquatic system and the overall ecosystem, and hence the environment.Some of the effluent compounds can moreover trigger mutagenic and genotoxic effects, and some of the polychlorinated compounds are even considered to be EDCs.To overcome those severe issues, several lignin modifying enzymes, laccases, and peroxidases such as LiP, MnP, VP, and not least the more recently described DyPs are eco-friendly natural biocatalysts, which could eliminate a broad range of these environmental contaminants, especially the phenolics.Other bioremediation approaches have also been examined, but to our knowledge, none of these are in use in practice at a very large scale.Newer computational biological pathway prediction methods combined with molecular biology and enzyme techniques such as CRISPR technology, protein engineering, and/or immobilization technologies for enzyme stabilization provide new powerful toolsets for developing efficient, sustainable, and robust enzymatic and biobased treatment methods.We anticipate and hope that these bioremediation technologies will be developed further to find a use for improved wastewater pollution control of high load wastewater emissions, such as those from the paper and pulp industry.

Declaration of competing interest
No conflict of interest exists as declared by the authors.
Novel bioremediation approaches for pollutant mitigation in wastewater • Oxidative catalytic potential of lignin modifying enzymes in wastewater treatment • Summary of forward-looking biotechnology tools to advance biobased remediation G R A P H I C A L A B S T R A C T a b s t r a c t a r t i c l e i n f o 1. Introduction

Fig. 1 .
Fig. 1.Schematic representation of a typical paper making process with emission of different pollutants from the paper industry.A: Raw material: Cellulose feedstock from plants.B: Different chemical process during paper manufacturing: Major processes Kraft pulping and bleaching.Black liquor is the major fluid, and it contains diverse chlorinated, phenolic, and complex pollutants, including potential EDCs.Lignin-modifying enzymes may help eliminate the pollutants in an eco-friendly manner.

Fig. 2 .
Fig. 2. Graphical representation of lignin and its different aspects.A: 2D depiction of three primary lignin polymer-forming unit (monomer; G; H; S units).B: Percentage of monomer units in a different types of lignin based on plant origin.C: Lignin polymer including respective percentage and constructive linkages.

Fig. 3 .
Fig.3.Negative environmental consequences and hazardous impacts on human health from pollutants from wastewaters.Certain pollutants can trigger undesirable endocrine effects and/ or cause human developmental deformities.

Fig. 5 .
Fig. 5. Explanatory illustration of bioremediation processes via use of different advanced bioremediation technologies: microbial, biostimulation, bioaugmentation, and phytoremediation.Hazardous compounds from wastewaters can be treated by employing all described techniques.The lignin-modifying enzymes can be used in combination with microbially mediated bioremediation.

Fig. 7 .
Fig. 7. Illustrative explanation of the in silico approach in bioremediation.Expert systems or algorithm mediated processes use for prediction of degradation pathways, environmental fate, and toxicity of concern pollutants with different toxicological endpoints.Other techniques used in predictive bioremediation include; molecular docking, molecular dynamics simulation, pathways prediction, and predictive toxicity.The predictions can help to plan remediation measures by understanding the fate and degradation paths of concern pollutants.
Table includes the individual contaminant compounds, their chemical properties and known toxicity or adverse effects.

Table 2
Detailed potential lignin-modifying enzyme producers acting on different substrates, incl.model lignin compounds and industrial dyes.

Table 3
Overview of chemical structures and elementary attributes of primary lignin monomers (p-coumaryl alcohol, coniferyl alcohol, sinapyl alcohol) and lignin model compounds.

Table 4
Molecular properties of lignin-modifying enzymes, based on a representative PDB ID for each type of enzyme: for each enzyme type, the number of constituent amino acid residues, molecular weight, and co-factor metal type are given.