Bioremediation of pulp and paper mill effluent by Phanerochaete chrysosporium

1.5.Bioremediation- An emerging Biotechnology for Environmental Clean Up

Biotechnology has emerged as an applied discipline of biological research with its applications in Agriculture, forestry, Industry as well as Medicine. An equally important area of global concern that has emerged only recently is environmental applications of biotechnology. In today’s condition where the industrialization is spreading its wings and the increasing population needs more and more requirements of Food and allied substances leading to more and more degradation of environment through the various pollutants. The main problem is due to the pollutants which are non biodegradable, which are resistant to biodegradation (recalcitrant) leads to the problems of Biomagnifications (P.D. Sharma : Environment and Ecology).The new opportunities were provided by the biotechnology for the cleanup of these pollutants through the use of microorganisms either in their natural form or by genetically modifying them. Hence a new Branch of biotechnology called as Environmental Biotechnology has emerged as a ray of light for pollution control. 

1.5.1. Various Definitions of Bioremediation:

1) "Remediate" means to solve a problem, and "bio-remediate" means to use biological organisms to solve an environmental problem. (ei.cornell.edu/student/glossary.asp)

2) The use of living organisms (e.g., bacteria) to clean up oil spills or remove other pollutants from soil, water, and wastewater, use of organisms such as non-harmful insects to remove agricultural pests or counteract diseases of trees, plants, and garden soil.
www.nsc.org/ehc/glossary.htm

3) A natural process in which environmental problems are solved by the use of bacteria or other microorganisms that break down a problem substance, such as oil, into harmless molecules.(www.deh.gov.au/industry/biotechnology/glossary.html)

4) The use of biological organisms such as plants or microbes to aid in removing hazardous substances from an area. (Source: Human Genome ProjectInformation
www.genomecanada.ca/GCglossaire/glossaire/index.asp)

5) Use of microorganisms to remove or detoxify toxic or unwanted chemicals from an environment. (library.thinkquest.org/3564/glossary/b.htm)

6) Use of biological organisms to remove or detoxify pollutants from a contaminated area.
(highered.mcgraw-hill.com/sites/0070294267/student_view0/glossary_a-d.html)

7)   The use of biological systems, usually microorganisms, to clean up a contaminated            site (www.biotechnology.vic.gov.au/info/glossary.asp).

8)  Simply, the use of biological techniques to clean up pollution. More specifically, the use of specialized, naturally-occurring micro-organisms with unique biological characteristics, appetites, and metabolisms as a form of waste cleanup. A critical underpinning of this process is the ability to economically generate a sufficient biomass of the appropriate microbes to accomplish in weeks or months what would normally take nature years to do. Typically, this is done either by applying a sufficient concentration of such microbes directly to the polluted area or by applying various concentrations of chemicals which, in turn, stimulate and foster the rapid growth (www.battle-creek.net/glossaryb.html)

            9) Exploiting the metabolic activity of microorganisms to transform or destroy contaminants.(hydram.epfl.ch/e-drologie/english/mod_0/glossary.htm)

10) The remediation or decontamination of any contaminated matter by the use of processesinvolving biological organisms.
(www.epa.nsw.gov.au/waste/envguidlns/compostingglossary.htm)

11) Bioremediation can be defined as any process that uses microorganisms or their enzymes to return the environment altered by contaminants to its original condition. Bioremediation may be employed in order to attack specific contaminants, such as chlorinated pesticides that are degraded by bacteria, or a more general approach may be taken, such as oil spills that are broken down using multiple techniques including the addition of fertilizer to facilitate the decomposition of crude oil by bacteria. (en.wikipedia.org/wiki/Bioremediation)

1.5.2. Principles of Bioremediation:

            Bioremediation is based on the idea that all the microorganisms remove substances form the environment to carry out growth and metabolism .Bacteria, Protista and Fungi are very good at degrading complex molecules and incorporating the breakdown products into their metabolisms. The resultant metabolic wastes that they produce are generally safe and somehow recycled into the other organisms. Fungi are especially good at digesting complex organic compounds that are normally not degraded by other organisms. The ability to degrade a pollutant is dependant on enzymes produced by organisms.

            Bioremediation does not involve only the degradation of pollutants. Sometimes it is sufficient to remove the pollutant from the environment without degrading it. Bacteria in particular take up large amounts of metals and minerals to ensure adequate recourses fro binary fission. The fungi act on the pollutants by mean of the metabolic enzymes which either modify the pollutant or completely degrade the pollutants like lignin and other higher molecular compounds.

 1.5.3. Types of Bioremediation:

            The bioremediation is the process by the degradation of polluting compounds occurs as a result of biochemical activity of microorganisms. Two options are available to utilize the biodegradative potential of microorganisms in polluted environment. These are:

1)      In situ Bioremediation: In this type the activity of microorganisms already presents in particular environment is targeted. More effective biodegradation results from enhanced activity of such microbes either by increasing there activity, e.g. by the addition of suitable additional nutrients which were otherwise limiting their activity and or by increasing their number so that there activity can be more effectively manifested in the particular system(Mason et al.,1992)

2)      Ex Situ Bioremediation: In this type specific microorganisms which possess a known specific degradative or other potential activity are introduced into the affected environment. The basis of this technique is to target the contaminating chemical pollutant with a microorganism able to degrade it. The strategy of introducing microorganisms into the environments alien to those where they are usually found is surprisingly common in environmental biology and parallel can be seen for example, in use of biological pest control agents, in composting, and in biofertilisers.

1.5.4. Technology of Bioremediation:

                        Bioremediation can be used to clean unwanted substances from air, soil, water and raw materials from industrial processing. Living organisms or just there enzymes can be used to accomplished this task. One unusual application involves the use of fungi to remove excess lignin from pulp and paper .Lignin is not one compound, but. a group of polyaromatic chemicals that harden wood. It is normally difficult to extract from pulp and ends up in a pollutant once it is removed. Most bioremediation technologies are designed to remove a pollutant once it is generated or released into the environment although some types of bioremediation remove chemicals before they become pollutants. Technologies using bioremediation treatment include bioaugmentation, biofilters, bioreactors, biostimulation, bioventing, compost and land farming. The technique used in the present investigation is the bioaugmentation due to the suitability in studying. The all mentioned above are:

1)      Bioaugmentation: This is the technique where the addition of microorganisms or enzymes to the material to remove unwanted chemicals. Bioaugmentation is used to remove byproducts from raw materials and potential pollutants from waste. Bacteria are the most common bioaugmentation organisms used, an attempt to use the fungi as a bioaugmentation organism was done.

2)       Biofilters: The removal of organic gases by passing air through compost or soil containing microorganisms capable of degrading the gases. It has been used to remove volatile organic compounds (VOC’s) from air.

3)      Bioreactors: The treatment of a contaminated substance in a large tank containing microorganisms or enzymes. Bioreactors are commonly used to remove toxic pollutants from solid waste and soil.

4)      Biostimulation: The use of nutrient or substrates to stimulant the naturally occurring organisms that can perform bioremediation. Fertilizers and growth supplements are the common stimulants. The presence of small amounts of the pollutant can also act as a stimulant by turning on operons for the bioremediation enzymes.

5)      Bioventing: This is similar to biostimulation. It involves the venting of oxygen through soil to stimulate the growth or natural and introduced bioremediation organisms. This is used predominantly for soils contaminated with petroleum products. It is not suitable for removing halogenated gases that contribute to ozone layer damage.

6)      Composting: This involves mixing contaminated materials with compost containing bioremediation organisms. The mixture incubates under aerobic and warm conditions. The resultant compost can be used as a soil augmentation or be placed in a sanitary landfill.

7)      LandFarming: The use of farming tilling and soil amendment technique to encourage the growth if bioremediation organisms in a contaminated area. It has been used successfully to remove large petroleum spills in soil.(http://www.accessexcellence.org/LC/SC.st3bg.html)

1.5.5. Fungi in Bioremediation: Fungi are good in the accumulation of heavy metals such as cadmium, copper, mercury, lead and zinc. Systems using Rhizopus arrhizus have been developed for treating uranium and thorium (Teen-Seers et al., 1984).

             The ability of fungi to transform a wide variety of hazardous chemicals has aroused interest in using them in bioremediation.  (Alexander, 1994).The white rot fungi are unique among   eukaryotes for having evolved nonspecific methods for the degradation of lignin; curiously they do not use lignin as a carbon source for their growth (Kirk et al., 1976). Lignin degradation is, therefore, essentially a secondary metabolic process, not required for the main growth process. Lamar et al. (1993) compared the abilities of three lignin-degrading fungi, Phanerochaete chrysosporium, P. sordida and Tramates hirsuta to degrade PCP (Pentachlorophenyl) and creosote in soil. Inoculation of soil with 10% (wt/wt) Phanerochaete sordida resulted in the greatest decrease of PCP and creosote.  P. sordida was also most useful in the degradation of PAHs (Polycyclic aromatic hydrocarbons) from soil. Davis et al. (1993) showed that P. sordida was capable of degrading efficiently the three ring PAHs, but less efficiently the four-ring PAHs.

                  Phanerochaete chrysosporium  has been shown to degrade a number of toxic xenobiotics   such  as aromatic hydrocarbons ( Benzo alpha pyrene, Phenanthrene, Pyrene) chlorinated organics (Alkyl halide insecticides,Chloroanilines, DDT, Pentachlorophenols, Trichlorophenol, Polychlorinated biphenyls, Trichlorophenoxyacetic acid),  nitrogen aromatics ( 2,4-Dinitrotoluene, 2,4,6-Trinitrotoluene-TNT) and several miscellaneous compounds such as sulfonated azodyes. Several enzymes which are released such as laccases, polyphenol oxidases, lignin peroxidases etc. play a role in the degradative process. In addition, a variety of intracellular enzymes such as  reductases, methyl transferases and cytochrome oxygenases are known to play a role in xenobiotic degradation (Barr & Aust, 1994).

         Phanerochaete chrysosporium  has been shown to  effect the bioleaching of  organic dyes ( Nigam et al. ,1995). Pauli ollikka et al. (1993) have also shown the decolorization of azo-triphenyl methane dyes by lignin peroxidase produced by  P. chrysosporium. Sami and Radhaune (1995) have demonstrated the role of  lignin peroxidase and manganese peroxidase from  P. chrysosporium  in the decolorization of olive mill waste water.  The work carried out in our laboratory ( Asoka, Manjunath & Sullia, 2000; Asoka, Geetha & Sullia,2002 ) has shown that Phanerochaete chrysosporium and microbial consortia were effective in color removal from  textile dye effluents   The fungus caused  80% decolorization in  broth containing  2.5% of effluent. There was reduction in BOD and COD values. A local isolate of  Fusarium sp.  caused various degrees of decolorization ranging from 35 to 85 %.

            Among the fungal systems, Phanerochaete chrysosporium is emerging as the model system for bioremediation. The basidiomycetous fungus Pleurotus ostreatus  has been shown to produce an extracellular hydrogen peroxide dependent lignolytic enzyme which removes the color due to remozol brilliant blue.  Oxidative enzymes play a very major role in biodegradation. Other fungi which can be used in bioremediation are obviously the members of Zygomycetes e.g., the mcoraceous fungi and the arbuscular mycorrhizal fungi. Aquatic fungi and anaerobic fungi are the other candidates for bioremediation.

            Among other fungi used in bioremediation, the yeasts, e.g., Candida tropicalis, Saccharomyces cerevisiae, S. carlbergensis and Candida utilis are important in clearing industrial effluents of unwanted chemicals.  Agaricus bisporus and Lentinus oloides are important in lignocellulose decomposition.  Corius versicolor is important in cleaning up pulp and paper mill wastes. Consortia of fungi and bacteria (usually uncharacterised) are used in composting, the most useful waste disposal practice. Phenolic azo dyes have been shown to be oxidized by the enzyme laccase produced by Pyricularia oryzae( Chivukula and Renganathan, 1995).

1.5.6. Applications of Bioremediation:

1)      To remove hazardous substances from environment.

2)      To restore the groundwater quality.

3)      To generate biogas from waste.

4)      Applied to remediate contaminated soil.

5)      Remove the oil spill problem from petroleum refineries.

6)      For Degradation of chlorinated pesticides.

7)      For Decomposition of crude oil in refineries.

8)      To remove heavy metals like Cadmium and Mercury from environment.

9)      To degrade the naturally no biodegrading substances which are persistent in environment.

10)  To clean up the sites contaminated by the industrial pollutants like wastewater which is discharged into the stream, river.

1.5.7. Limitations of Bioremediation:

                        There are several limitations to bioremediation. One major limitation has to do with the nature of organisms. The removal of pollutants is not benevolent gesture. Rather, it is a strategy for survival. Most bioremediation organisms do there job under environmental conditions that suite their needs. Consequently, some type of environmental modification is needed to encourage the organisms to degrade or take up the pollutant at an acceptable rate. In many instances the organism must be presented with low levels of pollutant over a period of time. This induces the organisms to produce the metabolic pathways needed to digest the pollutant. When using bacteria and fungi, it is usually necessary to add fertiliser or oxygen to the material containing the pollutant. This can be disruptive to the other organisms when done in situ. In situations where simple compounds and metals are being taken up it is likely that these pollutants are at toxic levels fro the organisms. Overall, the organisms do not always live as well on the pollutant diet as on other nutrients  found more commonly in there environment. This is problematical when doing in situ remediation.

                        Two other limitations concern cost benefit ratios: Cost versus overall environmental impact. Neither the government nor industry wants to spend large amount of money to clean up pollution. Industry in particular likes to keep cost down. The impact of genetically engineered micro organisms is less understood.

1.6. Test Organism:

Name

Phanerochaete chrysosporium Burdsall 1974.

Synonyms

Phanerochaete macrocystidiata Hallenb 1978.
Sporotrichum pulverulentum Novobranova 1972.
Chrysosporium lignorum Bergman & Nilsson 1966.

Taxonomy

Phanerochaetaceae, Polyporales, Agaricomycetidae, Basidiomycetes, Basidiomycota, Eumycota.

Description

Resupinate basidiomycetes belonging to the family Corticiaceae typically form effused, very flat fruiting bodies that appear in nature as no more than a crust on the underside of a log. In the case of Phanerochaete chrysosporium , this crust comprises white to ochraceous-salmon colored basidiocarps often less than 0.25 mm thick that are interspersed over a thin mat of white monomitic hyphae. The effuse almost cobweb-like appearance of this crust frequently makes this species difficult to spot in its natural habitat. P. chrysosporium displays both homothallic and heterothallic-bipolar sexuality. Microscopic examination of 2-week mycelial mats typically reveals simple septate hyphae ranging from 3-9 µm in diameter with sparse to moderate branching as well as the presence of thick-walled terminal or intercalary chlamydospores 50-60 µm in diameter. The blastoconidia are round to ellipsoid in appearance, 6-9 µm in diameter and are borne by poorly differentiated branched conidiophores.

Ecology

Phanerochaete chrysosporium is a secondary decomposer of both hardwood and softwood branches/logs, and can be found in temperate forests throughout North America , Europe and Iran. This species is the best-studied representative of the white-rot fungi which are so named for their ability to degrade the lignin component in wood and effectively bleach out the brown pigmentation associated with this biopolymer. This ability has generated much interest in the pulp and paper industry as an environmentally benign alternative to the chemical bleaching of pulp. The lack of subtrate specificity of the peroxidase-based ligninolytic system of P. chrysosporium can be exploited for the bioremediation of numerous recalcitrant organic waste byproducts.

Interesting Features

• Unlike many other white-rot fungi, the ligninolytic system of Phanerochaete chrysosporium does not involve the participation of phenol oxidases such as laccase.
• The extracellular ligninolytic system of P. chrysosporium comprises a myriad of interesting enzymes and biochemical intermediates. These include: lignin peroxidases (LIP), manganese peroxidases (MNP), cellobiohydrolases, endoglucanases, β-glucosidases, glyoxal oxidase, xylanases, xylosidases, α-galactosidase, pyranose 2-oxidase, superoxide dismutases and mannose-6-phosphatases.
• Several publications describe the design and implementation of bioreactors for the large-scale production of the ligninolytic enzymes of P. chrysosporium.
• LIP and MNP enzymes isolated from P. chrysosporium are useful in the bioremediation of a wide variety of organic waste byproducts including: textile dyes polyethylene, pesticides and herbicides, dynamite, PAHs, dioxins and oil-contaminated soil.

1.6.1. Enzymes Secreted by Phanerochaete chrysosporium:

The basidiomycete, P. chrysosporium, belongs to the white rot class of wood-rotting fungi. It produces different extracellular enzymes involved in lignin degradation. The first extracelular enzyme discovered to depolymerize lignin and lignin-substructured compounds in vitro was produced by this organism. The enzyme has been described variously as ‘ligninase’, ‘diary/propane oxygenase’ and ‘lignin peroxidase’(LIP) (Aitken and Irvine, 1989). A second class of enzyme also produced by P.chrysosporium is manganese peroxidase(MNP). Because of the requirement of this fungus for divalent manganese in carrying out peroxidase reactions, this enzyme is known as manganese- dependent peroxidase or manganese peroxidases (MNP) (Aitken and Irvine, 1989). Manganese is known to catalyze several oxidation reactions important in lignin degradation, including decarboxylation and demeth(ox)ylation of aromatic substrates. It plays an important role in the degradation of phenol units and nonphenol units, acting together with lipids. In contrast, lignin peroxidase degrades only the non-phenol units and acts with the hydrogen peroxide (Hatakka, 2001). Lignase actually comprises a series of isoenzymic peroxidases. The need for manganese in the MNP enzyme results from the enzyme’s ability to catalyze the oxidation of Mn(II) to Mn(III) in the presence of Mn(III) stabilizing ligands. The resulting Mn(III) complexes can then oxidize the organic substrate (Hatakka, 2001). These enzyme systems are responsible for the aggressive decomposition of lignin by P. chrysosporium. Mnperoxidase can catalyze the oxidation of several aromatic dyes (Barr and Aust, 1994) and monoaromatic phenols, (Barr and Aust, 1994) but these reactions depend on the existence of specific reaction conditions which includes the presence of both divalent Manganese and certain types of buffers (pH and redox potentials) (Atlas and Bartha, 1998). P. chrysoporium has also been shown to mineralize a variety of recalcitrant aromatic pollutants. Liginase has been shown to catalyze limited oxidation of benzo-pyrene and other polycyclic aromatics, as well as a number of phenolic pollutants (Aitken and Irvine, 1987).

The fungus can degrade various other xenobiotics such as polyaromatic hydrocarbons and chlorinated aromatic compounds, and also pollutants, which are covalently bound to humic substances (Pointing, 2001). Humic substances consist of aromatic rings connected by flexible and rather long aliphatic chains (Tuomela, 2002).This structure is formed by oxidative ring opening lignin, loss of phenolic and methoxyl groups and an increase in carboxyl and carbonyl group. Humic substances are thus less aromatic and have fewer methoxyl and more carboxyl groups than lignin. During the degradation of xenobiotics, the white rot fungi often polymerize or convert substantial amounts of compounds to humic bound products. The white rot fungi technology is very different from other well-established methods of bioremediation (e.g. bacterial systems). The differences are primarily due to the unusual mechanisms which nature has provided them with and several advantages for pollutant degradation. One distinct advantage these fungi have over bacterial systems is that they do not require preconditioning to the particular pollutant. Bacteria usually must be pre-exposed to a pollutant to allow the enzymes that degrade the pollutant to be induced. The pollutant also must be in a significant concentration; otherwise, induction of enzyme synthesis cannot occur. Thus, there is a finite level to which bacteria can degrade pollutants. Also because the induction of the degrading enzyme is not dependent on the pollutant in the fungi, the pollutant can be degraded to a near non-detectable level. In contrast to the bacterial system, the degradative enzymes of white rot fungi are induced by nutrient limitation. Thus, cultivation of the white rot fungi on a nutrient-limited substrate, will initiate the process (Tuomela, 2002; Aust, 1995). The basidiomycete produce different other extracellular enzymes involved in pollutant degradation. They use a variety of mechanisms to accomplish the complete degradation of lignin and a wide variety of other environmental pollutants. The fungi secrete a family of peroxidases to catalyse both direct an indirect oxidation of chemicals. The peroxidases can also catalyze reductions using electron donors to generate reductive radicals. The general, biodegradative ability of this fungus is related to the ability of the fungus to degrade lignin. This ability, which is unique to the group of fungi, is thought to be dependent on the family of peroxidases they secrete (Tien and Kirk, 1984). Through this, they can solubilize highly polymeric substances more complex than lignin (Barr and Aust, 1987). The fungus that degrades lignin is also able to degrade xenobiotics because their enzyme system is unspecific due to the heterogeneous nature of the lignin polymer and because xenobiotics are often aromatic and thus resemble lignin

or its degradation products (Orth et al., 1994; Paszozynski and Crawford, 1995; Pointing, 2001). In general, white rot fungi have been found to degrade or oxidize polycyclic aromatic hydrocarbons, chlorinated organic compounds, polychlorinated biphenyls, nitrosubstituted compounds, fluorinated aromatic compounds munitions waste, such as trinitrotoluene (TNT), synthetic dyes, synthetic polymers (plastics) and are humic bound synthetic compounds (Fernando and Aust, 1994; Orth et al., 1994; Paszozynski and Crawford, 1995; Pointing, 2001). The lignin peroxidases are somewhat unique in that they have higher oxidation potentials than do most peroxidases (Aust, 1995; Mills et al., 1989). In this way these enzymes have somewhat greater range of chemicals that they can oxidize.