Abstract:
A method of treating wastewater sources such as those from pulp and paper mills which contain lignin involves adding white rot fungi in the presence of glucose. While glucose may be directly added to the wastewater source, it has been found to be more economical to reduce or substantially remove color from the wastewater by additionally adding brown rot fungi in the presence of cellulose and hemi-cellulose. The addition of bacteria has also been found helpful to improve the reduction in color, biological oxygen demand, as well as chemical oxygen demand in wastewater.

Description:
FIELD OF THE INVENTION 
     The present invention relates to the treatment of wastewater. More particularly, the present invention relates to lignin-degradation in wastewater, and in particular to the combined use of brown rot and white rot fungi to reduce color, Biological Oxygen Demand (BOD), and/or Chemical Oxygen Demand (COD) in wastewater, especially in industrial environments such as pulp and paper manufacture and processing wastewater. 
     BACKGROUND OF THE INVENTION 
     The source of color in pulp and paper wastewater is primarily lignin. Lignin is a sticky substance that binds the cell structures of plants together giving them strength, rigidity and hardness. Without lignin, trees and other vegetation would not have the necessary rigidity to stand erect. Lignin becomes soluble when wood pulp is processed with strong caustic and acidic chemical solutions. The lignin may be separated and removed in several washing processes. 
     Lignin is very difficult to biodegrade. It is a complex polymer consisting of phenylpropane units. The phenylpropane units are highly cross-linked, perhaps as many as 500 times. This complexity inhibits microbial degradation for most organisms except primarily white rot fungus. 
     White rot fungi are a classification of microorganisms that are able to degrade lignin. Their ability to degrade lignin is a result of their secretion of highly oxidative ligninonlytic enzymes consisting primarily of phenoloxidases. The phenoloxidase enzymes believed to be of importance in this process are manganese peroxidase, lignin peroxidase, manganese-independent-peroxidase and laccase. These enzymes break the phenylpropane links of lignin creating much smaller compounds. The smaller compounds are called degradation by-products and are easier for bacteria to use as a food source to further degrade into carbon dioxide and water. The peroxidase enzymes may also assist in degrading cellulose and hemi-cellulose, which are structurally more complex than cellulose. It is known that white rot fungi preferentially produce phenoloxideses over the other enzymes the fungi normally produce when in the presence of glucose. 
     Normally aerobic measures are undertaken to reduce colorants and biochemical oxygen demand (BOD) in wastewater. Aerobic technologies include trickling filter, activated sludge, rotating biological contactors, oxidation ditch, sequencing batch reactor and even controlled wetlands. By introducing air into the wastewater stream, pumps and other mechanical systems are required which consume significant amounts of electricity. The use of electricity obviously increases the costs of treating wastewater. 
     Accordingly, an anaerobic, or anaerobic-friendly type of technology is believed to be somewhat advantageous to reduce the costs associated with treating the wastewater. Anaerobic technologies currently available are high-rate systems including continuous-flow stirred tank reactors, contact reactors, upflow sludge blankets, anaerobic filters (upflow and downflow), expanded or fluidized bed and two-stage systems that separate the acid-forming and the methane-forming phases of the anaerobic process. In many prior art systems, aerobic and anaerobic processes are combined into a treatment system. Anaerobic treatment may be used for removing organic matter in high concentration streams, and aerobic treatment may be used on lower concentration streams or as a polishing step to further remove residual organic matter and nutrients from wastewater. 
     There are numerous problems and difficulties with the prior art systems. First, start up and restart problems can occur with the prior art processes. Secondly, biomass separation, sludge settlement, foaming and scum formation can occur. Odors may be associated with the treatment, excessive biomass may be generated, nitrogen and phosphorous may need to be removed, and any number of other problems have been found with the various prior art systems. 
     Accordingly, a need exists to provide a more effective color removing method for use with wastewater streams. 
     Another need exists for an anaerobic process for removing colorants and/or otherwise treating wastewater. 
     SUMMARY OF THE INVENTION 
     An object of the present invention is to provide an improved method of anaerobically treating wastewater. 
     Another object of the present invention is to provide a method for treating wastewater utilizing white rot and brown rot fungi. 
     Another object of the present invention is to provide a method for enhancing the ability for white rot fungi to selectively extract beneficial enzymes for use in clarifying wastewater streams. 
     Accordingly, since white rot fungi increases production of phenoloxydases when in the presence of glucose, a need exists to provide a source of glucose to the wastewater stream. Although dissolving sugar of various varieties directly into the wastewater stream has been tried and does work, the cost of the sugar can be relatively expensive. Accordingly, there is a need to provide an inexpensive source of glucose or other simple sugar for a significant population of white rot fungi to preferentially produce the desired enzymes while possibly inhibiting the production of others. 
     Pulp and paper wastewater environments typically comprise a facultative regime. Normally a sludge layer is located on the bottom of a wastewater lagoon. The sludge consists of unprocessed wood, particularly processed cellulose pulp, cellulose pulp, hemi-cellulose pulp and biomass. 
     When brown rot fungus is introduced into the wastewater stream in the presence of cellulose and/or hemi-cellulose, the brown rot fungus produces a network of roots called hyphae. Along the hyphae network, the fungus secretes digestive acids. The acids dissolve the sludge of cellulose and/or hemi-cellulose material breaking them down into absorbable food. The wood, cellulose and hemi-cellulose components of the sludge are dissolved by these acids and broken down into glucose. 
     Glucose is a sugar. It is the primary nutrient required by white rot fungus to grow and produce phenoloxidase enzymes. Glucose is also a rich food source for wastewater bacteria populations. Glucose enables these bacteria populations to grow faster and consume more organic materials thereby assisting in further and faster degradation of color and biological oxygen demand (BOD) products. Furthermore, bacteria are also believed to assist in reducing chemical oxygen demand (COD) products. Accordingly, when brown rot fungus is introduced in the presence of cellulose or hemi-cellulose, the BOD and COD along with the lignin can be significantly reduced. 
     It has been discovered that many of the organic materials in wastewater are lignin and lignin degradation by-products. The bacteria populations which are feeding on the high glucose concentrations metabolize the lignin degradation by-products converting them to carbon dioxide and water thereby further reducing color, BOD and/or COD. Additionally, helpful bacteria may be added in addition to the white rot and brown rot fungus to further expedite and improve the efficiency of the process. 
     In order to treat wastewater, the applicants have developed a process whereby a combination of white rot and brown rot fungi are introduced into the wastewater. While aerobic conditions could be maintained in the wastewater, it is actually preferred that anaerobic conditions be established. Accordingly, all aeration equipment may be secured. 
     The brown rot fungi is introduced to the wastewater source. If there is no sludge bed or low concentrations of wood fiber and cellulose in the wastewater, timber, wood pieces or alternative cellulose sources may be floated or otherwise deposited into the water source to provide a food source for the brown rot fungi. At least  trichoderma ressei  and  trichoderma hazanium  have been found to be suitable brown rot fungi varieties, however, other varieties may also be utilized as will be explained in further detail hereinafter. Preferably a large population such as 10 12  CFU/ml or greater of the brown rot fungi may be allowed to accumulate in the waste water supply after repetitive seeding steps. Moreover, smaller populations may also be sufficient to provide adequate supplies of glucose depending on the flow of the waste water source. 
     Some bacteria is also useful in degrading cellulose into glucose. Accordingly,  clostridium cellulolyticum  and  clostridium celluloverans  can be added along with the brown rot fungi to assist in producing glucose. These bacteria may build up into a large population of at least about 10 16  CFU/ml. Moreover, smaller populations may also be utilized in some embodiments. 
     Although it is preferable to wait until after the brown rot fungi has established a suitable population, it is not absolutely necessary to wait until a large population of brown rot fungi has established itself before adding the white rot fungi.  Phanerochaete chrysosporium, phanerochaete sorida  and  pluerotis ostreatus  are white rot fungi that are known to have a suitable production level of manganese peroxidase, lignin peroxidase and laccase enzymes. 
     Glucose is an important feature for white rot fungi. The white rot fungi converts glucose to pyruvate. Pyruvate is believed to be an essential “building block” for the white rot fungus (life cycle). As the white rot bacteria builds up to a large population such as about 10 12  CFU/ml or larger, the coordinated combination of the microbials has been found effective to significantly reduce color in pulp and paper wastewater streams. In 150 hours in a test environment of a wastewater stream, over an 85% reduction in color was achieved through the combination of these populations while a similar size population of white rot fungi alone reduced the color levels less than 20%. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The particular features and advantages of the invention as well as other objects will become apparent from the following description taken in connection with the accompanying drawing in which: 
       The FIGURE is a graph showing oxygen uptake in milligrams of various test cultures over time expressed in hours. 
     
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     In order to treat wastewater whether it be in the form of wastewater streams, lagoons, pools or other wastewater systems, the applicants have devised and trusted a method of reducing colorants, BOD and/or COD in the wastewater sources. While the method developed by the applicants is primarily related to the paper and pulp industry, it could also be utilized in other wastewater streams and environments. 
     Specifically, in order to begin the method, it is preferable that all, or at least a part of the aeration systems in the wastewater environment are secured. This may encompass such steps as removing step aerators, turning off all surface aerators and/or securing all air diffusers. Since all this equipment draws electricity to motors, the electrical savings provide a huge benefit to the user of the applicant&#39;s method. In some applications, it may be desirable to utilize some aeration equipment, and the use may depend on the particular application. 
     The wastewater source, either with or without the aeration sources as secured, may then be seeded or otherwise supplied with a quantity of brown rot fungi. Typically, small populations of brown rot fungi are introduced into the wastewater system and are allowed to grow to large populations. Small populations are defined less than 10 12  CFU/ml while large populations of fungi are defined as 10 12  CFU/ml or greater. A preferred range may be anywhere from 10 6 –10 16  CFU/ml. Of course, smaller populations could also work if longer time periods are available. In other embodiments the quantity significant enough to constitute a large population may vary either inside or outside the preferred range depending on the particular application and need. 
     Upon introduction of brown rot fungi to the wastewater, the brown rot fungi decompose wood fibers, cellulose and hemi-cellulose. While two particular brown rot fungi, namely,  trichoderma ressei  and  trichoderma hazanium  have been found to be particularly effective, it is also anticipated that  aspergillus niger, aspergillus versicolor, chaetomium bolichotlitum, fusarium, solani , etc., may also be utilized. The selected brown rot fungi were chosen for their speed of degradation of cellulose and hemi-cellulose or other wood products into glucose. 
     The brown rot fungi attacks the wood fibers and cellulosic materials either in the sludgebed or otherwise to create glucose. In wastewater sources having no sludgebed or low concentrations of wood fiber, cellulose or hemi-cellulose, timber, wood pieces or alternative cellulose sources may be floated or otherwise placed in the water source to provide a habitat for the brown rot fungi. 
     While initial seeding of the brown rot fungi may prove inadequate for growing from a small to a large population, depending upon the size of the wastewater source and its flow, if any, additional inoculations of the brown rot fungi species may be added to the watersource over time. Additionally, multiple strains of brown rot fungi may be combined together and added in a single or in several/multiple additions. 
     The addition of two anaerobic bacteria, namely,  chlostridium cellulolyticum  and  clostridium cellulovorans  can also be beneficial when added to the water source. These anaerobic bacteria are believed to functionally work similarly to brown rot fungi in that they are believed to assist in degrading cellulose into glucose. These bacteria can assist brown rot fungi in continuous production of glucose. 
     It is important to remember that glucose is an important food source for the white rot fungi. The white rot fungus converts glucose to pyruvate. Pyruvate is believed to be one of the essential “food building blocks” for the white rot fungus&#39; life cycle. Furthermore, as has been discussed above, the presence of glucose environment accelerates the production of the phenoloxydate enzymes in the white rot fungi. In fact, other enzymatic activity may be reduced in the presence of glucose. 
     One or more additions of the anaerobic bacteria may be added to the water source to shorten the time to achieve a large population of bacteria. A large population of these bacteria is about 10 16  CFU/ml or greater. A preferred population is in the range of 10 8 –10 16  CFU/ml. Of course, other populations may be useful in some embodiments. Furthermore, while seeding of the bacteria is desirable, of course, in other embodiments larger populations may be added depending on their availability and time constraints involved. 
     In one preferred embodiment of the method, the brown rot fungi and bacteria are preconditioned in a water supply for approximately two months prior to the addition of any white rot fungi. Of course, in other embodiments the precondition step may be omitted, lowered even significantly, or reduced depending upon the specific objectives and conditions of the water source. Two months allowed for the growth of a small population to a large population in the test environment. Furthermore, upon the addition of white rot it may take several months, such as one or two months, and even repeated seeding to grow the white rot fungi to an acceptable population level to produce a desired reduction in color, BOD and/or COD, especially in a flow of wastewater. 
     The white rot fungi is preferably added to the water source in the presence of glucose so that the phenyloxidase enzymes are preferentially produced. In testing environments,  phanerochaete chrysosporium, phanerochaete sorida  and  pluerotis ostreatus  are utilized together and seeded at intervals. Of course, one or more of these separate fungi strains can be utilized depending upon the particular objectives. Additionally, other white rot fungi are capable of degrading lignin. Some of these additional white rot fungi strains include  bjerkandera adusta, Pycnoporus cinnabarinus, trametes versicolor, stagonospora gigaspora , etc. The particular strains of white rot fungi have been selected due to their anticipated levels of production of manganese peroxidase, lignin peroxidase and laccase enzymes. Of course, other white rot fungi may produce these three enzymes in suitable quantities to perform the desired color clarification and/or reduction of BOD and COD. 
     Furthermore, while seeding is the preferred technique through several inoculations or additions of white rot fungi to the water source, it may be desirable to achieve a large population through direct addition. A large population is 10 12  CFU/ml or greater, but a preferred range is 10 7 –10 16  CFU/ml Of course, small populations may also be useful and perform satisfactorily depending upon the objectives of the treatment desired. In non-flow through water sources, small populations of all the organisms are believed to work, it just may take substantially more time. 
     Initially, the applicants tested the theory that white rot fungi would preferentially produce the phenoloxidaxes in the presence of glucose by seeding white rot fungi into a wastewater lagoon and dumping about two tons of sugar in the lagoon daily. As one can examine the cost of this test was relatively significant although promising results were observed by the relatively rapid growing of the population of white rot fungi. The color reduction experienced led to the investigation for a cheaper source of sugar. 
     Another series of tests were conducted using a respirometer, an instrument measuring biological reactions by measuring an exchange of gas. The tests were performed to assist the applicants in determining whether pulp laced wastewater stream could be treated as proposed. The FIGURE shows oxygen intake in milligrams relative to hours in a wastewater stream. The lower line  2  represents an untreated wastewater supply. There is no change in oxygen uptake and no increase in oxygen uptake when no fungi were present. The average color measurement for this control test was 1504 Pt-Co units. The next test line  4  represents the addition of white rot fungi in the flow of mixture of  P. chrysosporium, P. sorida , and  Pluerotis Ostreatus . No bacteria is added to this solution. This resulted in a modest color reduction of 17.28% and a slight increase in oxygen uptake over a 150 hour period. The average color measurement was 1244 Pt-Co units. 
     As the FIGURE represents, the next test line  6  was conducted with both brown and white rot fungi with no bacteria added. As is reflected in the graph, the oxygen uptake substantially tripled compared to the use of white rot alone. A 79.25% reduction in color was experienced over a 150 hour test. The average color measurement was 312 Pt-Co units. The specific strains of fungi added where  P. chrysosporium, P. sorida, Pluerotis Ostreatus, T. hazanium  and  T. resseii . While these results were very good and a dramatic improvement over the prior art, the addition of the bacteria described above to the mixture results in even further color reduction of 87.76%. The average color measurement was 184 Pt-Co units. Test line  8  was conducted while both brown rot and white rot fungi and bacteria show that in the first 30 or 40 hours the presence of the bacteria reduced the oxygen uptake, because the oxygen uptake increased more exponentially as opposed to linearly as was done with the use of white and brown rot fungi alone. Accordingly, by the 150 hour point, the oxygen intake was more than double that of when using only the white rot and brown rot combinations together. 
     While the color reduction percentages are extremely important and significant, the increase in oxygen uptake is similarly significant as the higher the oxygen uptake, the higher the degree of biodegradation and the further reduction of BOD and COD are believed to be occurring during this process. 
     Numerous alterations of the structure herein disclosed will suggest themselves to those skilled in the art. However, it is to be understood that the present disclosure relates to the preferred embodiment of the invention which is for purposes of illustration only and not to be construed as a limitation of the invention. All such modifications which do not depart from the spirit of the invention are intended to be included within the scope of the appended claims.