Decolorization by Thanatephorus Cucumeris Dec 1

As a dye-decolorizing microorganism, a fungus, identified as Geotrichum candidum Dec 1, was isolated from the soil. Re-identification of this fungus revealed it to be Thanatephorus cucumeris Dec 1 abbreviated as Dec 1. A crude extracellular enzyme solution was prepared from the culture of Dec 1, and the enzyme solution showed a broad decolorization spectrum of dyes in the presence of H2O2 , indicating the existence of multiple enzymes, including peroxidases. Dec 1 decolorized approximately 12 g/1 of the dye, Reactive Blue 5 (RB5), without a decline in the decolorizing activity at the inhibitory concentration for most of the microorganisms, indicating the resistant property of Dec 1 to a high concentration of the dye RB5. Dec 1 expressed decolorizing activity in the presence of carbon sources and oxygen.

A peroxidase, DyP, produced by the fungus Dec 1 having the ability to decolorize dyes was purified. DyP contains 17% sugar comprising GlcNAc and Man. The molecular mass of DyP was estimated to be 60 kDa and the isoelectric point (pl) was determined to be 3.8. DyP degraded various dyes, and also phenolic compounds of 2,6-dimethoxyphenol and guaiacol. However, veratryl alcohol, abbreviated VA, a substrate of lignin peroxidase, was not degraded by DyP.

Molasses was utilized by Dec 1 to produce DyP as carbon and energy source. Within 10 g/l of molasses concentration, decolorization activity of Dec 1 toward RB5 gradually increased. However, at more than 20 g/l of molasses concentration, the inhibitory effect on the decolorizin g activity of Dec 1 was observed. As the activity of purified DyP was inhibited by 10 g/l of molasses concentration, this indicates that molasses has a stimulative effect on the decolorization activity of Dec 1, but has an inhibitory effect on the DyP activity.

Dec 1 also produced Aryl Alcohol Oxidase (AAO). The role of AAO in the Dec 1 decolorization process of dyes has been observed to be as follows: the first role was that H2O2 was produced by AAO oxidation of Veratryl Alcohol (VA) to veratraldehyde and then utilized by the peroxidase DyP. In the cultivation of Dec l, the presence of H2O2 and veratraldehyde has been detected. The second role was that the polymerization of products produced by DyP oxidation of a simplified RB5, AQ-2’, was prevented by AAO. This was confirmed by the result that the molecular weight of the products was reduced in the mixed decolorization of DyP and AAO.

A new versatile peroxidase named TcVP1 was purified from the Dec 1 culture. Purified TcVP1 behaved as Manganese Peroxidase (MnP) at pH 5, and the enzyme functioned as a Lignin Peroxidase (LiP) at pH 3. As TcVP1 decolorized preferentially azo dyes, co-application of TcVP1 and DyP conducted complete decolorization of anthraquinone dye, RB5, in vitro to colorless products.

A novel peroxidase, DyP gene, dyp, was cloned from a cDNA library of a newly isolated fungus, Dec 1. The open reading frame consisting of 1494 nucleotides indicated a primary translation product of 498 amino acids, and the Molecular mass (Mr) was estimated as 53,306. X-ray diffraction data using crystallized DyP revealed DyP to have a unique tertiary structure differing from that of most other well-known peroxidases. The analyzed amino acid sequence of DyP did not share homology with any other peroxidases except that of a peroxidase derived from Polyporaceae sp.

Mature cDNA encoding dyp was fused with the Aspergillus oryzae α-amylase promoter, amyB, and recombinant DyP was produced. The total activity of the purified recombinant DyP, rDyP, was produced. The total activity of rDyP, was about 400-fold higher than that of the native DyP derived from Dec 1.

The further productivity enhancement of rDyP was carried out using the following different cultivation methods and different media. Recombinant A. oryzae holding a DyP gene, dyp, was grown using the repeated batch method. When a synthetic liquid medium containing maltose as a carbon source was used in repeated batch culture, production of high-level rDyP activity continued for 26 repeated cycles of every 1-day batch.

When the production of rDyP by A. oryzae was carried out using complex media containing rice bran powder both in liquid repeated-batch and in fed-batch cultures, average rDyP productivities were similar in the two batch cultures.

The Solid-state Culture (SSC) was also attempted for the production of rDyP by recombinant A. oryzae using wheat bran as a solid medium and the productivity of rDyP was compared to that in the liquid cultures. The maximum productivity of rDyP in SSC reached 5.3g per kg wheat bran, and this productivity value was equivalent to the productivity using a 56 kg liquid culture. When the unit of the productivity per gram carbon of the medium was introduced, the productivity in SSC was 4.1-fold higher than that in the liquid cultures.

In order to overcome the disadvantages of SSC and liquid culture, Dec 1 was grown in an Air Membrane Surface (AMS) reactor. Although the growth of Dec 1 in AMS culture was almost the same as that in liquid culture at optimum temperatures, DyP productivity, DyP activity, and Aryl Alcohol Oxidase (AAO) activity in AMS culture were 18-, 232-, and 108-fold higher than those in liquid culture, respectively. The advantage of AMS culture of Dec 1 1over SSC and liquid culture has been proven, particularly in the production of DyP.

A biofilm was formed in the AMS culture of Dec 1 and the relationship between biofilm formation and DyP and MnP was analyzed. Two new DyP isozymes, DyP2 and DyP3, were purified from the Dec 1 culture in the AMS reactor, and they were characterized and compared with the characteristics of rDyP.

For an efficient application of the soluble recombinant enzyme of rDyP, immobilization of the enzyme was caried out to enhance and stabilize catalytic efficiency of rDyP. Although several conventional immobilization methods have been attempted for rDyP, no methods have been successful. Therefore, new catalysts developed for exhaust gas removal were employed. They were silica-based mesocellular foams and two silica-based porous materials, FSM-16 and AISBA-15, which were chemically synthesized. Immobilization of rDyP on them was carried out and immobilization efficiency was assessed. The overall efficiency was defined as adsorption efficiency x activity efficiency to find the maximum efficiency. The efficiency of rDyP immobilized on FSM-16 and AISBA-15 was maximum at pH 5 and pH 4, respectively. FSM-16 showed advantages over AISBA-15 in terms of stronger affinity for rDyP due to its anionic surface and much lower leaching of rDyP from FSM-16. When the rDyP immobilized on FSM-16, an anthraquinone dye, RBBR, was decolorized in repeated-batch mode, and eight sequential batches were possible, while rDyP immobilized on AISBA-15 enabled only two batches.

For evaluation of the practical potential of rDyP, the turnover capacity of rDyP was introduced. In order to minimize H2O2 inactivation for rDyP activity, four H2O2 supply methods were attempted and the turnover capacity of each method was compared. The continuous fed-batch supply of H2O2 and the stepwise fed-batch supply of the dye gave the maximum turnover capacity of 20.4. At this turnover capacity, one liter of crude rDyP solution containing 5,000 U could decolorize up to 102 g dye in 10h.

Molasses is a primary carbon source, especially in the microbial industry; however, molasses includes many colored substances, like melanoidins, which become concentrated by the Maillard reaction after sterilization [41]. Thus, these remain in the Molasses Wastewater (MWW) after use. For effective treatment of MWW, biological methods are attracting attention. Section 1-10, in the previous Chapter 3, showed molasses to be available as a carbon source for the growth of Dec 1, and that partial color removal of molasses by Dec 1 was possible. The enhanced color removal of molasses by Dec 1 was conducted using a jar fermenter system consisting of fan-type agitators and a pressure swing adsorption oxygen generator. The oxygen-enriched air supply was effective not only in obtaining the highest decolorization degree of molasses, but also the highest activity of peroxidase, DyP for the decolorization of several dyes.

By simultaneous decolorization of molasses and an anthraquinone dye, RB5, the degree of decolorization of molasses reached 87%, and thus, the maximum decolorization rate of the dye, RB5, was achieved. However, the decolorizing activity of purufied DyP toward molasses was significantly lower than that of culture broth of Dec 1 due to the inhibitory effects of molasses on DyP, but the inhibition was reduced in the progress of degradation of molasses by growing Dec 1 concentration. Dec 1 degraded molasses containing substances with a wide range of molecular weights prepared by ultrafiltered fractions of molasses. As Dec 1 was not able to utilize sucrose, sucrose in the molasses was hydrolyzed with invertase to utilize all sugars in molasses. As a result, the decolorization of molasses and rate of decolorization of the dye, RB5, by Dec 1 reached the highest level.

A long and stable decolorization of molasses was attempted using both suspended and immobilized cells of Dec 1. In semi-batch cultivation using suspended cells of Dec 1, 80% decolorization of molasses and a stable DyP activity were maintained for approximately four weeks. When repeated batch cultivation of Dec 1 cells immobilized on polyurethane foam was applied, a longer and stable decolorization of molasses as well as stable DyP activity lasted for more than eight weeks.

Dec 1 was applied for the decolorization of kraft pulp bleaching effluent, abbreviated as E-effluent when glucose was supplemented. The color removal of E-effluent and the reduced amount of Absorbable Organic Halogens (AOX) reached 78% and 43%, respectively. The average molecular weight of colored substances in molasses was reduced to less than 3000 from the original 5600. The contribution of extracellular enzymes, such as Peroxidase (DyP) and Manganese Peroxidase (MnP), to the decolorization of the kraft pulp bleaching effluent was observed in the later stage of decolorization.

Dec 1 decolorized up to 72% of Oxygen-delignified Bleaching Effluent (OBE). Biobleaching of Oxygen-delignified Kraft Pulp (OKP) was conducted at 2% pulp concentration. The brightness of OKP increased by 13% and the kappa value of OKP decreased by 4 points only for 3 days. However, at 25% of pulp concentration, the brightness of OKP increased only by 4% and the kappa value decreased by 3 points during a 12-day incubation period mainly because of oxygen limitation. When the culture after OBE decolorization was used for bleaching of OKP, the brightness of OKP increased to 62.7% at 2% pulp concentration. In the decolorization and biobleaching, the involvement of DyP and MnP was confirmed. From these results, the potentiality of Dec 1 for the decolorization of kraft pulp wastewater and biobleaching of kraft pulp in paper mills can be observed.