Polycyclic aromatic hydrocarbon degradation by the white rot fungus Bjerkandera sp. strain BOS55

Outline of this thesisIn this thesis the conditions for optimal PAH oxidation by the white rot fungus Bjerkandera sp. strain BOS55 were evaluated. In Chapter 2, culture conditions like aeration and cosubstrate concentrations, which influenced the oxidation of the PAH compound anthracene and the ligninolytic indicator dye Poly R-478 by the white rot fungus, were studied. Two parameters were identified as the most important PAH oxidation rate-limiting factors: the hydrogen peroxide production rate by the fungal cultures, needed for full activity of the peroxidases as described in Chapter 3, and the PAH bioavailability as described in Chapter 4. When these rate-limiting parameters were eliminated, extremely high PAH oxidation rates could be observed. In Chapter 5, the oxidation and mineralization of the 5-ring PAH benzo[ a ]pyrene to CO 2 was monitored using 14C-labeled benzo[ a ]pyrene. The accumulated metabolites were subjected to further mineralization by indigenous soil and sediment microflora. The elimination of the highly mutagenic potential of benzo[ a ]pyrene was also monitored. This thesis is concluded in Chapter 6, where the results of this study are discussed in relation to the use of white rot fungi for the bioremediation of PAH-polluted soils.Summary and concluding remarksAn alternative approach for bioremediation of PAH polluted soils has been investigated in this thesis. The approach is based on the ability of white rot fungi to oxidize PAHs with an extracellular oxidative enzyme system. In this chapter, the main results of this study are discussed and compared to literature data.First, this chapter will show the evidence that PAHs are oxidized extracellularly by the ligninolytic enzymes of the white rot fungus Bjerkandera sp. strain BOS55. The parameters which influence the PAH degrading capacity of the white rot fungus are then discussed. The results concerning the degradation of PAHs with respect to mineralization, accumulation of metabolites and effect on mutagenicity are summarized. Finally, the future prospects of white rot fungal bioremediation of PAH polluted soils are discussed briefly.PAH DEGRADATION BY WHITE ROT FUNGISoon after the initial observation that PAHs are oxidized by white rot fungi, the involvement of the extracellular ligninolytic enzyme system was indicated. Direct oxidation of PAH by the extracellular ligninolytic enzymes was first observed for LiP (Hammel et al., 1986; Haemmerli, 1986), and later for MnP (Moen and Hammel, 1994; Field et al., 1996b; Sack et al., 1997) and laccase (Collins et al., 1996; Johannes et al., 1997).In this study, further evidence was obtained demonstrating that extracellular oxidation by peroxidases is the main mechanism of PAH metabolism in white rot fungi (Chapter 2, 3, 4 and 5). The rate of PAH oxidation was similar in extracellular culture fluids as in the whole cultures. Furthermore, the same metabolite of anthracene oxidation, anthraquinone, was observed in similar yields in both extracellular culture fluids and whole cultures. Anthraquinone is a well-known metabolite of anthracene oxidation by ligninolytic enzymes (Haemmerli 1988; Hammel et al., 1991, Field et al., 1996b; Sack et al., 1997). Additionally, the oxidation of the ligninolytic indicator dye Poly R-478 was found to be correlated to PAH oxidation. The aromatic polymer Poly R-478 is a substrate of peroxidases and can only be oxidized extracellularly due to its excessive size. The involvement of peroxidases was also indicated by the stimulation of PAH and Poly R-478 oxidation by increasing the hydrogen peroxide production rate.Oxidation of PAH by non-ligninolytic cultures of white rot fungi has also been reported (Sutherland et al., 1991). The PAH metabolites observed, trans-dihydrodiols, suggested intracellular oxidation by P450 monooxygenases. White rot fungi have cytochrome P450 monooxygenases, which are capable of oxidizing PAH under physiological conditions when ligninolytic enzymes were not expressed (Masaphy et al. 1996; Bezalel et al., 1997). In this study, no significant participation of intracellular monooxygenases in the oxidation of PAH by ligninolytic cultures of Bjerkandera sp. strain BOS55 could be demonstrated.ENHANCEMENT OF THE PAH OXIDATION BY WHITE ROT FUNGIIn this study, the effect of several parameters, both physiological and non-physiological, on the PAH oxidation by Bjerkandera sp. strain BOS55 was monitored.Physiological Parameters Limiting PAH Oxidation Biomass.Since PAHs are not sole E- or C-sources for white rot fungi, a suitable cosubstrate is required for biomass production and ligninolytic activity. Consequently, oxidation of PAHs by white rot fungi is only observed in the presence of a cosubstrate (Aust, 1990; Morgan et al., 1993). Both biomass production and mineralization of the 5-ring PAH benzo[ a ]pyrene in soil by several white rot fungi was stimulated by increasing concentrations of carbon sources like wood chips and wheat straw (Morgan et al., 1993). Likewise, the PAH degradation by the white rot fungus Bjerkandera sp. strain BOS55 also depended on a suitable cosubstrate. This cosubstrate could either be a complex lignocellulose substrate such as hemp-stem-wood (HSW) or a simple substrate such as glucose (Chapter 2). The biomass production, the anthracene degradation as well as the decolorization of the ligninolytic indicator dye Poly R-478 increased with the glucose concentrations in the culture medium from 0 to 5 g liter -1(containing only 2.2 mM nitrogen). Above these concentrations, no increase in biomass nor ligninolytic activity was observed.Peroxidase titers.Stimulation of the degradative capacity of white rot fungi is often sought in selection of strains or culture conditions with higher peroxidase production (Orth et al., 1991; Kaal et al., 1993). Initially, N-limited media were thought to be a necessity for ligninolytic activity, since high-N repressed both the production of ligninolytic enzymes as well as the PAH degradation by the model white rot fungus Phanerochaete chrysosporium (Aust, 1990; Hammel 1992). The disadvantage of these media is the poor production of biomass and ligninolytic enzymes. Bjerkandera sp. strain BOS55, however, was shown to be N unregulated, high nutrient nitrogen concentrations did not repress ligninolytic enzyme production nor PAH degradation (Chapter 2). Instead, the use of high organic N-nutrients even dramatically improved the peroxidase titers (Kaal et al., 1993; Mester et al., 1996). Although the peroxidase titers were improved up to 30-fold in high organic N media compared to the N-limited media in this study, the rate of anthracene oxidation was not remarkably increased (Chapter 3). Clearly the low peroxidase titers in the N-limited media were not the rate limiting factor in the oxidation rate.Peroxidase profile.Manganese has an important impact on enzyme profiles and ligninolytic activity in white rot fungal cultures. The MnP titers in many white rot fungi are strongly stimulated by the presence of Mn (Bonnarme and Jeffries, 1990; Brown et al., 1990), and Mn can also severely decrease LiP titers (Bonnarme and Jeffries, 1990; Perez and Jeffries, 1992). In the absence of Mn, LiP titers were also increased in Bjerkandera sp. strain BOS55; whereas, the presence of Mn stimulated the MnP titers and partially repressed LiP titers (Mester et al., 1995). These different enzyme profiles clearly affected the anthracene oxidation rate (Chapter 3). In the absence of Mn, the anthracene oxidation rate was improved by up to 95%. However, no difference in the Poly R-478 oxidation rate was observed. These results suggest that LiP is a better anthracene oxidizing enzyme than MnP.Addition of Mn to 6-day-old Mn-deficient cultures simultaneously with anthracene decreased the anthracene oxidation as well as the anthraquinone accumulation (Chapter 3), suggesting Mn, in some way, also affected the activity of the existing ligninolytic enzymes. Mn can scavenge reduced oxygen radicals like superoxide (Rotschild et al., 1998), which might be disadvantageous for the oxidation of anthracene.Oxygen transfer.In static, low-nitrogen liquid cultures of Bjerkandera sp. strain BOS55, poor oxygen transfer into the cultures negatively affected the PAH oxidation rate (Chapter 2). Increasing the aeration by either increasing the surface area of the culture or applying an oxygen atmosphere dramatically improved the PAH oxidation rates. In high-N cultures, the PAH oxidation was limited by the oxygen transfer even in shallow cultures with high aeration surfaces (Chapter 3). Addition of an oxygen atmosphere to these high-N cultures enhanced the PAH oxidation rate up to 2.5-fold, resulting in an anthracene oxidation rate of 100 mg liter -1day -1. Apparently, the oxygen uptake rate by the metabolic activity of the fungus media interfered with the oxygen needed for PAH oxidation. High oxygen levels were shown previously to enhance the production of ligninolytic enzymes as well as ligninolytic activity (Reid and Seifert, 1982; Buswell 1991). During the short term experiments in this study, no effect of oxygen on the peroxidase titers was observed. Our study showed that improved aeration increased the endogenous hydrogen peroxide production rate in the fungal cultures, upon which the peroxidases are dependent for their oxidizing activity (Chapter 3).H 2 O 2 production rate.The effect of the hydrogen peroxide production rate on the PAH oxidation rate was further investigated. The endogenous hydrogen peroxide production rate in high-N cultures was enhanced 2.5-fold by improved aeration, which resulted in a 2.5-fold increase in PAH oxidation rate. A further 3.5-fold increase in the hydrogen peroxide production rate by an extra addition of glucose oxidase resulted in a 3.5-fold increase in anthracene oxidation rate up to 350 mg liter -1day -1(Chapter 3). Even in adequately aerated low N cultures with very low peroxidase titers, a small increase in hydrogen peroxide production rate by a small addition of glucose oxidase resulted in higher PAH oxidation rates. Apparently, the H 2 O 2 production rate was more rate limiting than the peroxidase titers under both N-limiting and non-limiting culture conditions. This could have physiological significance, since it is well known that peroxidases can be inactivated by high H 2 O 2 levels (Wariishi et al., 1989; Cai and Tien, 1992).The results of our study clearly show that of the physiological parameters that improve PAH oxidation by Bjerkandera sp. strain BOS55, the hydrogen peroxide production rate is the most important parameter.Non-physiological Parameters Limiting PAH OxidationThe most appealing feature of white rot fungi is the ability to degrade poorly bioavailable high molecular weight PAHs, with their extracellular enzyme system (Hammel et al., 1986; Field et al., 1993). However, during the optimization of physiological parameters for PAH oxidation, a discrepancy was observed between the increase in anthracene oxidation rate and the increase in the oxidation rate of the ligninolytic indicator Poly R-478. The oxidation rate of the water-soluble dye Poly R-478 was consistently improved to a greater extent than the oxidation rate of the poorly water-soluble PAH anthracene, which was present as colloidal suspension. This led us to believe that the oxidation rate of anthracene was limited by the low aqueous solubility of anthracene (low bioavailability).The hypothesis that the oxidation rate of PAHs by the ligninolytic enzymes was limited by the low bioavailability was confirmed by the 5-fold increase in the PAH oxidation rate when the bioavailability of the PAH was increased by the addition of surfactants to adequately aerated high-N cultures. The surfactants had no positive effect on the ligninolytic activity, instead, toxicity was observed. However, the partial loss of biocatalytic activity was clearly overcompensated by the increased bioavailability of PAH (Chapter 4).The role of surfactants in increasing the bioavailability was shown to be due to their effect on decreasing the particle size of the PAH precipitates and on increasing the apparent aqueous solubility of the PAHs. Both factors are shown to increase PAH bioavailability (Tiehm, 1994; Volkering et al., 1992; Rouse et al., 1994; Volkering et al., 1995; Field et al., 1996b). The addition of surfacants often fails to stimulate bacterial degradation of PAH, since many surfactants can have toxic effects above the CMC (Rouse et al., 1994) or preferential degradation of the surfactant occurs (Grimberg et al., 1996; Tiehm, 1994). Tween 80, a commonly used surfactant that showed both high PAH solubilizing activity as well as low toxicity towards Bjerkandera sp. strain BOS55, was degraded rapidly by the fungus. The stimulatory effect of Tween 80 on the benzo[ a ]pyrene oxidation rate, however, was still observed after degradation of the surfactant. Apparently, the reduction in benzo[ a ]pyrene particle size by Tween 80 accounted for the increased bioavailability and hence the oxidation rate (Chapter 4).The large effect of the two PAH oxidation rate limiting factors identified in this study, the hydrogen peroxide production rate and the PAH bioavailability, was demonstrated in extracellular culture fluids of high-N cultures of Bjerkandera sp. strain BOS55. By enhancing the hydrogen peroxide production rate with exogenous glucose oxidase and by enhancing the PAH bioavailability with surfactants, the anthracene oxidation rate could be increased 4- and 5-fold, respectively. The combination of both effects led to a 14-fold increase to a very high rate of anthracene oxidation of 1450 mg liter -1day -1. Under these conditions, the 5-ring PAH benzo[ a ]pyrene was oxidized at a rate of 450 mg liter -1day -1.FATE AND ENVIRONMENTAL IMPACT OF PAH DEGRADATION BY WHITE ROT FUNGIIn this study, the degradation of the 5-ring PAH benzo[ a ]pyrene by Bjerkandera sp. strain BOS55 was monitored in several ways. The extent of mineralization to CO 2 and the accumulation of metabolites, as well as the effect of oxidation on the highly mutagenic potential of benzo[ a ]pyrene was investigated (Chapter 5).PAH mineralization.White rot fungal degradation of PAHs by the ligninolytic enzyme system does not result in complete mineralization, in general the main effect is the accumulation of more polar products (Sanglard et al., 1986; Bumpus et al., 1989; Bogan and Lamar, 1996). In this study, benzo[ a ]pyrene was only mineralized to a maximum of 13%, but was oxidized for up to 73% to water-soluble products by Bjerkandera sp. strain BOS55. These water-soluble metabolites showed strong fluorescence when illuminated with UV light, indicating a polyaromatic structure still existed. Since the recovery of these metabolites by solvent extraction was enhanced by acidification of the medium, the increased solubility of these metabolites is tentatively attributed to the presence of carboxyl groups. Carboxyl groups have been identified previously in PAH metabolites after white rot fungal oxidation. For example, 2,2'-diphenic acid and phthalate have been observed after oxidation of phenanthrene and anthracene, respectively (Moen and Hammel, 1994; Hammel et al., 1991). Metabolites of benzo[ a ]pyrene oxidation by white rot fungi other than quinones have not yet been identified (Haemmerli et al., 1986). In whole cultures, more polar, unidentified metabolites of benzo[ a ]pyrene accumulate (Sanglard et al., 1986; Bogan and Lamar, 1996).PAH detoxification.The beneficial effect of white rot fungal biodegradation of PAHs has been questioned, since oxidized PAH metabolites accumulate. PAHs are well known examples of compounds that can be activated into mutagens by intracellular monooxygenases (Sutherland et al., 1992), and white rot fungi have monooxygenases capable of oxidizing PAHs (Masaphy et al., 1996; Bezalel et al., 1997). Therefore, the accumulation of PAH metabolites is considered not to be desirable. So far, the effect of white rot fungal oxidation of PAHs on the mutagenicity has not been studied in detail. The mutagenicity of benzo[ a ]pyrene-quinones, the only identified metabolites of white rot fungal benzo[ a ]pyrene oxidation so far, is largely reduced compared to the parent PAH (Thakker et al., 1985). This study indicated that the ligninolytic oxidation of the infamous PAH benzo[ a ]pyrene, a notorious example of a PAH which can be activated by intracellular monooxygenases to highly mutagenic metabolites, did not result in mutagenic activation. On the contrary, the highly mutagenic potential of benzo[ a ]pyrene was drastically decreased, and no direct mutagenic activity of the metabolites was observed in the Salmonella typhimurium revertant test (AMES test) (Chapter 5).Successive mineralization.To minimize the risks of accumulated PAH metabolites, further metabolism of the metabolites is desired. The PAH metabolites have higher aqueous solubilities than the parent PAH, therefore, these metabolites are likely to be better available for degradation by other microorganisms. This synergistic effect has been illustrated with anthraquinone, a well known dead-end metabolite of anthracene oxidation by some white rot fungi (Field et al., 1992, Anderson and Henrysson, 1996). Meulenberg et al. (1997) showed that this metabolite was degraded faster by non-adapted activated sludge and soil microflora than anthracene.In this study, the addition of natural occurring microflora in soils, sediment sludge and activated sludge to cultures of Bjerkandera sp. strain BOS55 with oxidized [ 14C]-radiolabeled benzo[ a ]pyrene resulted in a rapid increase in mineralization. A 21% higher recovery of 14CO 2 was observed upon addition of the natural occurring microflora, confirming that some of the metabolites had high bioavailability and biodegradability. These results also showed that the benzo[ a ]pyrene metabolites were mineralized faster and to a higher level by the added microflora than by the fungal culture itself. Table 1 summarizes the main findings of this study together with other studies in the literature concerning the combined action of white rot fungi and indigenous microflora.The results taken as a whole clearly show that the white rot fungal oxidized PAH metabolites are mineralized faster by indigenous microflora of different sources than their parent PAH. The metabolites of fungal oxidation are also better substrates for bacteria than for the white rot fungi themselves. The highest stimulatory effect of fungal "pre-oxidation" on the mineralization of PAHs is observed with benzo[ a ]pyrene, which also has the lowest bioavailability and degradability of the PAHs tested. Consequently, a faster and higher level of PAH mineralization is obtained by sequential treatment than by bacteria or fungi alone and the observed accumulation of white rot fungal PAH metabolites in pure cultures is therefore not likely to occur under non-sterile conditions, e.g. creosote contaminated soils.FUTURE PROSPECT OF WHITE ROT FUNGI IN THE BIOREMEDIATION OF PAH CONTAMINATED SOILSThe effect of white rot fungi on the PAH removal in aged industrially PAH contaminated soils has been monitored. These studies show an increased elimination of in particular the 4-ring PAHs like pyrene compared to the degradation by the indigenous microflora alone, but little or no increase in the elimination of 5- and 6-rings PAHs (Davis et al., 1993; Lamar et al., 1994; Field et al., 1996a). In spite of the improved overall PAH elimination, still high residual concentrations of PAHs were observed in these studies. Field et al. (1996a) showed that the recalcitrance of PAHs in soils from an old creosote-facility was due to the low PAH bioavailability; pretreatments that increased the bioavailability (presoaking of the soil in acetone and subsequent rapid evaporation of the acetone) increased the PAH degradation. Weissenfels et al. (1992) also observed higher PAH degradation by PAH adapted bacteria in a soil from a tar-oil refinery after a similar pretreatment. In contrast, Bjerkandera sp. strain BOS55 can rapidly degrade PAH in artificially contaminated soils (Field et al., 1995) for which only low residual concentrations were observed.The large difference between the PAH bioavailability in artificially contaminated and aged industrially contaminated soils is probably mainly caused by the method of soil contamination. In artificially contaminated soils, PAHs are added to the soil dissolved in solvents. After evaporation of the solvent, PAH are likely to be present as precipitated particles. The degradation rate of this artificially added benzo[ a ]pyrene by Bjerkandera sp. strain BOS55 appeared to be i