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Factors affecting the mineralization of [U-14C]benzene in spent mushroom substrate

Kirk T Semple, Ngaire U Watts, Terry R Fermor
DOI: http://dx.doi.org/10.1111/j.1574-6968.1998.tb13104.x 317-321 First published online: 1 July 1998


The degradation of benzene in pasteurized spent mushroom substrate (SMS) was assessed. Following a 3 month enrichment in the presence of a variety of BTEX compounds, the extent of [U-14C]benzene mineralization in the pasteurized SMS increased with increasing incubation temperature (18°C<37°C<50°C). The concentration as well as the chemical composition used to enrich the compost's degradative activity was also shown to be involved in determining the extent of benzene mineralization. SMS induced on a 12.5 mM BTEX mixture mineralized more benzene than composts induced using 500 μM benzene, 500 μM o-xylene, or 2.5 mM BTEX mixture. In the absence of a pre-enrichment period, benzene mineralization was minimal.

Key words
  • Biodegradation
  • Compost
  • Temperature
  • BTEX
  • Bioremediation

1 Introduction

BTEX compounds (benzene, toluene, ethyl benzene and o-, m- and p-xylenes) are toxic, water soluble and mobile petroleum components found mainly in aviation fuel, diesel and refined petroleum products [1]. BTEX contaminants enter soils, sediments and waters through leakage from storage facilities, pipelines, accidental spillages and improper disposal practices [2]. The family of chemicals that is BTEX has been shown to be neurotoxic and carcinogenic [3] and as such are named on the US EPA priority pollutant list. In fact, benzene has both acute and chronic toxic effects, ranging from a skin irritant to leukaemia and, wherever possible, has been replaced by the less toxic toluene or xylenes [4]. Despite its apparent toxicity and carcinogenicity, benzene and its alkylated homologues are biodegradable and, in fact, this type of contamination is one of the most frequently biologically treated types of pollution [5]. The biodegradation of benzene and its alkylated homologues is well known and has been reviewed elsewhere [6].

Spent mushroom substrate (SMS) is a waste product of the mushroom industry which produces some 500 000 tonnes of SMS per annum as a waste product in the UK [7]. After mushroom cropping, SMS is pasteurized with steam and, therefore, does not contain any live mushroom mycelia or non-spore-forming pathogens [8]. SMS is of little economic value and an estimated 75–80% is landfilled. Previous studies have examined the degradation of organic pollutants in other composts. For example, Valo and Salkinoja-Salonen [9] showed that windrow composts were capable of mineralizing pentachlorophenol and that the rate of degradation could be accelerated with the addition of degrading microbes. Laine and J?rgensen [10, 11] and Semple and Fermor [12] also showed that chlorophenols, particularly pentachlorophenol, could be mineralized when incubated with composts after a suitable period of enrichment. Laine and J?rgensen [13] went on to investigate the potential for dimerization of chlorophenols in the composts. The investigators concluded that, at least in their experiments, the biogenic formation of more toxic metabolic products was not occurring in their composts. Kästner and Mahro [14] highlighted the importance of the solid organic matrix in composts to enhanced degradation of PAHs as soil/compost microflora exhibited much lower degrading activity than in the presence of solid organic material. This phenomenon was observed by Semple and Fermor [12, 15] where compost isolates did not degrade PCP in liquid culture to the extent that the composts did.

This study aimed to investigate the use of mushroom composts for the bioremediation of other environmental pollutants. The hypothesis for this project was that pre-enriched (induced) pasteurized SMS is capable of degrading benzene more rapidly than uninduced composts.

2 Materials and methods

2.1 Enrichment of SMS

SMS enrichments (10 g) were generated using screwcap flasks containing center wells (Fig. 1). For the pasteurized SMS, benzene (500 μM) or o-xylene (500 μM) was placed into the center well and allowed to volatilize and then condense within the composts. Pasteurized SMS was also spiked with a BTEX mixture containing benzene, toluene, ethylbenzene, o- and p-xylene, each at 500 μM or 2.5 mM, giving a total of 2.5 mM or 12.5 mM. An unpasteurized SMS was also enriched in the presence of the BTEX mixture (2.5 mM), as described above, providing a comparison between unpasteurized and pasteurized composts. Parallel unspiked, unpasteurized and pasteurized SMS (uninduced) were used as control composts. Incubation of the enrichments was at 37°C. Enrichments were tested after 3 months to determine the mineralization potential of each compost.

2.2 Radiorespirometric studies

A screw cap 250 ml Erlenmeyer flask with a center well was used to conduct experiments involving [U-14C]benzene (Sigma Chemical Co.). The CO2-trapping efficiency (>97%) was tested using Na214CO3 and [U-14C]glucose [12]. All samples were analyzed using Hi-Safe 3 scintillation cocktail (LKB, UK), measured using a scintillation counter (Packard model 2205). SMS enrichments (10 g) were spiked with 5 g kg−1 [U-14C]benzene (11.2 kBq). Sampling of the KOH-trapped 14CO2 was carried out at regular intervals and analyzed using liquid scintillation counting. The composts were prevented from drying out by placing a filter paper disk on top of the compost sample. This was rehydrated with sterile double-distilled H2O back to its original weight (minus the sample weight just taken), once a week. All incubations were in duplicate and each sample was analyzed twice by liquid scintillation.

3 Results

Composts that were enriched with BTEX compounds mineralized the [U-14C]benzene to a greater extent than composts that had not been induced. However, all the microbial isolates, extracted from SMS, were unable to mineralize [U-14C]benzene in pure culture. Mineralization of [U-14C]benzene to 14CO2 was measured at 14 days (Table 1). Pasteurized composts incubated at 18°C and 37°C showed little mineralization, with maxima of 1.5%. At 37°C, benzene enrichment promoted 2.8% mineralization in pasteurized composts. However, the unpasteurized compost enriched on 2.5 mM BTEX mixture mineralized benzene to a greater extent at these temperatures with 1.5% and 5.2% at 18°C and 37°C, respectively. At 50°C, the mineralization of [U-14C]benzene was higher than the other incubation temperatures. For the pasteurized composts, 12.5 mM BTEX mixture enrichment accounted for 44% mineralization, with benzene and o-xylene enriched composts mineralizing 18% and 11.5%, respectively at 14 days. The unpasteurized compost enriched on 2.5 mM BTEX mixture mineralized 31% of the [U-14C]benzene at 14 days. This approximated to 30 times that of the pasteurized 2.5 mM BTEX mixture enriched compost.

View this table:
Table 1

Enhanced mineralization (%) of [U-14C]benzene at 18°C, 37°C and 50°C after 14 days incubation with pasteurized and unpasteurized SMS enriched on BTEX compounds

Enriched SMSEnhanced mineralization (%)a of
[U-14C]benzene at different
temperatures after 14 daysb
500 μM benzene1 (66)2.8 (70)18 (90)
500 μM o-xylene1.5 (100)1.5 (75)11.5 (88)
2.5 mM BTEX mixture1.5 (120)1 (100)1 (11)
12.5 mM BTEX mixture2 (50)1 (25)44 (98)
2.5 mM BTEX mixture1.5 (100)5.2 (95)31 (100)
  • aAll values are means (n= 2) with a standard deviation of <7%.

  • bControl mineralization values have been subtracted from enhanced mineralization values (above). Control values for the pasteurized SMS were 1% for 18°C and 50°C and 3% for 37°C. Control mineralization values for the unpasteurized SMS were 1% for 18°C, 0.8% for 37°C and 7% for 50°C (where 1% mineralization=0.484 pmoles 14CO2).

  • Figures in brackets are the percentages of the total mineralization measured at 28 days (Table 2).

Mineralization of [U-14C]benzene by the various enriched composts was measured, again, at 28 days (Table 2). As with the 14 day measurements, the incubation temperature had a significant impact on the extent of mineralization, with overall maximal mineralization occurring at 50°C, followed by 37°C with the lowest mineralization values at 18°C. In terms of enrichment of pasteurized composts, 12.5 mM BTEX mixture proved to be the most effective, followed by benzene, o-xylene, the 2.5 mM BTEX mixture. The 12.5 mM BTEX mixture enrichments mineralized almost 45% more [U-14C]benzene than that of the benzene enrichment. Once again, the unpasteurized 2.5 mM BTEX enrichment was more than double that of its pasteurized compost, although, the difference between the two composts had reduced from the 14 day measurements. At first glance, it appears that no mineralization has occurred between 14 days and 28 days in the unpasteurized composts. However, this is misleading because the unenriched control composts' mineralization values increased between 14 days and 28 days. At 28 days, 7% of the [U-14C]benzene had been mineralized by the unenriched, unpasteurized composts, as compared to 1% for the pasteurized SMS controls. By comparing the levels of mineralization at 14 and 28 days, it is clear that, in most incubations, the majority of the mineralization activity occurred within the first 14 days. Unenriched/uninduced pasteurized and unpasteurized composts were incubated under identical conditions to the enriched composts. The mineralization values, measured, were subtracted from the enriched SMS values recorded in Tables 1 and 2.

View this table:
Table 2

Enhanced mineralization (%) of [U-14C]benzene at 18°C, 37°C and 50°C after 28 days incubation with pasteurized and unpasteurized SMS enriched on BTEX compounds

Enriched compost typeEnhanced mineralization (%)a
of [U-14C]benzene at different
temperatures after 28 daysb
500 μM benzene1.5420
500 μM o-xylene1.5213
2.5 mM BTEX mixture1.2519
12.5 mM BTEX mixture4445
2.5 mM BTEX mixture1.55.531
  • aAll values are means (n= 2) with a standard deviation of <8%.

  • bControl mineralization values have been subtracted from enhanced mineralization values (above). Control values for the pasteurized SMS were 1% for 18°C and 50°C and 3% for 37°C. Control mineralization values for the unpasteurized SMS were 1% for 18°C, 1% for 37°C and 7% for 50°C (where 1% mineralization=0.484 pmoles 14CO2).

4 Discussion

Composting is an aerobic process that allows the rapid proliferation of a variety of microbial groups including aerobic actinomycetes, bacilli, pseudomonads and fungi. Ultimately, the role of the SMS would be to act as a readily available carrier for the xenobiotic-degrading microorganisms and as a nutrient source for the inoculant in contaminated soils. In general, biodegradation of aromatic compounds is achieved most effectively through the oxidative reactions of aerobic microorganisms, as is the case for benzene [6]. However, there have been conflicting reports about the degradation of BTEX compounds in the presence of co-substrates.

Benzene has been found in mixed culture incubations to be stimulated by the presence of either toluene or o-xylene, however, the effect was found to be inhibitory if both were present [16]. This study has shown that composts, which have been suitably enriched, were capable of degrading benzene. Interestingly, the extent to which the benzene is mineralized is dependent on the aromatic compounds used, the mixtures and also the concentrations of the chemicals used to induced catabolic activity. For example, the composts enriched with the 12.5 mM BTEX mixture mineralized benzene to a greater extent than the other composts, including benzene itself.

It is also apparent that the incubation temperature is an important factor in determining the extent of mineralization. Although benzene is volatile (H=557 Pa m3 mol−1), the C-14 labelled compound was not trapped in the KOH due to the absence of radioactivity in the uninduced, control incubations. In this case, incubations at 50°C achieved better mineralization than that of 37°C which, in turn, proved more effective than incubations at 18°C. During the course of mushroom compost production, thermophilic actinomycete populations increase, including species of Thermomonospora, Thermoactinomyces, Saccharmonospora and Streptomyces[17]. Virtually all other bacteria isolated from such composts at 50°C, and exhibiting a non-hyphal growth form, are Bacillus spp. [17]. Optimum temperatures for growth and activity of these thermophilic microorganisms are between 45–55°C with little or no activity below 25°C. This relationship between catabolic activity and temperature is clearly observed in this study.

Pasteurization of SMS, prior to disposal, is extremely important for mushroom farm hygiene. From this study, it is evident that the pasteurization process effects the rate to which benzene is degraded. How this heat treatment affects the benzene-degrading microbial communities with the compost are unclear at this point. However, all spore-forming bacteria are able to survive mushroom compost pasteurization in limited numbers. The end of crop pasteurization process (70°C, >12 h) eliminates fungi (the mushroom Agaricus bisporus will not survive at temperatures greater than 34°C) and also nematodes, mites and flies [8].

An important phenomenon, which should be considered when applying a bioremediative technology, is chemical ageing and its relationship to bioavailability of the contaminant to the microflora [18]. The longer an organic compound is in a given soil environment, the more likely it is to be affected by chemical and physical processes, such as diffusion into soil micropores, partitioning into soil organic matter, strong surface sorption and covalent binding to soil constituents [1922]. These processes result in a decrease in the bioavailability of the compound to the degradative microorganisms, thus reducing the effectiveness of the treatment. However, it has been suggested that desorption of compounds (previously unavailable), leading to an increase in bioavailability, may occur at elevated temperatures typically generated under composting conditions [23]. This will require further investigation to understand the processes involved and to characterize its effects on the degradation of environmental pollutants by mushroom composts.

SMS is a relatively untapped source for the bioremediation of xenobiotic contaminated soil, warranting further investigation. Development of the bioremediation potential of SMS, coupled with its well-documented environmental benefits (provision of plant nutrients, neutralization of soil acidity, improvement of water quality), should serve to enhance the value of SMS as an additional source of income to the mushroom grower [24].


This work was supported by Natural Environment Research Council ROPA grant GR3/R95-20.


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