Ligninolytic fungi in bioremediation: extracellular enzyme production and degradation rate

Ligninolytic fungi can be used for remediation of pollutants in water and soil. Extracellular peroxidases and laccases have been shown to oxidize recalcitrant compounds in vitro but the likely significance of individual enzyme levels in vivo remains unclear. This study documents the amounts and activities of Mn-dependent peroxidase (MnP), lignin peroxidase and laccase (LAC) in various species of ligninolytic fungi grown in liquid medium and soil and their effect on degradation of polycyclic aromatic hydrocarbons (anthracene and pyrene), a polychlorinated biphenyl mixture (Delor 106) and a number of synthetic dyes. Stationary cultures of a highly degradative strain Irpex lacteus exhibited 380-fold and 2-fold increase in production of MnP and LAC, respectively, compared to submerged cultures. Addition of Tween 80 to the submerged culture increased MnP levels 260-fold. High levels of MnP correlated with efficient decolorization of Reactive Orange 16 azo dye but not of Remazol Brilliant Blue R anthraquinone dye. Degradation of anthracene and pyrene in spiked soil by straw-grown explorative mycelium of Phanerochaete chrysosporium, Trametes versicolor and Pleurotus ostreatus showed the importance of MnP and LAC levels secreted into the soil. The importance of high fungal enzyme levels for efficient degradation of recalcitrant compounds was better demonstrated in liquid media compared to the same strains growing in soil.     

Enhanced bioremediation of PAH-contaminated soil by immobilized bacteria with plant residue and biochar as carriers

Purpose

Polycyclic aromatic hydrocarbons (PAHs) are largely accumulated in soils in China. The immobilized-microorganism technique (IMT) is a potential approach for abating soil contamination with PAHs. However, few studies about the application of IMT to contaminated soil remediation were reported. Due to recalcitrance to decomposition, biochar application to soil may enhance soil carbon sequestration, but few studies on the application of biochars to remediation of contaminated soil were reported. In this study, we illustrated enhanced bioremediation of soil having a long history of PAH contamination by IMT using plant residues and biochars as carriers.

Materials and methods

Two PAH-degrading bacteria, Pseudomonas putida and an unidentified indigenous bacterium, were selected for IMT. The extractability and biodegradation of 15 PAHs in solution and an actual PAH-contaminated soil amended with immobilized-bacteria materials were investigated under different incubation periods. The effects of carriers and the molecular weight of PAHs on bioremediation efficiency were determined to illustrate their different bio-dissipation mechanisms of PAHs in soil.

Results and discussion

The IMT can considerably enhance the removal of PAHs. Carriers impose different effects on PAH bio-dissipation by amended soil with immobilized-bacteria, which can directly degrade the carrier-associated PAHs. The removal of PAHs from soil depended on PAH molecular weight and carrier types. Enhanced bio-dissipation by IMT was much stronger for 4- and 5-ring PAHs than for 3- and 6-ring ones in soil. Only P400 biochar-immobilized bacteria enhanced bio-dissipation of all PAHs in contaminated soil after a 90-day incubation.

Conclusions

Biochar can promote bioremediation of contaminated soil as microbial carriers of IMT. It is vital to select an appropriate biochar as an immobilized carrier to stimulate biodegradation. It is feasible to use adsorption carriers with high sorptive capabilities to concentrate PAHs as well as microorganisms and thereby enhance dissipation of PAHs and mitigate soil pollution.     

酸碱调控对泥浆反应去除土壤中多环芳烃的影响研究

采用摇瓶试验模拟研究了酸碱调控对泥浆反应去除污染土壤中多环芳烃的影响,结果表明,泥浆反应对污染土壤中的多环芳烃具有一定的去除效果,长期污染土壤中多环芳烃的去除率为10.6% ~ 20.7%,模拟污染土壤中的去除率为37.4% ~ 42.1%;酸碱调控对不同性质的多环芳烃的去除影响不同,整体上看,酸性条件有利于高环（五环和六环）多环芳烃的去除,而中性条件有利于低环（三环和四环）多环芳烃的去除。在实际修复中,根据污染土壤中多环芳烃的组成进行适当的酸碱调控,可以促进污染土壤的快速修复。     

Biochar reduces uptake and accumulation of polycyclic aromatic hydrocarbons (PAHs) in winter wheat on a PAH-contaminated soil

For years, biochar has been successfully used for the remediation of polycyclic aromatic hydrocarbons(PAHs) in contaminated soils, not only for improving their removal from soil but also for reducing their uptake by crops. However, the underlying mechanism of biochar application reducing PAH uptake and accumulation in winter wheat remains unclear. Pot trials were conducted on a PAH-contaminated soil amended with bamboo biochar, coconut shell biochar,and maize straw biochar(MSB) for an entire gro...     

Rapid, semimicro method for determination of polycyclic aromatic hydrocarbons in shellfish by automated gel permeation/liquid chromatography

A simple, rapid, easily automated method is described for the determination of polycyclic aromatic hydrocarbons (PAHs) in shellfish such as American lobster (Homarus americanus) and blue mussel (Mytilus edulis). PAHs are extracted from small amounts (1-8 g) of tissue by saponification in 1N ethanolic potassium hydroxide followed by partitioning into 2,2,4-trimethylpentane. This solution is evaporated just to dryness by rotary evaporation and the residue is dissolved in cyclohexane-dichloromethane (1 + 1) for gel permeation chromatography (GPC) on Bio-Beads SX-3. The GPC procedure is ideal as a screening method in the range 25-18 000 ng PAHs/g tissue. If individual PAH measurements are required, the appropriate GPC fraction is collected and PAHs are separated by reverse phase liquid chromatography (LC) with fluorometric detection. Individual PAHs at concentrations as low as 0.25-10 ng/g can be determined. Recoveries of added fluoranthene, pyrene, benz[a]anthracene, chrysene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene, benzo[ghi]perylene, and indeno[1,2,3-cd]pyrene were quantitative, with relative standard deviations ranging from 0.0 to 16.9%.     

农药企业搬迁土地多环芳烃的风险评价

以常州市某农药厂搬迁土地为研究对象,在监测分析土壤中16种多环芳烃（PAHs）的基础上,对该区域土壤进行健康风险和生态风险评价。结果表明,研究区域土壤中∑PAHs的含量范围为0～1.546mg·kg-1,优势化合物中萘、菲等低环化合物含量大于高环的荧蒽、苯并[k]荧蒽和芘等化合物,且土壤中PAHs可能来源于石油源。健康风险评价结果在可接受的10-6～10-4范围内,而生态风险评价表明,尽管研究区域土壤中的多环芳烃不存在严重的生态风险,但是化合物苊和芴含量超出了风险评价低值（ER-L和ISQV-L）,存在着对生物的潜在危害。     

Removal of polycyclic aromatic hydrocarbons from soil amended with biosolid or vermicompost in the presence of earthworms (Eisenia fetida)

Earthworms burrow through the soil thereby accumulating many lipophilic organic pollutants from the surrounding environment, so they could be used to remove polycyclic aromatic hydrocarbons (PAHs) from soil. Sterilized and unsterilized soil was contaminated with phenanthrene (Phen), anthracene (Anth) and benzo[a]pyrene (BaP), with or without added Eisenia fetida and biosolid or vermicompost. Concentrations of PAHs were monitored in soil and earthworms for 70 days. Removal of PAHs increased in soil with earthworms added as 91% of Anth, 16% BaP and 99% Phen was dissipated compared to 42%, 3% and 95% in unamended soil. The microorganisms in the gut of the earthworm contributed to PAHs removal and 100% of Phen, 63% of Anth and 58% of BaP was removed from sterilized soil with E. fetida added. Biosolid and to lesser extent vermicompost accelerated removal of PAHs from soil. Applying earthworms to a contaminated site might be an environmentally friendly way to remove hydrocarbons from soil. However, a limitation might be the cost of the large amounts of earthworms required to remove PAHs from soil and the necessity to supply them with sufficient substrate while maintaining the water content of the soil high enough for their normal functioning.     

Dissipation of polycyclic aromatic hydrocarbons (PAHs) in soil microcosms amended with mushroom cultivation substrate

The potential of mushroom cultivation substrate (MCS) in bioremediation was examined in polycyclic aromatic hydrocarbon (PAH)-contaminated soil. After a 60-day incubation, 32.9% dissipation of the 15 studied PAHs was observed in MCS-amended microcosms, with anthracene, benzo(a)pyrene and benzo(a)anthracene being the most degradable PAHs. MCS significantly increased the abundance and changed the community compositions of bacteria, fungi and aromatic hydrocarbon degraders. Two species belonging to the Sordariomycetes of the Ascomycota were enriched in all MCS-treated soil samples, and coupled with the unique changes in the PAH profile, this implies the involvement of laccase-like enzymes. Limited improvement was observed after adding Pleurotus ostreatus, possibly because of its poor colonization of the soil. In addition, alfalfa appeared to antagonize the bioremediation effects of MCS. The results of this study suggest that MCS can be a cost-effective and green biostimulation agent, thereby providing support for the development of MCS-based biostimulation of PAH-contaminated soil.     

真菌漆酶氧化蒽的影响因子及其产物毒性研究

The transformation profiles of polycyclic aromatic hydrocarbons （PAHs） by pure laccases from Trametes versicolor and Pycnoporus sanguineus, and the optimal reaction conditions （acetonitrile concentration, pH, temperature and incubation time） were determined. Anthracene was the most transformable PAH by both laccases, followed by benzo[a]pyrene, and benzo[a]anthracene. Laccase-mediator system （LMS） could not only improve the PAH oxidation but also extend the substrate types compared to laccase alone. 5e/0 or 10~ （v/v） of acetonitrile concentration, pH 4, temperature of 40 ~C, and incubation time of 24 h were most favorable for anthracene oxidation by laccase from T. versicolor or P. sanguineus. The gas chromatography-mass spectrometry analysis indicated that 9,10- anthraquinone was the main product of anthracene transformed by laccase from T. versicolor. Microtox test results showed that both anthracene and its laccase-transformation products were not acute toxic compounds, suggesting that laccase-treatment of anthracene would not increase the acute toxicity of contaminated site.     

Characterization and Sources of PAHs and Potentially Toxic Metals in Urban Environments of Sevilla (Southern Spain)

The purpose of this study was to determine the degree of PAH contamination and the association of PAHs with metals in urban soil samples from Sevilla (Spain). Fifteen polycyclic aromatic hydrocarbons-PAHs (naphthalene, acenaphthene, fluorene, phenanthrene, anthracene, fluoranthene, pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[g,h,i]perylene, indeno[1,2,3-c,d]pyrene) and seven metals (Cd, Cr, Cu, Mn, Ni, Pb, Zn) have been evaluated in representative urban soil samples. Forty-one top soils (0–10 cm) under different land use (garden, roadside, riverbank and agricultural allotment) were selected. PAHs from soil samples were extracted by sonication using dichloromethane. The simultaneous quantification of 15 different PAH compounds were carried out by HPLC using multiple wavelength shift in the fluorescence detector. For qualitative analysis a photo diode-array detector was used. Metal (pseudo-total) analysis was carried out by digestion of the soils with aqua regia in microwave oven. The mean concentration of each PAH in urban soils of Sevilla showed a wide range, they are not considered highly contaminated. The results of the sum of 15 PAHs in Sevilla soils are in the range 89.5–4004.2 μg kg^?1, but there seems not to be a correlation between the concentration of PAHs and the land use. Of the 15 PAHs examined, phenanthrene, fluoranthene and pyrene were present at the highest concentrations, being the sum of these PAHs about 40% of the total content. Although metal content were not especially high in most soils, there are significant hints of moderate pollution in some particular spots. Such spots are mainly related with some gardens within the historic quarters of the city. The associations among metals and PAHs content in the soil samples was checked by principal components analysis (PCA). The largest values both for ‘urban’ metals (Pb, Cu and Zn) and for PAHs were mainly found in sites close to the historic quarters of the city in which a heavy traffic of motor vehicles is suffered from years.     

A highly effective polycyclic aromatic hydrocarbon-degrading bacterium, Paracoccus sp. HPD-2, shows opposite remediation potential in two soil types

Bioaugmentation is an efficient and eco-friendly strategy for the bioremediation of polycyclic aromatic hydrocarbons (PAHs). Since the degrading abilities of soils can greatly alter the abilities of PAH-degrading bacteria, illustrating the potential and mechanism of highly efficient degrading bacteria in different soil environments is of great importance for bioremediation. A PAH-degrading bacterium, Paracoccus aminovorans HPD-2, and two soil types, red and paddy soils, with distinct PAH-degrading abilities, were selected for this study. A soil microcosm experiment was performed by adding pyrene (PYR) and benzo[a]pyrene (B[a]P). Illumina sequencing was used to examine bacterial community structure. The results showed that inoculation with HPD-2 significantly elevated PYR and B[a]P degradation rates by 44.7% and 30.7%, respectively, in the red soil, while it only improved the degradation rates by 1.9% and 11%, respectively, in the paddy soil. To investigate the underlying mechanism, the fate of strain HPD-2 and the response of the indigenous bacterial communities were determined. Strain HPD-2 occupied certain niches in both soils, and the addition of the bacterium changed the native community structure more noticeably in the red soil than in the paddy soil. The addition of PAHs and strain HPD-2 significantly changed the abundances of 7 phyla among the 15 detected phyla in the red soil. In the paddy soil, 5 of the 12 dominant phyla were significantly affected by PAHs and the inoculation of HPD-2, while 6 new phyla were detected in the low-abundance phyla (< 0.1%). The abundances of Massilia, Burkholderia, and Rhodococcus genera with PAH degradation efficiency were significantly increased by the inoculation of HPD-2 in the red soil during 42 d of incubation. Meanwhile, in the paddy soil, the most dominant effective genus, Massilia, was reduced by HPD-2 inoculation. This research revealed the remediation ability and inherent mechanism of the highly effective PAH-degrading strain HPD-2 in two different soil types, which would provide a theoretical basis for the application of degrading bacteria in different soils.     

Remediation of contaminated soil using soil washing and biopile methodologies at a field level

Background, Aims and Scope  An out-of-service oil distribution and storage station (ODSS), which operated from 1966 to 2000 in Mexico, is contaminated mainly by gasoline and diesel, showing the presence of methyl-tert-butyl-ether, benzene, toluene, ethyl benzene, and xylenes. Nine of the 16 polycyclic aromatic hydrocarbons were found, as well as Fe, Pb, V, and Zn. The health risk assessment suggested the necessity of reducing of three PAHs [benzo(a)anthracene, benzo(a)pyrene, and benzo-(b)fluoranthene], and vanadium. The aim of this work is to show that soil washing (on-site) and biopiles are excellent remediation methodologies to treat soils contaminated with petroleum derivates and metals. Applying them, it is possible to reach the goal value of 2,000 mg TPH/kg in a few months, as requested by Mexican legislation. Methods  More than 140 m³ were excavated from the ODSS. Three soil-washing dishes were built. 1540 m³ were treated by soil washing using a nonionic surfactant. A 100 m³ biopile was built to study the system capabilities in the biodegradation of around 4,500 mg/kg of TPH using the autochthonous microflora. Results and Discussion  The soil washing, average TPH-removal value was 83%, but values up to ca. 93% were observed. Removal values resulted in a function of the TPH initial values. Biopile (100 m³) worked during 66 days, reaching a TPH-removal value of 85%. At the end of the processes, no PAHs were detected. The contaminated soil was treated successfully, reaching the legislation limits (TPH values under 2,000 mg/kg, and a significant reduction in PAH concentrations). Conclusion and Recommendation  Both systems are suitable for remediation purposes, achieving high removal efficiencies at short and medium stages. It is highly recommended to proceed with soil washing studies, identifying new products, and mixtures, which could reduce costs and assure optimum operation.     

Improved zinc tolerance in Eucalyptus globulus inoculated with Glomus deserticola and Trametes versicolor or Coriolopsis rigida

The potential of interactions between saprophytic and arbuscular mycorrhizal (AM) fungi to improve Eucalyptus globulus grown in soil contaminated with Zn were investigated. The presence of 100 mg kg ⁻¹ Zn decreased the shoot and root dry weight of E. globulus colonized with Glomus deserticola less than in plants not colonized with AM. Zn also decreased the extent of root length colonization by AM and the AM fungus metabolic activity, measured as succinate dehydrogenase (SDH) activity of the fungal mycelium inside the E. globulus root. The saprophytic fungi Trametes versicolor and Coriolopsis rigida increased the shoot dry weight and the tolerance of E. globulus to Zn when these plants were AM-colonized. Both saprophytic fungi increased the percentage of AM root length colonization and elevated G. deserticola SDH activity in the presence of all Zn concentrations applied to the soil. In the presence of 500 and 1000 mg kg⁻¹ Zn, there were higher metal concentrations in roots and shoots of AM than in non-AM plants; furthermore, both saprophytic fungi increased Zn uptake by E. globulus colonized by G. deserticola. The higher root to shoot metal ratio observed in mycorrhizal E. globulus plants indicates that G. deserticola enhanced Zn uptake and accumulation in the root system, playing a filtering/sequestering role in the presence of Zn. However, saprophytic fungi did not increase the root to shoot Zn ratio in mycorrhizal E. globulus plants. The effect of the saprophytic fungi on the tolerance and the accumulation of Zn in E. globulus was mediated by its effect on the colonization and metabolic activity of the AM fungi.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

雷公藤根际微生物特征研究

根际微生物的代谢作用, 直接促进或抑制根的营养吸收和生长, 也影响根际土壤中的物质转化, 雷公藤根系发达且多与其他树种混交栽培, 其根际微生物活性对雷公藤的生长和土壤肥力均有不可忽视的影响。以福建省泰宁县3 种不同栽培模式雷公藤林(野生雷公藤林、杉木雷公藤混交林、厚朴雷公藤混交林)为研究对象, 通过稀释平板法测定3 种不同雷公藤林分根际土壤和非根际土壤中细菌、真菌、放线菌的数量。结果表明: 根际土的微生物数量大于非根际土的微生物数量; 3 种林分, 无论是在根际土壤中, 还是在非根际土壤中, 均表现为细菌数量＞放线菌数量＞真菌数量; 根际微生物(R)比非根际微生物(S)更活跃, 3 种林分的3 大类微生物的R/S 数量比值均大于1; 3 种林分的微生物活性表现为厚朴雷公藤混交林＞杉木雷公藤混交林＞野生雷公藤林, 表明混交方式可促进雷公藤根际微生物活性。     

Fungal Bioremediation of Creosote-Contaminated Soil: A Laboratory Scale Bioremediation Study Using Indigenous Soil Fungi

The aim of the study is to determine the efficacy of indigenous soil fungi in removing (PAHs) from creosote-contaminated soil with a view to developing a bioremediation strategy for creosote-contaminated soil. Five fungal isolates, Cladosporium, Fusarium, Penicillium, Aspergillus and Pleurotus, were separatelyinoculated onto sterile barley grains and incubated in the dark. Thecolonized barley was inoculated onto creosote-contaminated (250 000 mg kg^?1) soil in 18 duplicate treatments and incubated at 25 °C forseventy days. The soil was amended with nutrient supplements to give a C:N:Pratio of 25:5:1 and tilled weekly. Creosote removal was higher (between 78and 94%) in nutrient supplemented treatments than in the un-supplementedones (between 65 and 88%). A mixed population of fungi was more effective(94.1% in the nutrient amended treatment) in creosote removal than singlepopulations wit a maximum of 88%. Barley supported better fungal growthand PAH removal. Pleurotus sp. removed the creosote more than the other isolates. Two andthree-ring PAHs were more susceptible to removal than the 4- and 5-ringPAHs, which continued to remain in small amounts to the end of thetreatment. Reduction of creosote in the present study was higher than wasobserved in an earlier experiment using a consortium of microorganisms, mainly bacteria, on the same contaminated soil (Atagana, 2003).     

Hydrocarbon status of soils under atmospheric pollution from a local industrial source

Contents and compositions of bitumoids, polycyclic aromatic hydrocarbons (PAHs), and free and retained hydrocarbon gases in soils along a transect at different distances from the local industrial source of atmospheric pollution with soot emissions have been studied. The reserves of PAHs progressively decrease when the distance from the source increases. Among the individual PAHs, the most significant decrease is observed for benzo[a]pyrene, tetraphene, pyrene, chrysene, and anthracene. On plowlands, the share of heavy PAHs—benzo[ghi]perylene, benzo[a]pyrene, perylene, etc.—is lower than in the forest soils. In automorphic soils of the park zone adjacent to the industrial zone, the penetration depth of four-, five-, and sixring PAHs from the atmosphere is no more than 25 cm. In soils under natural forest vegetation, heavy PAHs do not penetrate deeper than 5 cm; in tilled soils, their penetration depth coincides with the lower boundary of plow horizons. Analysis of free gases in the soil air revealed hydrocarbons only under forest. From the quantitative and qualitative parameters of the content, reserves, and compositions of different hydrocarbons, the following modification types of hydrocarbon status in the studied soils were revealed: injection, atmosedimentation–injection, atmosedimentation–impact, atmosedimentation–distant, and biogeochemical types.     

Fraxinus-Glomus-Pisolithus symbiosis: Plant growth and soil aggregation effects

It has been established that arbuscular mycorrhizal (AM) fungi are involved in the conservation of soil structure. However, the effect of ectomycorrhizal (EM) fungi alone or in interaction with AM fungi in soil structure has been much less studied. This experiment evaluated EM and AM fungi effects on soil aggregation and plant growth. Ash plants (Fraxinus uhdei) were grown in pots, and were inoculated with Glomus intraradices and Pisolithus tinctorius separately but also in combination. Our results showed that F. uhdei established a symbiotic association with EM and AM fungi, and that these organisms, when interacting, showed synergistic and additive effects on plant growth compared to singly inoculated treatments. EM and AM fungi prompted changes in root morphology and increased water-stable aggregates. AM fungi affect mainly small-sized macroaggregates, while EM and EM-AM fungi interaction mainly affected aggregates bigger than 0.5 mm diameter. These results suggest that ectomyccorrhizal as well as arbuscular mycorrhizal fungi should be considered in restoration programs with Fraxinus plants.     

Energy efficiency of the mycoparasite Sporidesmium sclerotivorum in vitro and in soil

The energy content of the mycoparasite Sporidesmium sclerotivorum mycelium was 18,389 J g^?1 and 16,334 J g^?1 for macroconidia on a dry weight basis. The energy content of Sclerotinia minor sclerotia, the host of the mycoparasite, was 16,485 J g^?1. In liquid culture, the economic coefficient for the conversion of glucose to mycelium (mycelial dry wt ÷ glucose consumed × 100) was 51–60 whereas the mycelial energy coefficient, [mycelial energy (J) ÷ substrate energy (J) × 100] was 65–75. In soil, the conidial energy coefficient [conidial energy (J) ÷ substrate energy (J) × 100] for the conversion of host sclerotial energy to the macroconidia of the mycoparasite was 19.8, which was 2–9 times that for the conversion of glucose in liquid culture. The conidial energy coefficient when grown on a liquid medium on vermiculite was 23.0. S. sclerotivorum, as an obligate parasite of sclerotia in soil, was most efficient in the conversion of energy in a system where there was a high surface: energy ratio. In liquid culture S. sclerotivorum is more efficient than most other fungi.