Rainer Martens

Screening of white-rot fungi for their ability to mineralize polycyclic aromatic hydrocarbons in soil.

Soil samples from an agricultural field contaminated with 10 ppm14C-benz(a)anthracene in glass tubes were brought into contact with cultures of wood-rotting fungi, precultivated on wheat straw substrate. Forty-five strains of white-rot fungi and four brown-rot fungi were tested for their ability to colonize the soil and to mineralize14C-benz(a)anthracene to14CO2 within a 20-week incubation time. Twenty-two white-rot fungi and all brown-rot fungi were unable to colonize the soil. Twenty-three strains of white-rot fungi, all belonging to the genusPleurotus, colonized the soil. During the experiment the noncolonizing fungi and their substrate disintegrated more and more to a nonstructured pulp from which water diffused into the soil. The same phenomenon was observed in the control which contained only straw without fungus and contaminated soil. In samples with colonizing fungi the substrate as well as the mycelia in the soil remained visibly unchanged during the entire experiment. Surprisingly, most samples with fungi not colonizing the soil and the control without fungus liberated between 40 and 58 % of the applied radioactivity as14CO2 whereas the samples with the colonizing fungi respired only 15–25 % as14CO2. This was 3–5 times more14CO2 than that liberated from the control (4.9 %) which contained only contaminated soil without straw and fungus. A similar result was obtained with selected colonizing and noncolonizing fungi and soil contaminated with 10 ppm14C-pyrene. However, in pure culture studies in which14C-pyrene was added to the straw substrate,Pleurotus sp. (P2), as a representative of the colonizing fungi, mineralized 40.3 % of the added radioactivity to14CO2. The noncolonizing fungiDichomitus squalens andFlammulina velutipes liberated only 17.2 or 1.7 %, respectively, as14CO2. These results lead to the hypothesis that the native soil microflora stimulated by the formed products of straw lysis is responsible for high degradation rates found with noncolonizing fungi.

Degradation of eight highly condensed polycyclic aromatic hydrocarbons by Pleurotus sp. Florida in solid wheat straw substrate

Abstract The degradation of eight unlabeled highly condensed polycyclic aromatic hydrocarbons (PAH) and the mineralization of three 14C-labeled PAH by the white-rot fungus Pleurotus sp. Florida was investigated. Three concentrations containing 50, 250 or 1250 μg each unlabeled PAH/5u2009g straw were added to sterile sea sand. Selected treatments were added subsequently with 14C-labeled pyrene, benzo[a]anthracene or benzo[a]pyrene. The PAH-loaded sea sand was then mixed into straw substrate and incubated. The disappearance of the unlabeled four-to six-ring PAH: pyrene, benzo[a]anthracene, chrysene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[a]pyrene, dibenz[a,h]anthracene and benzo[ghi]perylene, was determined by high-performance liquid chromatography. After 15 weeks of incubation, the recoveries were less than 25% for initial amounts of 50u2009μg (controlsu2009aboveu200985%). The recoveries of unlabeled PAH increased in the inoculated samples with increasing concentrations applied. No correlation could be determined between the number of condensed rings of the PAH and the recoveries of added PAH. Pleurotus sp. Florida mineralized 53% [14C]pyrene, 25% [14C]benzo[a]anthracene and 39% [14C]benzo[a]pyrene to 14CO2 in the presence of eight unlabeled PAH (50u2009μg applied) within 15 weeks. During the course of cultivation, Pleurotus sp. Florida degraded more than 40% of the wheat straw substrate. Variation of the initial concentration of PAH did not influence the extent of degradation of the organic matter.

Origin and Diversity of Metabolically Active Gut Bacteria from Laboratory-Bred Larvae of Manduca sexta (Sphingidae, Lepidoptera, Insecta)

ABSTRACT Cultivation-independent analyses based on genetic profiling of partial bacterial 16S rRNA genes by PCR-single-strand conformation polymorphism (PCR-SSCP), reverse transcriptase (RT)-PCR-SSCP of the 16S rRNA itself, and stable isotope probing (SIP), followed by RT-PCR-SSCP, were applied to characterize the diversity of metabolically active bacteria in the larval gut of Manduca sexta bred on tobacco leaves under greenhouse conditions. For SIP, hatching larvae were fed with leaves from tobacco plants grown in a 13CO2-enriched atmosphere. Dominant SSCP bands were sequenced and phylogenetically analyzed. Only one major gut colonizer, an Enterococcus relative, was detected; it occurred in the heavy RNA fraction, demonstrating its metabolic activity, and it originated from eggs, where its metabolic activity was also indicated by rRNA-based SSCP profiles. In contrast, a Citrobacter sedlakii relative was detected on eggs by DNA-SSCP, but rRNA-SSCP and SIP-rRNA-SSCP were negative, suggesting that these bacterial cells were inactive. A Burkholderia relative was dominant and metabolically active on the tobacco leaves but inactive inside the gut, where it was also quantitatively reduced, as suggested by lower band intensities in the DNA-based SSCP profiles. SIP-RNA-SSCP detected another metabolically active gut bacterium (Enterobacter sp.) and more bacteria in the light RNA fraction, indicating low or no metabolic activity of the latter inside the gut. We conclude that the larval gut supported only a low diversity of metabolically active bacteria.

Response of microbial communities to long‐term fertilization depends on their microhabitat

The objective of this study was to characterize the microbial communities attached to clay (<xa02xa0μm), fine silt (2-20xa0μm), coarse silt (20-63xa0μm) and sand-sized fractions [>xa063xa0μm; including particulate organic matter (POM)] of an arable soil and analyse their response to more than 100xa0years of two different fertilization regimes. Mild ultrasonic dispersal, wet-sieving and centrifugation allowed the separation of soil particles with the majority of bacterial cells and DNA still attached. Fertilizations increased soil organic carbon (SOC), total DNA and the abundance of bacterial, archaeal and fungal rRNA genes more strongly in the larger-sized fractions than in fine silt, and no effect was seen with clay, the latter representing above 70% of the total microbial populations. A highly positive correlation was found between microbial rRNA genes and the surface area provided by the particles, while the correlation with SOC was lower, indicating a particle-size-specific heterogeneous effect of SOC. The prokaryotic diversity responded more strongly to fertilization in the larger particles but not with clay. Overall, these results demonstrate that microbial responsiveness to long-term fertilization declined with smaller particle sizes and that especially clay fractions exhibit a high buffering capacity protecting microbial cells against changes even after 100xa0years under different agricultural management.

Depolymerization of Straw Lignin by Manganese Peroxidase from Nematoloma frowardii is Accompanied by Release of Carbon Dioxide

Summary Manganese peroxidase preparations (MnP) from the white-rot fungus Nematoloma frowardii were able to release 14CO2 directly from 14C-labeled milled wheat straw (MWS; total lignin fraction) and milled straw lignin (MSL; dioxane soluble part of MWS). Apart from the formation of 14CO2 (4–10 %) the treatment of insoluble MWS and MSL with MnP resulted in the formation of water-soluble 14C-lignin fragments (lignin solubilization, 14–25%). Analyses with gel permeation chromatography (GPC) demonstrated the formation of lignin fragments with predominant molecular masses around 1 kDa. The extent of MWS mineralization and solubilization was enhanced in the presence of reduced glutathione (GSH) acting as thiol mediator, whereas MSL mineralization was not stimulated by GSH. The principle of direct extracellular mineralization of lignin catalyzed by the MnP system may make a significant contribution to the formation of carbon dioxide in lignincellulose containing habitats.

Importance of soil organic matter for the diversity of microorganisms involved in the degradation of organic pollutants

Many organic pollutants are readily degradable by microorganisms in soil, but the importance of soil organic matter for their transformation by specific microbial taxa is unknown. In this study, sorption and microbial degradation of phenol and 2,4-dichlorophenol (DCP) were characterized in three soil variants, generated by different long-term fertilization regimes. Compared with a non-fertilized control (NIL), a mineral-fertilized NPK variant showed 19% and a farmyard manure treated FYM variant 46% more soil organic carbon (SOC). Phenol sorption declined with overall increasing SOC because of altered affinities to the clay fraction (soil particles <2u2009mm in diameter). In contrast, DCP sorption correlated positively with particulate soil organic matter (present in the soil particle fractions of 63–2000u2009μm). Stable isotope probing identified Rhodococcus, Arthrobacter (both Actinobacteria) and Cryptococcus (Basidiomycota) as the main degraders of phenol. Rhodococcus and Cryptococcus were not affected by SOC, but the participation of Arthrobacter declined in NPK and even more in FYM. 14C-DCP was hardly metabolized in the NIL variant, more efficiently in FYM and most in NPK. In NPK, Burkholderia was the main degrader and in FYM Variovorax. This study demonstrates a strong effect of SOC on the partitioning of organic pollutants to soil particle size fractions and indicates the profound consequences that this process could have for the diversity of bacteria involved in their degradation.

Initiation of [36Cl]hexachlorobenzene dechlorination in three different soils under artificially induced anaerobic conditions

Abstract The potential for reductive dechlorination of hexachlorobenzene was investigated in samples of three different, naturally oxic soils held under conditions of high oxygen deficiency. The soils were water-saturated and the influence on dechlorination of adding different electron donors, a surfactant and an anaerobic microbial consortium was tested. The influence of supplied electron donors seems to depend on the organic matter content of the soils. Dechlorination in the organic-matter-rich soil from Maulach was not affected by amendment with organic electron donors. A release of about 40% chloride within 140 days was observed for this soil in all biotic-treated assays. By contrast, the organic-matter-poor soil of Eppingen showed no dechlorination in unamended assays. However, when it was supplemented with organic electron donors dechlorination of 2%–37% occurred within 140 days, depending on the type of electron donor. Complex substrate (wheat strawdust), from which carbon is slowly liberated, gave the best results. These two soils had an indigenous dechlorinating anaerobic microflora, whereas the third soil (Rastatt) required inoculation with an anaerobic consortium for dechlorination. The addition of electron donors alone did not cause dechlorination in this sandy soil. The addition of a surfactant (Tween 80) to increase the bioavailability of hexachlorobenzene did not enhance dechlorination. This process was not inhibited by inherent alternative electron acceptors in soil (NO3−, SO42−, Fe3+). The dechlorination did not require methanogenic conditions.

Enhancing the mineralization of [U-14C]dibenzo-p-dioxin in three different soils by addition of organic substrate or inoculation with white-rot fungi

Abstract The potential for aerobic mineralization of [U-14C]dibenzo-p-dioxin (DD) was investigated in samples of three different agricultural soils already contaminated with polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by industrial activities. The influence of amendments, i.e. wheat straw and compost, and of soil treatment by inoculation with lignolytic fungi, grown on wheat straw substrate, was tested. All the soils tested contained an indigenous DD-mineralizing microflora. The soil characterized by the highest organic matter content and the highest content of soil microbial biomass displayed the best DD mineralization of 36.6% within 70 days, compared with the two organic-matter-poor soils with an endogenous DD mineralization of 19.5% and 23.3% respectively. Amendments with compost increased DD mineralization up to 28% in both soils with low organic matter and microbial biomass content, but did not affect mineralization in the organic-matter-rich soil. Addition of wheat straw had no constant influence on DD mineralization in the soils tested. The best DD mineralization resulted from inoculation with lignolytic white-rot fungi (Phanerochaete chrysosporium, Pleurotus sp. Florida, Dichomitus squalens) and with an unidentified lignolytic fungus, which was isolated originally from a long-term PCDD/F-contaminated soil. A mineralization of up to 50% within 70 days was reached by this treatment. The influence of inoculated fungi on mineralization differed between the soils investigated.

Production of the 14C-labeled insecticidal protein Cry1Ab for soil metabolic studies using a recombinant Escherichia coli in small-scale batch fermentations

Insecticidal Cry proteins naturally produced by Bacillus thuringiensis are a major recombinant trait expressed by genetically modified crops. They are released into the soil during and after cropping. The objective of this study was to produce 14C-labeled Cry1Ab proteins for soil metabolic studies in scope of their environmental risk assessment. Cry1Ab was synthesized as a protoxin by Escherichia coli HB101 pMP in 200-mL liquid batch culture fermentations and purified from inclusion bodies after trypsin digestion. For cultivation, U-14C-glycerol was the main carbon source. Inclusion bodies were smaller and Cry1Ab yield was lower when the initial amount of total organic carbon in the cultivation broth was below 6.4xa0mg C L−1. Concentrations of 12.6xa0g 14C-labeled glycerol L−1 (1xa0%u2009v/v) resulted in the production of 17.1xa0mg 14C-Cry1Ab L−1 cultivation medium. 14C mass balances showed that approx. 50xa0% of the label was lost by respiration and 20xa0% remained in the growth media, while the residual activity was associated with biomass. Depending on the production batch, 0.01 to 0.05xa0% of the total 14C originated from Cry1Ab. In the presence of 2.04xa0MBq 14C-labeled carbon sources, a specific activity of up to 268xa0Bqxa0mg−114C-Cry1Ab was obtained. A more than threefold higher specific activity was achieved with 4.63xa0MBq and an extended cultivation period of 144xa0h. This study demonstrates that 14C-labeled Cry1Ab can be obtained from batch fermentations with E. coli in the presence of a simple 14C-labeled carbon source. It also provides a general strategy to produce 14C-labeled proteins useful for soil metabolic studies.