Matthias Kästner

Biodegradation of ciprofloxacin in water and soil and its effects on the microbial communities

While antibiotics are frequently found in the environment, their biodegradability and ecotoxicological effects are not well understood. Ciprofloxacin inhibits active and growing microorganisms and therefore can represent an important risk for the environment, especially for soil microbial ecology and microbial ecosystem services. We investigated the biodegradation of (14)C-ciprofloxacin in water and soil following OECD tests (301B, 307) to compare its fate in both systems. Ciprofloxacin is recalcitrant to biodegradation and transformation in the aqueous system. However, some mineralisation was observed in soil. The lower bioavailability of ciprofloxacin seems to reduce the compounds toxicity against microorganisms and allows its biodegradation. Moreover, ciprofloxacin strongly inhibits the microbial activities in both systems. Higher inhibition was observed in water than in soil and although its antimicrobial potency is reduced by sorption and aging in soil, ciprofloxacin remains biologically active over time. Therefore sorption does not completely eliminate the effects of this compound.

Current approaches for the assessment of in situ biodegradation

Considering the high costs and technical difficulties associated with conventional remediation strategies, in situ biodegradation has become a promising approach for cleaning up contaminated aquifers. To verify if in situ biodegradation of organic contaminants is taking place at a contaminated site and to determine if these processes are efficient enough to replace conventional cleanup technologies, a comprehensive characterization of site-specific biodegradation processes is essential. In recent years, several strategies including geochemical analyses, microbial and molecular methods, tracer tests, metabolite analysis, compound-specific isotope analysis, and in situ microcosms have been developed to investigate the relevance of biodegradation processes for cleaning up contaminated aquifers. In this review, we outline current approaches for the assessment of in situ biodegradation and discuss their potential and limitations. We also discuss the benefits of research strategies combining complementary methods to gain a more comprehensive understanding of the complex hydrogeological and microbial interactions governing contaminant biodegradation in the field.

Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin.

ABSTRACT Carbon stable isotope fractionation of tetrachloroethene (PCE) during reductive dechlorination by whole cells and crude extracts of Sulfurospirillum multivorans and Desulfitobacterium sp. strain PCE-S and the abiotic reaction with cyanocobalamin (vitamin B12) was studied. Fractionation was largest during the reaction with cyanocobalamin with αC = 1.0132. Stable isotope fractionation was lower but still in a similar order of magnitude for Desulfitobacterium sp. PCE-S (αC = 1.0052 to 1.0098). The isotope fractionation of PCE during dehalogenation by S. multivorans was lower by 1 order of magnitude (αC = 1.00042 to 1.0017). Additionally, an increase in isotope fractionation was observed with a decrease in cell integrity for both strains. For Desulfitobacterium sp. strain PCE-S, the carbon stable isotope fractionation factors were 1.0052 and 1.0089 for growing cells and crude extracts, respectively. For S. multivorans, αC values were 1.00042, 1.00097, and 1.0017 for growing cells, crude extracts, and the purified PCE reductive dehalogenase, respectively. For the field application of stable isotope fractionation, care is needed as fractionation may vary by more than an order of magnitude depending on the bacteria present, responsible for degradation.

Classification and Modelling of Nonextractable Residue (NER) Formation of Xenobiotics in Soil – A Synthesis

This review provides a comprehensive overview about nonextractable residue (NER) formation and attempts to classify the various types. Xenobiotic NER derived from parent pesticides (or other environmental contaminants) and primary metabolites sorbed or entrapped within the soil organic matter (Type I) or covalently bound (Type II) pose a considerably higher risk than those derived from productive biodegradation. However, biogenic nonextractable residues (bioNER) (Type III) resulting from conversion of carbon (or nitrogen) from the compounds into microbial biomass molecules do not pose any risk. Experimental approaches to clearly distinguish between the types are provided, and a model to prospectively estimate bioNER formation in soil is proposed.

Formation and Fate of Bound Residues from Microbial Biomass during 2,4-D Degradation in Soil

During organic contaminant degradation in soil, bound or nonextractable residues (NER) are formed. Part of these residues may be biogenic, because degrading microorganisms assimilate carbon derived from the pollutant and mineralized CO(2) to form cellular components for example, [fatty acids (FA) and amino acids (AA)], which are subsequently stabilized within soil organic matter (SOM). We investigated the formation and fate of FA and AA from biodegradation of (13)C(6)-2,4-D in soil and the incorporation of the (13)C-label into living biomass via (13)CO(2) fixation. After 64 days of incubation, (13)C-AA in SOM indicated that 44% of the initially applied (13)C(6)-2,4-D equivalents had been converted to microbial biomass and finally to biogenic residues. The intermediate maximum of (13)C-FA in SOM indicated a 20% conversion of (13)C(6)-2,4-D to biomass, but (13)C-FA decreased to 50% of that value whereas (13)C-AA in the SOM remained stable. We provide the first evidence that nearly all bound residues from 2,4-D are biogenic, containing natural microbial residues stabilized in SOM. Because of biogenic residue formation, the potential risk of bound residues from readily metabolized xenobiotics in soils is highly overestimated. Hence, the formation of biogenic residues must be considered in general when performing mass balances of pollutant biodegradation in soils.

Removal of dichloromethane from waste gases in one- and two-liquid-phase stirred tank bioreactors and biotrickling filters

The removal of dichloromethane (DCM) from polluted air was studied both in biotrickling filters and in continuous stirred tank bioreactors, using either a single-liquid aqueous phase or a combination of an aqueous-organic liquid phase. The presence of the organic phase, i.e. silicone oil, at a volume ratio of 10% of the liquid phase, increased the maximum EC by about 25% in the BTF, reaching 200 gm(3)/h, and by as much as 300% in the CSTB, reaching 350 gm(3)/h. Based on data of chloride release in the aqueous phase and carbon dioxide production in the gas phase, complete dechlorination and mineralization of the pollutant could be confirmed. When applying shock loads, a more stable behaviour was observed in the presence of the organic phase. Generally, the completely mixed reactors were also more stable than the plug-flow biotrickling filters, irrespective of the presence of the organic phase. The use of molecular techniques allowed showing that the originally inoculated DCM-degrading Hyphomicrobium strains remained present, although not dominant, after long-term bioreactor operation. Different new bacterial populations did also appear in the systems, some of which were unable to degrade DCM.

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [(13)C]-alpha-toluene or [(13)C(7)]-toluene as growth substrates. Cells incubated with [(13)C]-alpha-toluene or [(13)C(7)]-toluene incorporated 8-15 at.%(13)C and 51-57 at.%(13)C into total lipid fatty acids, respectively, indicating a lower specific incorporation of (13)C from [(13)C(7)]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [(13)C]-alpha-toluene and [(13)C(7)]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.

Non-phototrophic CO2 fixation by soil microorganisms

Although soils are generally known to be a net source of CO2 due to microbial respiration, CO2 fixation may also be an important process. The non-phototrophic fixation of CO2 was investigated in a tracer experiment with 14CO2 in order to obtain information about the extent and the mechanisms of this process. Soils were incubated for up to 91 days in the dark. In three independent incubation experiments, a significant transfer of radioactivity from 14CO2 to soil organic matter was observed. The process was related to microbial activity and could be enhanced by the addition of readily available substrates such as acetate. CO2 fixation exhibited biphasic kinetics and was linearly related to respiration during the first phase of incubation (about 20–40 days). The fixation amounted to 3–5% of the net respiration. After this phase, the CO2 fixation decreased to 1–2% of the respiration. The amount of carbon fixed by an agricultural soil corresponded to 0.05% of the organic carbon present in the soil at the beginning of the experiment, and virtually all of the fixed CO2 was converted to organic compounds. Many autotrophic and heterotrophic biochemical processes result in the fixation of CO2. However, the enhancement of the fixation by addition of readily available substrates and the linear correlation with respiration suggested that the process is mainly driven by aerobic heterotrophic microorganisms. We conclude that heterotrophic CO2 fixation represents a significant factor of microbial activity in soils.

Different influences of DNA purity indices and quantity on PCR-based DGGE and functional gene microarray in soil microbial community study

Based on the comparative study of the DNA extracts from two soil samples obtained by three commercial DNA extraction kits, we evaluated the influence of the DNA quantity and purity indices (the absorbance ratios A260/280 and A260/230, as well as the absorbance value A320 indicating the amount of humic substances) on polymerase chain reaction (PCR)-based denaturing gradient gel electrophoresis (DGGE) and a functional gene microarray used in the study of microbial communities. Numbers and intensities of the DGGE bands are more affected by the A260/280 and A320 values than by the ratio A260/230 and conditionally affected by the DNA yield. Moreover, we demonstrated that the DGGE band pattern was also affected by the preferential extraction due to chemical agents applied in the extraction. Unlike DGGE, microarray is more affected by the A260/230 and A320 values. Until now, the successful PCR performance is the mostly used criterion for soil DNA purity. However, since PCR was more influenced by the A260/280 ratio than by A260/230, it is not accurate enough any more for microbial community assessed by non-PCR-based methods such as microarray. This study provides some useful hints on how to choose effective DNA extraction method for the subsequent assessment of microbial community.

In vivo emission of dinitrogen by earthworms via denitrifying bacteria in the gut

ABSTRACT Earthworms emit the greenhouse gas nitrous oxide (N2O), and ingested denitrifiers in the gut appear to be the main source of this N2O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N2), the end product of complete denitrification. When [15N]nitrate was injected into the gut, the earthworms Aporrectodea caliginosa and Lumbricus terrestris emitted labeled N2 (and also labeled N2O) under in vivo conditions; emission of N2 by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N2 per h per g (fresh weight), respectively. Isolated gut contents also produced [15N]nitrate-derived N2 and N2O under anoxic conditions. N2 is formed by N2O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [15N]nitrate-derived N2 by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N2O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N2 (i.e., without added nitrate) by A. caliginosa and L. terrestris were 1.1 and 1.5 nmol N2 per h per g (fresh weight), respectively; these emission rates approximated that of N2O. These collective observations indicate that (i) earthworms emit N2 concomitant with the emission of N2O via the in situ activity of denitrifying bacteria in the gut and (ii) N2O is quantitatively an important denitrification-derived end product under in situ conditions.