Unraveling the effects of acrylonitrile butadiene styrene (ABS) microplastic ageing on the sorption and toxicity of ionic liquids with 2,4-D and glyphosate herbicides.

Microplastics represent a novel category of environmental pollutants, and understanding their interactions with typical xenobiotics is crucial. In this study, we investigated the impact of ionic liquids (ILs) containing herbicidal anions, namely glyphosate [Glyph] and 2,4-dichlorophenoxyacetate [2,4-D], and the surfactant cation - dodecyltrimethylammonium [C12TMA] on acrylonitrile butadiene styrene (ABS) microplastics. The aim of the study was to assess the sorption capacity of microplastics that were present in both untreated and aged form using standard and modified Fenton methods. In addition, impact on toxicity and stress adaptation of the model soil bacterium Pseudomonas putida KT2440 was measured. Upon ageing, ABS microplastics underwent a fivefold increase in BET surface area and total pore volume (from 0.001 to 0.004 cm3/g) which lead to a dramatic increase in adsorption of the cations on ABS microplastics from 40 to 45% for virgin ABS to 75-80% for aged ABS. Toxicity was mainly attributed to hydrophobic cations in ILs (EC50 ∼ 60-65 mg/dm3), which was also mitigated by sorption on ABS. Furthermore, both cations and anions behaved similarly across different ILs, corresponding chlorides, and substrates used in the ILs synthesis. These findings highlight microplastics potential as hazardous sorbents, contributing to the accumulation of xenobiotics in the environment.

Production of (hydroxy)benzoate-derived polyketides by engineered Pseudomonas with in situ extraction.

Polyketides from (hydroxy)benzoates are an interesting group of plant polyphenolic compounds, whose biotechnological production is so far underrepresented due to their challenging heterologous biosynthesis. Efficient heterologous production of 2,4,6-tri- and 2,3',4,6-tetrahydroxybenzophenone, 3,5-dihydroxybiphenyl, and 4-hydroxycoumarin by whole-cell biocatalysis in combination with in situ product extraction with an organic solvent was demonstrated. Production was highly dependent on the used CoA ligase and polyketide synthase type III. Therefore, different combinations of polyketide synthases and benzoate-CoA ligases were evaluated for their biosynthesis performance in the solvent-tolerant Pseudomonas taiwanensis VLB120. A solvent screening yielded 2-undecanone as biocompatible, extraction-efficient solvent with good phase separation. In aqueous-organic two-phase cultivations, this solvent extraction circumvents product instability in the aqueous cultivation medium, and it increases yields by reducing inhibitory effects. Complete de novo synthesis from glucose of all (hydroxy)benzoate-derived polyketides was achieved in two-phase cultivations with metabolically engineered strains. Additionally, mutasynthesis was applied to obtain fluorinated benzophenone derivatives.

An optimized method for RNA extraction from the polyurethane oligomer degrading strain Pseudomonas capeferrum TDA1 growing on aromatic substrates such as phenol and 2,4-diaminotoluene.

Bacterial degradation of xenobiotic compounds is an intense field of research already for decades. Lately, this research is complemented by downstream applications including Next Generation Sequencing (NGS), RT-PCR, qPCR, and RNA-seq. For most of these molecular applications, high-quality RNA is a fundamental necessity. However, during the degradation of aromatic substrates, phenolic or polyphenolic compounds such as polycatechols are formed and interact irreversibly with nucleic acids, making RNA extraction from these sources a major challenge. Therefore, we established a method for total RNA extraction from the aromatic degrading Pseudomonas capeferrum TDA1 based on RNAzol® RT, glycogen and a final cleaning step. It yields a high-quality RNA from cells grown on TDA1 and on phenol compared to standard assays conducted in the study. To our knowledge, this is the first report tackling the problem of polyphenolic compound interference with total RNA isolation in bacteria. It might be considered as a guideline to improve total RNA extraction from other bacterial species.

Membrane Fatty Acid Composition and Cell Surface Hydrophobicity of Marine Hydrocarbonoclastic Alcanivorax borkumensis SK2 Grown on Diesel, Biodiesel and Rapeseed Oil as Carbon Sources.

The marine hydrocarbonoclastic bacterium Alcanivorax borkumensis is well known for its ability to successfully degrade various mixtures of n-alkanes occurring in marine oil spills. For effective growth on these compounds, the bacteria possess the unique capability not only to incorporate but also to modify fatty intermediates derived from the alkane degradation pathway. High efficiency of both these processes provides better competitiveness for a single bacteria species among hydrocarbon degraders. To examine the efficiency of A. borkumensis to cope with different sources of fatty acid intermediates, we studied the growth rates and membrane fatty acid patterns of this bacterium cultivated on diesel, biodiesel and rapeseed oil as carbon and energy source. Obtained results revealed significant differences in both parameters depending on growth substrate. Highest growth rates were observed with biodiesel, while growth rates on rapeseed oil and diesel were lower than on the standard reference compound (hexadecane). The most remarkable observation is that cells grown on rapeseed oil, biodiesel, and diesel showed significant amounts of the two polyunsaturated fatty acids linoleic acid and linolenic acid in their membrane. By direct incorporation of these external fatty acids, the bacteria save energy allowing them to degrade those pollutants in a more efficient way. Such fast adaptation may increase resilience of A. borkumensis and allow them to strive and maintain populations in more complex hydrocarbon degrading microbial communities.

Formulation and stabilization of an Arthrobacter strain with good storage stability and 4-chlorophenol-degradation activity for bioremediation.

Chlorophenols are widespread and of environmental concern due to their toxic and carcinogenic properties. Development of less costly and less technically challenging remediation methods are needed; therefore, we developed a formulation based on micronized vermiculite that, when air-dried, resulted in a granular product containing the 4-chlorophenol (4-CP)-degrading Gram-positive bacterium Arthrobacter chlorophenolicus A6. This formulation and stabilization method yielded survival rates of about 60% that remained stable in storage for at least 3 months at 4 °C. The 4-CP degradation by the formulated and desiccated A. chlorophenolicus A6 cells was compared to that of freshly grown cells in controlled-environment soil microcosms. The stabilized cells degraded 4-CP equally efficient as freshly grown cells in two different set-ups using both hygienized and non-treated soils. The desiccated microbial product was successfully employed in an outdoor pot trial showing its effectiveness under more realistic environmental conditions. No significant phytoremediation effects on 4-CP degradation were observed in the outdoor pot experiment. The 4-CP degradation kinetics from both the microcosms and the outdoor pot trial were used to generate a predictive model of 4-CP biodegradation potentially useful for larger-scale operations, enabling better bioremediation set-ups and saving of resources. This study also opens up the possibility of formulating and stabilizing also other Arthrobacter strains possessing different desirable pollutant-degrading capabilities.

Arsenite response in Coccomyxa sp. Carn explored by transcriptomic and non-targeted metabolomic approaches.

Arsenic is a toxic metalloid known to generate an important oxidative stress in cells. In the present study, we focused our attention on an alga related to the genus Coccomyxa, exhibiting an extraordinary capacity to resist high concentrations of arsenite and arsenate. The integrated analysis of high-throughput transcriptomic data and non-targeted metabolomic approaches highlighted multiple levels of protection against arsenite. Indeed, Coccomyxa sp. Carn induced a set of transporters potentially preventing the accumulation of this metalloid in the cells and presented a distinct arsenic metabolism in comparison to another species more sensitive to that compound, i.e. Euglena gracilis, especially in regard to arsenic methylation. Interestingly, Coccomyxa sp. Carn was characterized by a remarkable accumulation of the strong antioxidant glutathione (GSH). Such observation could explain the apparent low oxidative stress in the intracellular compartment, as suggested by the transcriptomic analysis. In particular, the high amount of GSH in the cell could play an important role for the tolerance to arsenate, as suggested by its partial oxidation into oxidized glutathione in presence of this metalloid. Our results therefore reveal that this alga has acquired multiple and original defence mechanisms allowing the colonization of extreme ecosystems such as acid mine drainages.

Genome sequence and functional genomic analysis of the oil-degrading bacterium Oleispira antarctica.

Ubiquitous bacteria from the genus Oleispira drive oil degradation in the largest environment on Earth, the cold and deep sea. Here we report the genome sequence of Oleispira antarctica and show that compared with Alcanivorax borkumensis--the paradigm of mesophilic hydrocarbonoclastic bacteria--O. antarctica has a larger genome that has witnessed massive gene-transfer events. We identify an array of alkane monooxygenases, osmoprotectants, siderophores and micronutrient-scavenging pathways. We also show that at low temperatures, the main protein-folding machine Cpn60 functions as a single heptameric barrel that uses larger proteins as substrates compared with the classical double-barrel structure observed at higher temperatures. With 11 protein crystal structures, we further report the largest set of structures from one psychrotolerant organism. The most common structural feature is an increased content of surface-exposed negatively charged residues compared to their mesophilic counterparts. Our findings are relevant in the context of microbial cold-adaptation mechanisms and the development of strategies for oil-spill mitigation in cold environments.

Bacterial metabolism of environmental arsenic--mechanisms and biotechnological applications.

Arsenic causes threats for environmental and human health in numerous places around the world mainly due to its carcinogenic potential at low doses. Removing arsenic from contaminated sites is hampered by the occurrence of several oxidation states with different physicochemical properties. The actual state of arsenic strongly depends on its environment whereby microorganisms play important roles in its geochemical cycle. Due to its toxicity, nearly all organisms possess metabolic mechanisms to resist its hazardous effects, mainly by active extrusion, but also by extracellular precipitation, chelation, and intracellular sequestration. Some microbes are even able to actively use various arsenic compounds in their metabolism, either as an electron donor or as a terminal electron acceptor for anaerobic respiration. Some microorganisms can also methylate inorganic arsenic, probably as a resistance mechanism, or demethylate organic arsenicals. Bioavailability of arsenic in water and sediments is strongly influenced by such microbial activities. Therefore, understanding microbial reactions to arsenic is of importance for the development of technologies for improved bioremediation of arsenic-contaminated waters and environments. This review gives an overview of the current knowledge on bacterial interactions with arsenic and on biotechnologies for its detoxification and removal.

Biostimulation by methanol enables the methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. to reveal high formaldehyde biodegradation potential as well as to adapt to this toxic pollutant.

The methylotrophic yeasts Hansenula polymorpha and Trichosporon sp. revealed enhanced biodegradation capability of exogenously applied formaldehyde (Fd) upon biostimulation achieved by the presence of methanol, as compared to glucose. Upon growth on either of the above substrates, the strains proved to produce the activity of glutathione-dependent formaldehyde dehydrogenase-the enzyme known to control the biooxidative step of Fd detoxification. However, in the absence of methanol, the yeasts' tolerance to Fd was decreased, and the elevated sensitivity was especially pronounced for Trichosporon sp. Both strains responded to the methanol and/or Fd treatment by increasing their unsaturation index (UI) at xenobiotic levels below minimal inhibitory concentrations. This indicated that the UI changes effected from the de novo synthesis of (poly) unsaturated fatty acids carried out by viable cells. It is concluded that the yeast cell response to Fd intoxication involves stress reaction at the level of membranes. Fluidization of the lipid bilayer as promoted by methanol is suggested as a significant adaptive mechanism increasing the overall fitness enabling to cope with the formaldehyde xenobiotic via biodegradative pathway of C1-compound metabolism.

Klebsiella sp. strain C2A isolated from olive oil mill waste is able to tolerate and degrade tannic acid in very high concentrations.

Four bacterial strains capable of growing in the presence of tannic acid as sole carbon and energy source were isolated from olive mill waste mixtures. 16S rRNA gene sequencing assigned them to the genus Klebsiella. The most efficient strain, Klebsiella sp. strain C2A, was able to degrade 3.5 g L(-1) tannic acid within 35 h with synthesizing gallic acid as main product. The capability of Klebsiella sp. strain C2A to produce tannase was evidenced at high concentrations of tannic acid up to 50 g L(-1) . The bacteria adapted to the toxicity of tannic acids by an increase in the membrane lipid fatty acids degree of saturation, especially in the presence of concentrations higher than 20 g L(-1) . The highly tolerant and adaptable bacterial strain characterized in this study could be used in bioremediation processes of wastes rich in polyphenols such as those derived from olive mills, winery or tanneries.

Membrane vesicle formation as a multiple-stress response mechanism enhances Pseudomonas putida DOT-T1E cell surface hydrophobicity and biofilm formation.

Among the adaptive responses of bacteria to rapid changes in environmental conditions, those of the cell envelope are known to be the most crucial. Therefore, several mechanisms with which bacteria change their cell surface and membranes in the presence of different environmental stresses have been elucidated. Among these mechanisms, the release of outer membrane vesicles (MV) in Gram-negative bacteria has attracted particular research interest because of its involvement in pathogenic processes, such as that of Pseudomonas aeruginosa biofilm formation in cystic fibrosis lungs. In this study, we investigated the role of MV formation as an adaptive response of Pseudomonas putida DOT-T1E to several environmental stress factors and correlated it to the formation of biofilms. In the presence of toxic concentrations of long-chain alcohols, under osmotic stress caused by NaCl, in the presence of EDTA, and after heat shock, cells of this strain released MV within 10 min in the presence of a stressor. The MV formed showed similar size and charge properties, as well as comparable compositions of proteins and fatty acids. MV release caused a significant increase in cell surface hydrophobicity, and an enhanced tendency to form biofilms was demonstrated in this study. Therefore, the release of MV as a stress response could be put in a physiological context.

Desulfitobacterium aromaticivorans sp. nov. and Geobacter toluenoxydans sp. nov., iron-reducing bacteria capable of anaerobic degradation of monoaromatic hydrocarbons.

Dissimilatory iron reduction plays a significant role in subsurface environments. Currently, it is assumed that members of the genus Geobacter constitute the majority of the iron-reducing micro-organisms that oxidize aromatic compounds in contaminated subsurface environments. Here, we report the isolation of two phylogenetically distinct pure cultures of iron-reducing degraders of monoaromatic hydrocarbons, strain TMJ1(T), which belongs to the genus Geobacter within the Deltaproteobacteria, and strain UKTL(T), belonging to the genus Desulfitobacterium within the Clostridia. Both strains utilize a wide range of substrates as carbon and energy sources, including the aromatic compounds toluene, phenol and p-cresol. Additionally, strain UKTL(T) utilizes o-xylene and TMJ1(T) utilizes m-cresol. Anaerobic degradation of toluene in both strains and o-xylene in strain UKTL(T) is initiated by activation with fumarate addition to the methyl group. The genomic DNA G+C contents of strains TMJ1(T) and UKTL(T) are 54.4 and 47.7 mol%, respectively. Based on a detailed physiological characterization and phylogenetic analysis of the 16S rRNA genes of both strains, we propose the names Desulfitobacterium aromaticivorans sp. nov. (type strain UKTL(T) =DSM 19510(T) =JCM 15765(T)) and Geobacter toluenoxydans sp. nov. (type strain TMJ1(T) =DSM 19350(T) =JCM 15764(T)) to accommodate these strains. To the best of our knowledge, strain UKTL(T) is the first described spore-forming, iron-reducing bacterium that can degrade aromatic hydrocarbons.

Energetics and surface properties of Pseudomonas putida DOT-T1E in a two-phase fermentation system with 1-decanol as second phase.

The solvent-tolerant strain Pseudomonas putida DOT-T1E was grown in batch fermentations in a 5-liter bioreactor in the presence and absence of 10% (vol/vol) of the organic solvent 1-decanol. The growth behavior and cellular energetics, such as the cellular ATP content and the energy charge, as well as the cell surface hydrophobicity and charge, were measured in cells growing in the presence and absence of 1-decanol. Although the cells growing in the presence of 1-decanol showed an about 10% reduced growth rate and a 48% reduced growth yield, no significant differences were measured either in the ATP and potassium contents or in the energy charge, indicating that the cells adapted completely at the levels of membrane permeability and energetics. Although the bacteria needed additional energy for adaptation to the presence of the solvent, they were able to maintain or activate electron transport phosphorylation, allowing homeostasis of the ATP level and energy charge in the presence of the solvent, at the price of a reduced growth yield. On the other hand, significantly enhanced cell hydrophobicities and more negative cell surface charges were observed in cells grown in the presence of 1-decanol. Both reactions occurred within about 10 min after the addition of the solvent and were significantly different after killing of the cells with toxic concentrations of HgCl2. This adaptation of the surface properties of the bacterium to the presence of solvents seems to be very similar to previously observed reactions on the level of lipopolysaccharides, with which bacteria adapt to environmental stresses, such as heat shock, antibiotics, or low oxygen content. The results give clear physiological indications that the process with P. putida DOT-T1E as the biocatalyst and 1-decanol as the solvent is a stable system for two-phase biotransformations that will allow the production of fine chemicals in economically sound amounts.

The cis-trans isomerase of unsaturated fatty acids in Pseudomonas and Vibrio: biochemistry, molecular biology and physiological function of a unique stress adaptive mechanism.

Isomerization of cis to trans unsaturated fatty acids is a mechanism enabling Gram-negative bacteria belonging to the genera Pseudomonas and Vibrio to adapt to several forms of environmental stress. The extent of the isomerization apparently correlates with the fluidity effects caused, i.e. by an increase in temperature or the accumulation of membrane-toxic organic compounds. Trans fatty acids are generated by direct isomerization of the respective cis configuration of the double bond without a shift of its position. The conversion of cis unsaturated fatty acids to trans is apparently instrumental in the adaptation of membrane fluidity to changing chemical or physical parameters of the cellular environment. Such an adaptive mechanism appears to be an alternative way to regulate membrane fluidity when growth is inhibited, e.g. by high concentrations of toxic substances. The cis-trans isomerase (Cti) activity is constitutively present and is located in the periplasma, it requires neither ATP nor any other cofactor such as NAD(P)H or glutathione, and it operates in the absence of de novo synthesis of lipids. Its independence from ATP is in agreement with the negative free energy of the reaction. cti encodes a polypeptide with an N-terminal hydrophobic signal sequence, which is cleaved off during or shortly after the enzyme is transported across the cytoplasmic membrane to the periplasmic space. A functional heme-binding site of the cytochrome c-type was identified in the predicted Cti polypeptide and very recently, direct evidence was obtained that isomerization does not include a transient saturation of the double bond.