Toward the future of OECD/ISO biodegradability testing-new approaches and developments.

In the past decades, industrial and scientific communities have developed a complex standardized system (e.g., OECD, ISO, CEN) to evaluate the biodegradability of chemical substances. This system includes for OECD three levels of testing (ready and inherent biodegradability tests, simulation tests). It was adopted by many countries and is completely integrated into European legislation (registration, evaluation, authorization, and restriction of chemicals, REACH). Nevertheless, the different tests have certain deficiencies, and the question arises of how accurately these tests display the situation in the real environment and how the results can be used for predictions. This review will focus on the technical advantages and weaknesses of current tests concerning the technical setup, the inoculum characterization, and its biodegradation potential as well as the use of adequate reference compounds. A special focus of the article will be on combined test systems offering enhanced possibilities to predict biodegradation. The properties of microbial inocula are critically discussed, and a new concept concerning the biodegradation adaptation potential (BAP) of inocula is proposed. Furthermore, a probability model and different in silico QSAR (quantitative structure-activity relationships) models to predict biodegradation from chemical structures are reviewed. Another focus lies on the biodegradation of difficult single compounds and mixtures of chemicals like UVCBs (unknown or variable composition, complex reaction products, or biological materials) which will be an important challenge for the forthcoming decades. KEY POINTS: • There are many technical points to be improved in OECD/ISO biodegradation tests • The proper characterization of inocula is a crucial point in biodegradation tests • Combined biodegradation test systems offer extended possibilities for biodegradation tests.

Adaptation of the metolachlor-degrading fungus Trichoderma harzianum to the simultaneous presence of low-density polyethylene (LDPE) microplastics.

Although it is known that microplastics (MPs) in soils cause a threat to this complex environment, the actual effects of MPs on soil microorganisms and their catabolic activities, particularly with the biodegradation of herbicides, remain unclear. Hence, the objective of this study was to investigate the effects of a simultaneous presence of metolachlor and low-density polyethylene (LDPE) microplastics on growth inhibition and adaptive responses of Trichoderma harzianum in soil microcosms. Using ergosterol content as an indicator of fungal biomass, it was observed that MPs alone had a marginal inhibitory effect on the growth of the fungus, whereas MET exhibited a dose-dependent inhibitory effect on T. harzianum. However, the presence of MPs did not influence the fungal transforming activity toward the herbicide. Conversely, analysis of lipid profiles in the presence of MPs and herbicides revealed a reduction in the overall fluidity of phospholipid fatty acids, primarily attributed to an increase in lysophospholipids. The activities of six extracellular enzymes in the soil, measured using methylumbelliferone-linked substrates, were significantly enhanced in the presence of MET. These findings contribute to a broader understanding of the alterations in fungal activity in soil resulting from the influence of MPs and MET.

In vitro and in vivo lipidomics as a tool for probiotics evaluation.

The probiotic bacteria are helpful for nutritional and therapeutic purposes, and they are commercially available in various forms, such as capsules or powders. Increasing pieces of evidence indicate that different growth conditions and variability in manufacturing processes can determine the properties of probiotic products. In recent years, the lipidomic approach has become a useful tool to evaluate the impact that probiotics induce in host physiology. In this work, two probiotic formulations with identical species composition, produced in two different sites, the USA and Italy, were utilized to feed Caenorhabditis elegans, strains and alterations in lipid composition in the host and bacteria were investigated. Indeed, the multicellular organism C. elegans is considered a simple model to study the in vivo effects of probiotics. Nematodes fat metabolism was assessed by gene expression analysis and by mass spectrometry-based lipidomics. Lipid droplet analysis revealed a high accumulation of lipid droplets in worms fed US-made products, correlating with an increased expression of genes involved in the fatty acid synthesis. We also evaluated the lifespan of worms defective in genes involved in the insulin/IGF-1-mediated pathway and monitored the nuclear translocation of DAF-16. These data demonstrated the involvement of the signaling in C. elegans responses to the two diets. Lipidomics analysis of the two formulations was also conducted, and the results indicated differences in phosphatidylglycerol (PG) and phosphatidylcholine (PC) contents that, in turn, could influence nematode host physiology. Results demonstrated that different manufacturing processes could influence probiotics and host properties in terms of lipid composition. KEY POINTS: • Probiotic formulations impact on Caenorhabditis elegans lipid metabolism; • Lipidomic analysis highlighted phospholipid abundance in the two products; • Phosphocholines and phosphatidylglycerols were analyzed in worms fed the two probiotic formulations.

How to accurately assess surfactant biodegradation-impact of sorption on the validity of results.

Surfactants not only are widely used in biotechnological processes but also constitute significant contaminants of the modern world. Among many reports, there is a shortage of works which summarize the issue of surfactant sorption to biomass in a way that would elucidate the biological factors for analysts and analytical factors for microbiologists. The main factor, which is not as obvious as one would expect, is associated with the susceptibility of analytical approaches to errors resulting from incorrect handling of biomass. In case of several publications reviewed in the framework of this study, it was not possible to establish whether the decrease of the analytical signal observed by the authors actually resulted from biodegradation of the surfactant. This review emphasizes the necessity to consider the possibility of surfactant sorption to microbial cells, which may result in significant detection errors as well as conceptual inconsistency. In addition, a reference study regarding representative surfactants (cationic, anionic and non-ionic) as well as yeast, Gram-negative, Gram-positive bacteria, and activated sludge was provided to highlight the possible errors which may arise from disregarding sorption processes when determining degradation of surfactants. This particularly applies to systems which include ionic surfactants and activated sludge as sorption may account for 90% of the observed depletion of the surfactant. Therefore, a systematic approach was proposed in order to improve the credibility of the obtained results. Finally, the need to employ additional procedures was highlighted which may be required in order to verify that the decrease of surfactant concentration results from biodegradation processes.

Microbial Degradation of Hydrocarbons-Basic Principles for Bioremediation: A Review.

Crude oil-derived hydrocarbons constitute the largest group of environmental pollutants worldwide. The number of reports concerning their toxicity and emphasizing the ultimate need to remove them from marine and soil environments confirms the unceasing interest of scientists in this field. Among the various techniques used for clean-up actions, bioremediation seems to be the most acceptable and economically justified. Analysis of recent reports regarding unsuccessful bioremediation attempts indicates that there is a need to highlight the fundamental aspects of hydrocarbon microbiology in a clear and concise manner. Therefore, in this review, we would like to elucidate some crucial, but often overlooked, factors. First, the formation of crude oil and abundance of naturally occurring hydrocarbons is presented and compared with bacterial ability to not only survive but also to utilize such compounds as an attractive energy source. Then, the significance of nutrient limitation on biomass growth is underlined on the example of a specially designed experiment and discussed in context of bioremediation efficiency. Next, the formation of aerobic and anaerobic conditions, as well as the role of surfactants for maintaining appropriate C:N:P ratio during initial stages of biodegradation is explained. Finally, a summary of recent scientific reports focused on the removal of hydrocarbon contaminants using bioaugmentation, biostimulation and introduction of surfactants, as well as biosurfactants, is presented. This review was designed to be a comprehensive source of knowledge regarding the unique aspects of hydrocarbon microbiology that may be useful for planning future biodegradation experiments. In addition, it is a starting point for wider debate regarding the limitations and possible improvements of currently employed bioremediation strategies.

Quantification of outer membrane vesicles: a potential tool to compare response in Pseudomonas putida KT2440 to stress caused by alkanols.

The bacterial release of outer membrane vesicles (OMVs) is an important physiological mechanism of Gram-negative bacteria playing numerous key roles. One function of the release of OMVs is related to an increase in surface hydrophobicity. This phenomenon initiates biofilm formation, making bacteria more tolerant to environmental stressors. Recently, it was qualitatively shown for Pseudomonas putida that vesicle formation plays a crucial role in multiple stress responses. Yet, no quantification of OMVs for certain stress scenarios has been conducted. In this study, it is shown that the quantification of OMVs can serve as a simple and feasible tool, which allows a comparison of vesicle yields for different experimental setups, cell densities, and environmental stressors. Moreover, the obtained results provide insight to the underlying mechanism of vesicle formation as it was observed that n-alkanols, with a chain length of C7 and longer, caused a distinct and steep increase in vesiculation (12-19-fold), compared to shorter chain n-alkanols (2-4-fold increase).

Changes in bacterial diversity and catabolic gene abundance during the removal of dimethylphenol isomers in laboratory-scale constructed wetlands.

Constructed wetlands (CWs) are well-established wastewater treatment technologies and applied for bioremediation of contaminated water. Despite the optimal performance of CWs, the understanding of the bacterial processes in the rhizosphere, where mainly microbial degradation processes take place, is still limited. In the present study, laboratory-scale CWs planted with Juncus effusus and running under controlled conditions were studied in order to evaluate removal efficiency of dimethylphenols (DMPs), also in comparison to an unplanted bed. Next to removal rates, the bacterial community structure, diversity, and distribution, their correlation with physiochemical parameters, and abundance of the phenol hydroxylase gene were determined. As a result, better removal performance of DMP isomers (3,4-, 3,5-, and 2,6-DMP added as singles compounds or in mixtures) and ammonium loads, together with a higher diversity index, bacterial number, and phenol hydroxylase gene abundance in Juncus effusus CW in comparison with the non-planted CW, indicates a clear rhizosphere effect in the experimental CWs. An enhancement in the DMP removal and the recovery of the phenol hydroxylase gene were found during the fed with the DMP mixture. In addition, the shift of bacterial community in CWs was found to be DMP isomer dependent. Positive correlations were found between the bacteria harboring the phenol hydroxylase gene and communities present with 3,4-DMP and 3,5-DMP isomers, but not with the community developed with 2,6-DMP. These results indicate that CWs are highly dynamic ecosystems with rapid changes in bacterial communities harboring functional catabolic genes.

Hybrid electrochemical and biological treatment of herbicidal ionic liquids comprising the MCPA anion.

The present study was focused on the application of an electrochemical oxidation process combined with biodegradation for the removal of novel Herbicidal Ionic Liquids (HILs) -promising protection plant products which incorporate herbicidal anions and ammonium cations. The influence of carbon chain length (n = 8, 10, 12, 14, 16, 18) in the dialkyldimethylammonium cations on electrochemical oxidation kinetics, degradation efficiency and biodegradation by activated sludge was investigated. It was established that the applied cation influenced the heterogeneous rate constant and diffusion coefficient of electrochemical oxidation. The oxidation efficiency ranged from 17% in case of HILs with C8 alkyl chain to approx. 60% in case of HILs comprising C14 and C16 alkyl chains after 3 h of electrochemical treatment. Subsequent biodegradation studies revealed that electrochemical oxidation improved the mineralization efficiency of the studied HILs. The mineralization efficiency of electrochemically-treated HILs ranged from 28% in case of HILs comprising the C8 alkyl chain to 57% in case of HILs with C14 and C16 alkyl chains after 28 days. In case of untreated HILs, the corresponding mineralization efficiency ranged from 0 to 8%, respectively. This confirms the feasibility of a hybrid electrochemical-biological approach for treatment of herbicidal ionic liquids based on MCPA.

Delftia sp. LCW, a strain isolated from a constructed wetland shows novel properties for dimethylphenol isomers degradation.

BACKGROUND: Dimethylphenols (DMP) are toxic compounds with high environmental mobility in water and one of the main constituents of effluents from petro- and carbochemical industry. Over the last few decades, the use of constructed wetlands (CW) has been extended from domestic to industrial wastewater treatments, including petro-carbochemical effluents. In these systems, the main role during the transformation and mineralization of organic pollutants is played by microorganisms. Therefore, understanding the bacterial degradation processes of isolated strains from CWs is an important approach to further improvements of biodegradation processes in these treatment systems. RESULTS: In this study, bacterial isolation from a pilot scale constructed wetland fed with phenols led to the identification of Delftia sp. LCW as a DMP degrading strain. The strain was able to use the o-xylenols 3,4-DMP and 2,3-DMP as sole carbon and energy sources. In addition, 3,4-DMP provided as a co-substrate had an effect on the transformation of other four DMP isomers. Based on the detection of the genes, proteins, and the inferred phylogenetic relationships of the detected genes with other reported functional proteins, we found that the phenol hydroxylase of Delftia sp. LCW is induced by 3,4-DMP and it is responsible for the first oxidation of the aromatic ring of 3,4-, 2,3-, 2,4-, 2,5- and 3,5-DMP. The enzyme may also catalyze both monooxygenation reactions during the degradation of benzene. Proteome data led to the identification of catechol meta cleavage pathway enzymes during the growth on ortho DMP, and validated that cleavage of the aromatic rings of 2,5- and 3,5-DMPs does not result in mineralization. In addition, the tolerance of the strain to high concentrations of DMP, especially to 3,4-DMP was higher than that of other reported microorganisms from activated sludge treating phenols. CONCLUSIONS: LCW strain was able to degraded complex aromatics compounds. DMPs and benzene are reported for the first time to be degraded by a member of Delftia genus. In addition, LCW degraded DMPs with a first oxidation of the aromatic rings by a phenol hydroxylase, followed by a further meta cleavage pathway. The higher resistance to DMP toxicity, the ability to degrade and transform DMP isomers and the origin as a rhizosphere bacterium from wastewater systems, make LCW a suitable candidate to be used in bioremediation of complex DMP mixtures in CWs systems.

Immediate response mechanisms of Gram-negative solvent-tolerant bacteria to cope with environmental stress: cis-trans isomerization of unsaturated fatty acids and outer membrane vesicle secretion.

Bacteria have evolved an array of adaptive mechanisms enabling them to survive and grow in the presence of different environmental stresses. These mechanisms include either modifications of the membrane or changes in the overall energy status, cell morphology, and cell surface properties. Long-term adaptations are dependent on transcriptional regulation, the induction of anabolic pathways, and cell growth. However, to survive sudden environmental changes, bacterial short-term responses are essential to keep the cells alive after the occurrence of an environmental stress factor such as heat shock or the presence of toxic organic solvents. Thus far, two main short-term responses are known. On the one hand, a fast isomerization of cis into trans unsaturated fatty leads to a quick rigidification of the cell membrane, a mechanism known in some genera of Gram-negative bacteria. On the other hand, a fast, effective, and ubiquitously present countermeasure is the release of outer membrane vesicles (OMVs) from the cell surface leading to a rapid increase in cell surface hydrophobicity and finally to the formation of cell aggregates and biofilms. These immediate response mechanisms just allow the bacteria to stay physiologically active and to employ long-term responses to assure viability upon changing environmental conditions. Here, we provide insight into the two aforementioned rapid adaptive mechanisms affecting ultimately the cell envelope of Gram-negative bacteria.

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.

Biodiversity of soil bacteria exposed to sub-lethal concentrations of phosphonium-based ionic liquids: Effects of toxicity and biodegradation.

Little is known about the effect of ionic liquids (ILs) on the structure of soil microbial communities and resulting biodiversity. Therefore, we studied the influence of six trihexyl(tetradecyl)phosphonium ILs (with either bromide or various organic anions) at sublethal concentrations on the structure of microbial community present in an urban park soil in 100-day microcosm experiments. The biodiversity decreased in all samples (Shannon's index decreased from 1.75 down to 0.74 and OTU's number decreased from 1399 down to 965) with the largest decrease observed in the microcosms spiked with ILs where biodegradation extent was higher than 80%. (i.e. [P66614][Br] and [P66614][2,4,4]). Despite this general decrease in biodiversity, which can be explained by ecotoxic effect of the ILs, the microbial community in the microcosms was enriched with Gram-negative hydrocarbon-degrading genera e.g. Sphingomonas. It is hypothesized that, in addition to toxicity, the observed decrease in biodiversity and change in the microbial community structure may be explained by the primary biodegradation of the ILs or their metabolites by the mentioned genera, which outcompeted other microorganisms unable to degrade ILs or their metabolites. Thus, the introduction of phosphonium-based ILs into soils at sub-lethal concentrations may result not only in a decrease in biodiversity due to toxic effects, but also in enrichment with ILs-degrading bacteria.

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.

Functional Characterization of a 28-Kilobase Catabolic Island from Pseudomonas sp. Strain M1 Involved in Biotransformation of ß-Myrcene and Related Plant-Derived Volatiles.

Pseudomonas sp. strain M1 is able to mineralize highly hydrophobic and recalcitrant compounds, such as benzene, phenol, and their methylated/halogenated derivatives, as well as the backbone of several monoterpenes. The ability to use such a spectrum of compounds as the sole carbon source is, most probably, associated with a genetic background evolved under different environmental constraints. The outstanding performance of strain M1 regarding ß-myrcene catabolism was elucidated in this work, with a focus on the biocatalytical potential of the ß-myrcene-associated core code, comprised in a 28-kb genomic island (GI), predicted to be organized in 8 transcriptional units. Functional characterization of this locus with promoter probes and analytical approaches validated the genetic organization predicted in silico and associated the ß-myrcene-induced promoter activity to the production of ß-myrcene derivatives. Notably, by using a whole-genome mutagenesis strategy, different genotypes of the 28-kb GI were generated, resulting in the identification of a novel putative ß-myrcene hydroxylase, responsible for the initial oxidation of ß-myrcene into myrcen-8-ol, and a sensor-like regulatory protein, whose inactivation abolished the myr+ trait of M1 cells. Moreover, it was demonstrated that the range of monoterpene substrates of the M1 enzymatic repertoire, besides ß-myrcene, also includes other acyclic (e.g., ß-linalool) and cyclic [e.g., R-(+)-limonene and (-)-ß-pinene] molecules. Our findings are the cornerstone for following metabolic engineering approaches and hint at a major role of the 28-kb GI in the biotransformation of a broad monoterpene backbone spectrum for its future biotechnological applications.IMPORTANCE Information regarding microbial systems able to biotransform monoterpenes, especially ß-myrcene, is limited and focused mainly on nonsystematic metabolite identification. Complete and detailed knowledge at the genetic, protein, metabolite, and regulatory levels is essential in order to set a model organism or a catabolic system as a biotechnology tool. Moreover, molecular characterization of reported systems is scarce, almost nonexistent, limiting advances in the development of optimized cell factories with strategies based on the new generation of metabolic engineering platforms. This study provides new insights into the intricate molecular functionalities associated with ß-myrcene catabolism in Pseudomonas, envisaging the production of a molecular knowledge base about the underlying catalytic and regulatory mechanisms of plant-derived volatile catabolic pathways.

Monaibacterium marinum, gen. nov, sp. nov, a new member of the Alphaproteobacteria isolated from seawater of Menai Straits, Wales, UK.

The novel Gram-negative, aerobic, non-motile, non-spore-forming, short-rod bacterium, strain C7T, was isolated from a seawater sample from Menai Straits (Wales, UK) and characterized. Phylogenetic analysis of 16S rRNA gene sequences showed that this strain represented a distinct lineage within the Roseobacterclade of family Rhodobacteracea within Alphaproteobacteria. The members of the genera Pontivivens (Pontivivensinsulae GYSW-23T), Celeribacter (Celeribactermanganoxidans DY2-5T), Donghicola (Donghicola eburneus SW-277T), Roseovarius (Roseovariushalotolerans HJ50T and Roseovariuspacificus 81-2T), Cribrihabitans (Cribrihabitansmarinus CZ-AM5T) and Aestuariihabitans (Aestuariihabitansbeolgyonensis BB-MW15T) were the closest relatives with 16S rRNA gene sequence identities between 93.4 and 95.6 %. Strain C7T could utilize a restricted number of complex substrates with a preference for yeast extract and tryptone, which is consistent with earlier observations that peptides may serve as an important energy and carbon source for bacteria from the Roseobacterclade. Growth occurred in the absence of sodium ions. The isolate C7T is a mesophilic bacterium that optimally grows at 20 °C. The strain can grow under microaerophilic conditions. The major fatty acid was C18 : 1cis d11. The only detected ubiquinone was Q10. The polar lipids of strain C7T were phosphatidylglycerol, two unknown aminolipids and three unknown lipids. The DNA G+C content of the strain was 60.0 mol%. Based on the results of the morphological, physiological and phylogenetic analyses, the new genus, Monaibacterium gen. nov., to include the new species Monaibacterium marinum sp. nov., is proposed. Strain C7T (=DSM 100241T, =LMG 28800T) is the type and only strain of M. marinum.

Rectinema cohabitans gen. nov., sp. nov., a rod-shaped spirochaete isolated from an anaerobic naphthalene-degrading enrichment culture.

The anaerobic, non-motile strain HMT was isolated from the naphthalene-degrading, sulfate-reducing enrichment culture N47. For 20 years, strain HMT has been a stable member of culture N47 although it is neither able to degrade naphthalene nor able to reduce sulfate in pure culture. The highest similarity of the 16S rRNA gene sequence of strain HMT (89 %) is with a cultivated member of the family Spirochaetaceae, Treponema caldariumstrain H1T (=DSM 7334T), an obligately anaerobic, thermophilic spirochaete isolated from cyanobacterial mat samples collected at a freshwater hot spring in Oregon, USA. In contrast to this strain and the majority of spirochaete species described, strain HMT showed a rod-shaped morphology. Growth occurred at temperatures between 12 and 50 °C (optimum 37 °C) but the isolate was not able to grow at 60 °C. The strain fermented various sugars including d-glucose, d-fructose, lactose and sucrose. Addition of 0.1 % (w/v) yeast extract or 0.1 % (w/v) tryptone to the culture medium was essential for growth and could not be replaced by either the vitamin solutions tested or by 0.1 % (w/v) peptone or 0.1 % (w/v) casamino acids. The DNA G+C content of the isolate was 51.5 mol%. The major fatty acids were C14 : 0, C18 : 1ω13c, C16 : 1ω9t, C16 : 1ω11c and C16 : 1ω9c. Based on the unique morphology and the phylogenetic distance from the closest cultivated relative, a novel genus and species, Rectinema cohabitans gen. nov., sp. nov., is proposed. The type strain is strain HMT (=DSM 100378T=JCM 30982T).

Physiological and Transcriptome Response of the Polycyclic Aromatic Hydrocarbon Degrading Novosphingobium sp. LH128 after Inoculation in Soil.

Soil bioaugmentation involves the inoculation of pollutant-degrading bacteria to accelerate pollutant degradation. Often the inoculum shows a dramatic decrease in Colony Forming Units (CFU) upon soil inoculation but this behavior is not well-understood. In this study, the physiology and transcriptomic response of a GFP tagged variant of Novosphingobium sp. LH128 was examined after inoculation into phenanthrene spiked soil. Four hours after inoculation, strain LH128-GFP showed about 99% reduction in CFU while microscopic counts of GFP-expressing cells were identical to the expected initial cell density, indicating that the reduction in CFU number is explained by cells entering into a Viable But Non-Culturable (VBNC)-like state and not by cell death. Transcriptome analysis showed a remarkably higher expression of phenanthrene degradation genes 4 h after inoculation, compared to the inoculum suspension concomitant with an increased expression of genes involved in stress response. This indicates that the cells were active in phenanthrene degradation while experiencing stress. Between 4 h and 10 days, CFU numbers increased to numbers comparable to the inoculated cell density. Our results suggest that strain LH128-GFP enters a VBNC-like state upon inoculation into soil but is metabolically active and that VBNC cells should be taken into account in evaluating bioaugmentation approaches.

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.

In situ†protein-SIP highlights Burkholderiaceae as key players degrading toluene by para ring hydroxylation in a constructed wetland model.

In constructed wetlands, organic pollutants are mainly degraded via microbial processes. Helophytes, plants that are commonly used in these systems, provide oxygen and root exudates to the rhizosphere, stimulating microbial degradation. While the treatment performance of constructed wetlands can be remarkable, a mechanistic understanding of microbial degradation processes in the rhizosphere is still limited. We investigated microbial toluene removal in a constructed wetland model system combining 16S rRNA gene sequencing, metaproteomics and (13) C-toluene in situ protein-based stable isotope probing (protein-SIP). The rhizospheric bacterial community was dominated by Burkholderiales and Rhizobiales, each contributing about 20% to total taxon abundance. Protein-SIP data revealed that the members of Burkholderiaceae, the proteins of which showed about 73% of (13) C-incorporation, were the main degraders of toluene in the planted system, while the members of Comamonadaceae were involved to a lesser extent in degradation (about 64% (13) C-incorporation). Among the Burkholderiaceae, one of the key players of toluene degradation could be assigned to Ralstonia pickettii. We observed that the main pathway of toluene degradation occurred via two subsequent monooxygenations of the aromatic ring. Our study provides a suitable approach to assess the key processes and microbes that are involved in the degradation of organic pollutants in complex rhizospheric ecosystems.

Aerobic Toluene Degraders in the Rhizosphere of a Constructed Wetland Model Show Diurnal Polyhydroxyalkanoate Metabolism.

UNLABELLED: Constructed wetlands (CWs) are successfully applied for the treatment of waters contaminated with aromatic compounds. In these systems, plants provide oxygen and root exudates to the rhizosphere and thereby stimulate microbial degradation processes. Root exudation of oxygen and organic compounds depends on photosynthetic activity and thus may show day-night fluctuations. While diurnal changes in CW effluent composition have been observed, information on respective fluctuations of bacterial activity are scarce. We investigated microbial processes in a CW model system treating toluene-contaminated water which showed diurnal oscillations of oxygen concentrations using metaproteomics. Quantitative real-time PCR was applied to assess diurnal expression patterns of genes involved in aerobic and anaerobic toluene degradation. We observed stable aerobic toluene turnover by Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis was upregulated in these bacteria during the day, suggesting that they additionally feed on organic root exudates while reutilizing the stored carbon compounds during the night via the glyoxylate cycle. Although mRNA copies encoding the anaerobic enzyme benzylsuccinate synthase (bssA) were relatively abundant and increased slightly at night, the corresponding protein could not be detected in the CW model system. Our study provides insights into diurnal patterns of microbial processes occurring in the rhizosphere of an aquatic ecosystem. IMPORTANCE: Constructed wetlands are a well-established and cost-efficient option for the bioremediation of contaminated waters. While it is commonly accepted knowledge that the function of CWs is determined by the interplay of plants and microorganisms, the detailed molecular processes are considered a black box. Here, we used a well-characterized CW model system treating toluene-contaminated water to investigate the microbial processes influenced by diurnal plant root exudation. Our results indicated stable aerobic toluene degradation by members of the Burkholderiales during the day and night. Polyhydroxyalkanoate synthesis in these bacteria was higher during the day, suggesting that they additionally fed on organic root exudates and reutilized the stored carbon compounds during the night. Our study illuminates microbial processes occurring in the rhizosphere of an aquatic ecosystem.