Functional response of Acinetobacter guillouiae SFC 500-1A to tannery wastewater as revealed by a complementary proteomic approach.

Acinetobacter guillouiae SFC 500-1 A is a promising candidate for the bioremediation of tannery wastewater. In this study, we applied shotgun proteomic technology in conjunction with a gel-based assay (Gel-LC) to explore the strain's intracellular protein profile when grown in tannery wastewater as opposed to normal culture conditions. A total of 1775 proteins were identified, 52 of which were unique to the tannery wastewater treatment. Many of them were connected to the degradation of aromatic compounds and siderophore biosynthesis. On the other hand, 1598 proteins overlapped both conditions but were differentially expressed in each. Those that were upregulated in wastewater (109) were involved in the processes mentioned above, as well as in oxidative stress mitigation and intracellular redox state regulation. Particularly interesting were the downregulated proteins under the same treatment (318), which were diverse but mainly linked to the regulation of basic cellular functions (replication, transcription, translation, cell cycle, and wall biogenesis); metabolism (amino acids, lipids, sulphate, energetic processes); and other more complex responses (cell motility, exopolysaccharide production, biofilm formation, and quorum sensing). The findings suggest that SFC 500-1 A engages in survival and stress management strategies to cope with the toxic effects of tannery wastewater, and that such strategies may be mostly oriented at keeping metabolic processes to a minimum. Altogether, the results might be useful in the near future to improve the strain's effectiveness if it will be applied for bioremediation.

Sequential application of activated sludge and phytoremediation with aquatic macrophytes on tannery effluents: in search of a complete treatment.

Tannery effluents with a high organic matter load (indicated by their COD level) have to be treated before they are discharged, so as to minimize their negative impact on the environment. Using field mesocosm systems, this study evaluated the feasibility of treating such effluents through bioaugmentation with activated sludge, followed by phytoremediation with aquatic macrophytes (Lemnoideae subfamily). Regardless of its quality, the activated sludge was able to remove approximately 77% of the COD from effluents with a low initial organic load (up to 1500 mg/L). The macrophytes then enhanced removal (up to 86%), so the final COD values were permissible under the current legislation for effluent discharge. When the initial organic load in the undiluted effluents was higher (around 3000 mg/L), the COD values obtained after consecutive bioaugmentation and phytoremediation were close to the legally allowed limits (583 mg/L), which highlights the potential of phytoremediation as a tertiary treatment. This treatment also brought total coliform counts down to legally acceptable values, without plant biomass decreasing over time. Moreover, the plant biomass remained viable and capable of high COD removal efficiency (around 75%) throughout two additional reuse cycles. These findings indicate that the efficiency of the biological treatments assayed here depends largely on the initial organic load in the tannery effluents. In any case, the sequential application of activated sludge and aquatic macrophytes proved to be a successful alternative for remediation.

Proteomic analysis to unravel the biochemical mechanisms triggered by Bacillus toyonensis SFC 500-1E under chromium(VI) and phenol stress.

Bacillus toyonensis SFC 500-1E is a member of the consortium SFC 500-1 able to remove Cr(VI) and simultaneously tolerate high phenol concentrations. In order to elucidate mechanisms utilized by this strain during the bioremediation process, the differential expression pattern of proteins was analyzed when it grew with or without Cr(VI) (10 mg/L) and Cr(VI) + phenol (10 and 300 mg/L), through two complementary proteomic approaches: gel-based (Gel-LC) and gel-free (shotgun) nanoUHPLC-ESI-MS/MS. A total of 400 differentially expressed proteins were identified, out of which 152 proteins were down-regulated under Cr(VI) and 205 up-regulated in the presence of Cr(VI) + phenol, suggesting the extra effort made by the strain to adapt itself and keep growing when phenol was also added. The major metabolic pathways affected include carbohydrate and energetic metabolism, followed by lipid and amino acid metabolism. Particularly interesting were also ABC transporters and the iron-siderophore transporter as well as transcriptional regulators that can bind metals. Stress-associated global response involving the expression of thioredoxins, SOS response, and chaperones appears to be crucial for the survival of this strain under treatment with both contaminants. This research not only provided a deeper understanding of B. toyonensis SFC 500-1E metabolic role in Cr(VI) and phenol bioremediation process but also allowed us to complete an overview of the consortium SFC 500-1 behavior. This may contribute to an improvement in its use as a bioremediation strategy and also provides a baseline for further research.

Biohybrid membranes for effective bacterial vehiculation and simultaneous removal of hexavalent chromium (CrVI) and phenol.

The aim of the present study was to obtain an effective vehiculation system in which bacterial agents could maintain viability improving their removal capacity. Herein, we present a novel biohybrid membrane of polymeric nanofibers and free-living bacteria for the simultaneous removal of pollutants. In this system, bacteria are free within the pores between the nanofibers and adsorbed to the surface of the membranes. Association between bacteria and the membranes was performed through a self-formulated medium, and the presence of the bacteria in the polymeric matrix was evidenced through atomic force microscopy (AFM). Biohybrid membranes associated with the remediation agents Bacillus toyonensis SFC 500-1E and Acinetobacter guillouiae SFC 500-1A promoted a reduction of up to 2.5 mg/L of hexavalent chromium and up to 200 mg/L of phenol after 24 h of treatment in synthetic medium containing the contaminants. Similarly, more than 46% of the hexavalent chromium and all of the phenol content were removed after treatment of a tannery effluent with initial concentrations of 7 mg/L of Cr(VI) and 305 mg/L of phenol. Counts of the remediation agents from the membranes were always above 1.107 CFU/g, also in the reutilization assays performed without reinoculation. Biohybrid membranes were hydrolysis-resistant, reusable, and effective in the simultaneous removal of contaminants for more than 5 cycles. Viability of the microorganisms was maintained after long-term storage of the membranes at 4 °C, without the use of microbiological media or the addition of cryoprotectants. Graphical abstract KEY POINTS: • Polymeric membranes were effectively associated with the SFC 500-1 remediation consortium • Biohybrid membranes removed hexavalent chromium and phenol from different matrices • Removal of contaminants was achieved in many successive cycles without reinoculation.

Polyphasic characterization and identification of the bioremediation agent Bacillus sp. SFC 500-1E.

Bacillus sp. SFC 500-1E is used for the effective treatment of tannery effluents since it consistently removes hexavalent chromium from diverse contaminated matrices. The aim of the present study was to complete identification of the strain through a polyphasic characterization, which included the pattern of carbohydrate utilization, fatty acids profile, multilocus sequence analysis, multiplex PCR profile and the analysis of the complete genome sequence. Morpho-physiological and biochemical characterization results and analysis of 16S rRNA sequences were not conclusive. The strain formed a monophyletic clade with B. toyonensis BCT-7112, B. thuringiensis MC28 and B. cereus Rock 1-3. However, genomic comparisons with type strains of B. cereus and B. thuringiensis showed that the isolated belonged to a different species. Results of this study highlight the relevance of the genome sequence of this strain, identified as Bacillus toyonensis SFC 500-1E, to expand knowledge of its bioremediation potential and to explore unknown decontamination activities.

Impact assessment of bioaugmented tannery effluent discharge on the microbiota of water bodies.

Effluents are commonly discharged into water bodies, and in order for the process to be as environmentally sound as possible, the potential effects on native water communities must be assessed alongside the quality parameters of the effluents themselves. In the present work, changes in the bacterial diversity of streamwater receiving a tannery effluent were monitored by high-throughput MiSeq sequencing. Physico-chemical and microbiological parameters and acute toxicity were also evaluated through different bioassays. After the discharge of treated effluents that had been either naturally attenuated or bioaugmented, bacterial diversity decreased immediately in the streamwater samples, as evidenced by the over-representation of taxa such as Brachymonas, Arcobacter, Marinobacterium, Myroides, Paludibacter and Acinetobacter, typically found in tannery effluents. However, there were no remarkable changes in diversity over time (after 1 day). In terms of the physico-chemical and microbiological parameters analyzed, chemical oxygen demand and total bacterial count increased in response to discharge of the treated effluents. No lethal effects were observed in Lactuca sativa L. seeds or Rhinella arenarum embryos exposed to the streamwater that had received the treated effluents. All of these results contribute to the growing knowledge about the environmental safety of effluent discharge procedures.

Morphological and structural response of Bacillus sp. SFC 500-1E after Cr(VI) and phenol treatment.

Bacillus sp. SFC 500-1E, a bacterial strain isolated from tannery sediments, is able to remove Cr(VI) and simultaneously tolerate high concentrations of phenol. In this study, we used high-resolution microscopies, fluorescence polarization techniques, and several biochemical approaches to improve our understanding about the adaptive mechanisms of this strain to survive in the presence of Cr(VI) and phenol, both individually and simultaneously. Among adaptive strategies developed by Bacillus sp. SFC 500-1E, an increase in bacterial size, such as length, width, and height, and ultrastructural alterations, such as electron-dense precipitates, the presence of exopolymers, and cell lysis, are noteworthy. The exopolymers observed were consistent with the extensive biofilm formation and exopolysaccharides and extracellular protein quantification. At the cell membrane level, a rapid rigidity was induced in Cr(VI) + phenol treatment. This effect was counteracted after 16 h by changes at the level of phospholipids, mainly in the composition of fatty acids (FAs); in particular, an increase in the unsaturated fatty acid/saturated fatty acid ratio was detected. This study shows evidence of some adaptive responses displayed by Bacillus sp. SFC 500-1E, which allows it to survive in stressful conditions.

Membrane Rigidity and Phosphatidic Acid (PtdOH) Signal: Two Important Events in Acinetobacter guillouiae SFC 500-1A Exposed to Chromium(VI) and Phenol.

The remodeling of membrane lipids is a mechanism that allows microorganisms to survive in unfavorable environments such as industrial effluents, which often contain inorganic and organic pollutants, like chromium and phenol. In the present work, we evaluated the effect of Cr(VI) and phenol on the membrane of Acinetobacter guillouiae SFC 500-1A, a bacterial strain isolated from tannery sediments where such pollutants can be found. The presence of lipid kinases and phospholipases and the changes in their activities under exposure to these pollutants were determined. Cr(VI) and Cr(VI) + phenol caused the membrane to become more rigid for up to 16 h after exposure. This could be due to an increase in cardiolipin (Ptd2 Gro) and a decrease in phosphatidylethanolamine (PtdEtn), which are indicative of more order and rigidity in the membrane. Increased phospholipase A activity (PLA, EC 3.1.1.4) could be responsible for the decrease in PtdEtn levels. Moreover, our results indicate that Cr(VI) and Cr(VI) + phenol trigger the phosphatidic acid (PtdOH) signal. The finding of significantly increased phosphatidylinositol-4-phosphate (PtdIns-4-P) levels means this is likely achieved via PtdIns-PLC/DGK. This report provides the first evidence that A. guillouiae SFC 500-1A is able to sense Cr(VI) and phenol, transduce this signal through changes in the physical state of the membrane, and trigger lipid-signaling events.

How the bacterial community of a tannery effluent responds to bioaugmentation with the consortium SFC 500-1. Impact of environmental variables.

Bioaugmentation with the consortium SFC 500-1 is a promising alternative to remediate wastewaters, such as tannery effluents. With the aim of assessing the changes produced in response to bioaugmentation, bacterial 16S rDNA genes were sequenced with Illumina MiSeq Platform. Additionally, bacterial and fungal groups were analyzed through standard culture dependent methods. The impact of diverse physico-chemical and microbiological parameters on the prokaryotic diversity was also evaluated throughout. Bacteroidetes, Firmicutes and Proteobacteria, represented together up to 91% of the total number of sequences obtained from the tannery effluent. Diversity decreased immediately after inoculation, due to an increase in the representation of the taxa to which the added consortium belongs. However, bioaugmentation produced no greater variations since only a 10% of unique operational taxonomic units were found in the inoculated treatment. An increase in the abundance of Myroides and a reduction in the representation of Proteiniclasticum and Halomonas were major observed variations. On the other hand, pH and dissolved oxygen constituted main environmental factors affecting the structure of the prokaryotic communities. In all treatments yeasts increased over time, to the detriment of filamentous fungi. Together, data from this report may contribute to the development of improved bioremediation strategies of industrial wastewaters.

Laboratory and field microcosms as useful experimental systems to study the bioaugmentation treatment of tannery effluents.

Tannery effluents require effective treatment prior to their final disposal, and the use of native bacterial consortia could be an appropriate strategy for this purpose. In the present work, consortium SFC 500-1 was found to be highly tolerant to different metals, metalloids and aromatic compounds like phenols. It was also able to remove the black dye commonly used in tanneries. Moreover, it promoted a significant reduction in chemical oxygen demand and exhibited high capability for the simultaneous removal of Cr(VI) and phenol. However, the effectiveness of the remediation processes markedly varied from one experimental system (Erlenmeyer flasks) to another (field microcosm system), highlighting the importance of moving from a small-scale study system to one involving more realistic environmental scenarios. In addition, we found a decrease in the toxicity of the effluent treated with consortium SFC 500-1. Taken together, our results indicate that this consortium possesses great potential for the treatment of tannery effluents. We conclude that for the development of a bioremediation strategy, it is necessary to develop experiments at a larger scale under conditions similar to those of the original system, in order to complete the scenario first created by in vitro approaches.

Identification of the main mechanisms involved in the tolerance and bioremediation of Cr(VI) by Bacillus sp. SFC 500-1E.

Chromium pollution is a problem that affects different areas worldwide and, therefore, must be solved. Bioremediation is a promising alternative to treat environmental contamination, but finding bacterial strains able to tolerate and remove different contaminants is a major challenge, since most co-polluted sites contain mixtures of organic and inorganic substances. In the present work, Bacillus sp. SFC 500-1E, isolated from the bacterial consortium SFC 500-1 native to tannery sediments, showed tolerance to various concentrations of different phenolic compounds and heavy metals, such as Cr(VI). This strain was able to efficiently remove Cr(VI), even in the presence of phenol. The detection of the chrA gene suggested that Cr(VI) extrusion could be a mechanism that allowed this strain to tolerate the heavy metal. However, reduction through cytosolic NADH-dependent chromate reductases may be the main mechanism involved in the remediation. The information provided in this study about the mechanisms through which Bacillus sp. SFC 500-1E removes Cr(VI) should be taken into account for the future application of this strain as a possible candidate to remediate contaminated environments.

An approach to study ultrastructural changes and adaptive strategies displayed by Acinetobacter guillouiae SFC 500-1A under simultaneous Cr(VI) and phenol treatment.

Acinetobacter guillouiae SFC 500-1A, a native bacterial strain isolated from tannery sediments, is able to simultaneously remove high concentrations of Cr(VI) and phenol. In this complementary study, high-resolution microscopy techniques, such as atomic force microscopy (AFM) and transmission electron microscopy (TEM), were used to improve our understanding of some bacterial adaptive mechanisms that enhance their ability to survive. AFM contributed in gaining insight into changes in bacterial size and morphology. It allowed the unambiguous identification of pollutant-induced cellular disturbances and the visualization of bacterial cells with depth sensitivity. TEM analysis revealed that Cr(VI) produced changes mainly at the intracellular level, whereas phenol produced alterations at the membrane level. This strain tended to form more extensive biofilms after phenol treatment, which was consistent with microscopy images and the production of exopolysaccharides (EPSs). In addition, other exopolymeric substances (DNA, proteins) significantly increased under Cr(VI) and phenol treatment. These exopolymers are important for biofilm formation playing a key role in bacterial aggregate stability, being especially useful for bioremediation of environmental pollutants. This study yields the first direct evidences of a range of different changes in A. guillouiae SFC 500-1A which seems to be adaptive strategies to survive in stressful conditions.