Advancing sustainable biotechnology through protein engineering.

The push for industrial sustainability benefits from the use of enzymes as a replacement for traditional chemistry. Biological catalysts, especially those that have been engineered for increased activity, stability, or novel function, and are often greener than alternative chemical approaches. This Review highlights the role of engineered enzymes (and identifies directions for further engineering efforts) in the application areas of greenhouse gas sequestration, fuel production, bioremediation, and degradation of plastic wastes.

Bioremediation of mercury-polluted soil and water by the plant symbiotic fungus Metarhizium robertsii.

Fungi are central to every terrestrial and many aquatic ecosystems, but the mechanisms underlying fungal tolerance to mercury, a global pollutant, remain unknown. Here, we show that the plant symbiotic fungus Metarhizium robertsii degrades methylmercury and reduces divalent mercury, decreasing mercury accumulation in plants and greatly increasing their growth in contaminated soils. M. robertsii does this by demethylating methylmercury via a methylmercury demethylase (MMD) and using a mercury ion reductase (MIR) to reduce divalent mercury to volatile elemental mercury. M. robertsii can also remove methylmercury and divalent mercury from fresh and sea water even in the absence of added nutrients. Overexpression of MMD and MIR significantly improved the ability of M. robertsii to bioremediate soil and water contaminated with methylmercury and divalent mercury. MIR homologs, and thereby divalent mercury tolerance, are widespread in fungi. In contrast, MMD homologs were patchily distributed among the few plant associates and soil fungi that were also able to demethylate methylmercury. Phylogenetic analysis suggests that fungi could have acquired methylmercury demethylase genes from bacteria via two independent horizontal gene transfer events. Heterologous expression of MMD in fungi that lack MMD homologs enabled them to demethylate methylmercury. Our work reveals the mechanisms underlying mercury tolerance in fungi, and may provide a cheap and environmentally friendly means of cleaning up mercury pollution.

Utilization of diverse organophosphorus pollutants by marine bacteria.

Anthropogenic organophosphorus compounds (AOPCs), such as phosphotriesters, are used extensively as plasticizers, flame retardants, nerve agents, and pesticides. To date, only a handful of soil bacteria bearing a phosphotriesterase (PTE), the key enzyme in the AOPC degradation pathway, have been identified. Therefore, the extent to which bacteria are capable of utilizing AOPCs as a phosphorus source, and how widespread this adaptation may be, remains unclear. Marine environments with phosphorus limitation and increasing levels of pollution by AOPCs may drive the emergence of PTE activity. Here, we report the utilization of diverse AOPCs by four model marine bacteria and 17 bacterial isolates from the Mediterranean Sea and the Red Sea. To unravel the details of AOPC utilization, two PTEs from marine bacteria were isolated and characterized, with one of the enzymes belonging to a protein family that, to our knowledge, has never before been associated with PTE activity. When expressed in Escherichia coli with a phosphodiesterase, a PTE isolated from a marine bacterium enabled growth on a pesticide analog as the sole phosphorus source. Utilization of AOPCs may provide bacteria a source of phosphorus in depleted environments and offers a prospect for the bioremediation of a pervasive class of anthropogenic pollutants.

Unraveling the complex regulatory networks in biofilm formation in bacteria and relevance of biofilms in environmental remediation.

Biofilms are assemblages of bacteria embedded within a matrix of extracellular polymeric substances (EPS) attached to a substratum. The process of biofilm formation is a complex phenomenon regulated by the intracellular and intercellular signaling systems. Various secondary messenger molecules such as cyclic dimeric guanosine 3',5'-monophosphate (c-di-GMP), cyclic adenosine 3',5'-monophosphate (cAMP), and cyclic dimeric adenosine 3',5'-monophosphate (c-di-AMP) are involved in complex signaling networks to regulate biofilm development in several bacteria. Moreover, the cell to cell communication system known as Quorum Sensing (QS) also regulates biofilm formation via diverse mechanisms in various bacterial species. Bacteria often switch to the biofilm lifestyle in the presence of toxic pollutants to improve their survivability. Bacteria within a biofilm possess several advantages with regard to the degradation of harmful pollutants, such as increased protection within the biofilm to resist the toxic pollutants, synthesis of extracellular polymeric substances (EPS) that helps in the sequestration of pollutants, elevated catabolic gene expression within the biofilm microenvironment, higher cell density possessing a large pool of genetic resources, adhesion ability to a wide range of substrata, and metabolic heterogeneity. Therefore, a comprehensive account of the various factors regulating biofilm development would provide valuable insights to modulate biofilm formation for improved bioremediation practices. This review summarizes the complex regulatory networks that influence biofilm development in bacteria, with a major focus on the applications of bacterial biofilms for environmental restoration.

Efficient NADPH-dependent dehalogenation afforded by a self-sufficient reductive dehalogenase.

Reductive dehalogenases are corrinoid and iron-sulfur cluster-containing enzymes that catalyze the reductive removal of a halogen atom. The oxygen-sensitive and membrane-associated nature of the respiratory reductive dehalogenases has hindered their detailed kinetic study. In contrast, the evolutionarily related catabolic reductive dehalogenases are oxygen tolerant, with those that are naturally fused to a reductase domain with similarity to phthalate dioxygenase presenting attractive targets for further study. We present efficient heterologous expression of a self-sufficient catabolic reductive dehalogenase from Jhaorihella thermophila in Escherichia coli. Combining the use of maltose-binding protein as a solubility-enhancing tag with the btuCEDFB cobalamin uptake system affords up to 40% cobalamin occupancy and a full complement of iron-sulfur clusters. The enzyme is able to efficiently perform NADPH-dependent dehalogenation of brominated and iodinated phenolic compounds, including the flame retardant tetrabromobisphenol, under both anaerobic and aerobic conditions. NADPH consumption is tightly coupled to product formation. Surprisingly, corresponding chlorinated compounds only act as competitive inhibitors. Electron paramagnetic resonance spectroscopy reveals loss of the Co(II) signal observed in the resting state of the enzyme under steady-state conditions, suggesting accumulation of Co(I)/(III) species prior to the rate-limiting step. In vivo reductive debromination activity is readily observed, and when the enzyme is expressed in E. coli strain W, supports growth on 3-bromo-4-hydroxyphenylacetic as a sole carbon source. This demonstrates the potential for catabolic reductive dehalogenases for future application in bioremediation.

Molecular binding of different classes of organophosphates to methyl parathion hydrolase from Ochrobactrum species.

Methyl parathion hydrolase (MPH) is an enzyme of the metallo-ß-lactamase superfamily, which hydrolyses a wide range of organophosphates (OPs). Recently, MPH has attracted attention as a promising enzymatic bioremediator. The crystal structure of MPH enzyme shows a dimeric form, with each subunit containing a binuclear metal ion center. MPH also demonstrates metal ion-dependent selectivity patterns. The origins of these patterns remain unclear but are linked to open questions about the more general role of metal ions in functional evolution and divergence within enzyme superfamilies. We aimed to investigate and compare the binding of different OP pesticides to MPH with cobalt(II) metal ions. In this study, MPH was modeled from Ochrobactrum sp. with different OP pesticides bound, including methyl paraoxon and dichlorvos and profenofos. The docked structures for each substrate optimized by DFT calculation were selected and subjected to atomistic molecular dynamics simulations for 500 ns. It was found that alpha metal ions did not coordinate with all the pesticides. Rather, the pesticides coordinated with less buried beta metal ions. It was also observed that the coordination of beta metal ions was perturbed to accommodate the pesticides. The binding free energy calculations and structure-based pharmacophore model revealed that all the three substrates could bind well at the active site. However, profenofos exhibit a stronger binding affinity to MPH in comparison to the other two substrates. Therefore, our findings provide molecular insight on the binding of different OP pesticides which could help us design the enzyme for OP pesticides degradation.

In silico biotechnological potential of Bacillus sp. strain MHSD_37 bacterial endophyte.

BACKGROUND: Endophytic bacteria possess a range of unique characteristics that enable them to successfully interact with their host and survive in adverse environments. This study employed in silico analysis to identify genes, from Bacillus sp. strain MHSD_37, with potential biotechnological applications. RESULTS: The strain presented several endophytic lifestyle genes which encode for motility, quorum sensing, stress response, desiccation tolerance and root colonisation. The presence of plant growth promoting genes such as those involved in nitrogen fixation, nitrate assimilation, siderophores synthesis, seed germination and promotion of root nodule symbionts, was detected. Strain MHSD_37 also possessed genes involved in insect virulence and evasion of defence system. The genome analysis also identified the presence of genes involved in heavy metal tolerance, xenobiotic resistance, and the synthesis of siderophores involved in heavy metal tolerance. Furthermore, LC-MS analysis of the excretome identified secondary metabolites with biological activities such as anti-cancer, antimicrobial and applications as surfactants. CONCLUSIONS: Strain MHSD_37 thereby demonstrated potential biotechnological application in bioremediation, biofertilisation and biocontrol. Moreover, the strain presented genes encoding products with potential novel application in bio-nanotechnology and pharmaceuticals.

Adsorption of Hg2+/Cr6+ by metal-binding proteins heterologously expressed in Escherichia coli.

BACKGROUND: Removal of heavy metals from water and soil is a pressing challenge in environmental engineering, and biosorption by microorganisms is considered as one of the most cost-effective methods. In this study, the metal-binding proteins MerR and ChrB derived from Cupriavidus metallidurans were separately expressed in Escherichia coli BL21 to construct adsorption strains. To improve the adsorption performance, surface display and codon optimization were carried out. RESULTS: In this study, we constructed 24 adsorption engineering strains for Hg2+ and Cr6+, utilizing different strategies. Among these engineering strains, the M'-002 and B-008 had the strongest heavy metal ion absorption ability. The M'-002 used the flexible linker and INPN to display the merRopt at the surface of the E. coli BL21, whose maximal adsorption capacity reached 658.40 µmol/g cell dry weight under concentrations of 300 µM Hg2+. And the B-008 overexpressed the chrB in the intracellular, its maximal capacity was 46.84 µmol/g cell dry weight under concentrations 500 µM Cr6+. While in the case of mixed ions solution (including Pb2+, Cd2+, Cr6+ and Hg2+), the total amount of ions adsorbed by M'-002 and B-008 showed an increase of up to 1.14- and 4.09-folds, compared to the capacities in the single ion solution. CONCLUSION: The construction and optimization of heavy metal adsorption strains were carried out in this work. A comparison of the adsorption behavior between single bacteria and mixed bacteria systems was investigated in both a single ion and a mixed ion environment. The Hg2+ absorption capacity is reached the highest reported to date with the engineered strain M'-002, which displayed the merRopt at the surface of chassis cell, indicating the strain's potential for its application in practical environments.

Harnessing a Biocatalyst to Bioremediate the Purification of Alkylglycosides.

As the world moves towards net-zero carbon emissions, the development of sustainable chemical manufacturing processes is essential. Within manufacturing, purification by distillation is often used, however this process is energy intensive and methods that could obviate or reduce its use are desirable. Developed herein is an alternative, oxidative biocatalytic approach that enables purification of alkyl monoglucosides (essential bio-based surfactant components). Implementing an immobilised engineered alcohol oxidase, a long-chain alcohol by-product derived from alkyl monoglucoside synthesis (normally removed by distillation) is selectively oxidised to an aldehyde, conjugated to an amine resin and then removed by simple filtration. This affords recovery of the purified alkyl monoglucoside. The approach lays a blueprint for further development of sustainable alkylglycoside purification using biocatalysis and, importantly, for refining other important chemical feedstocks that currently rely on distillation.

Environmentally Friendly Degradation and Detoxification of Rifampicin by a Bacterial Laccase and Hydrogen Peroxide.

Antibiotics are micropollutants accumulating in our rivers and wastewaters, potentially leading to bacterial antibiotic resistance, a worldwide problem to which there is no current solution. Here, we have developed an environmentally friendly two-step process to transform the antibiotic rifampicin (RIF) into non-antimicrobial compounds. The process involves an enzymatic oxidation step by the bacterial CotA-laccase and a hydrogen peroxide bleaching step. NMR identified rifampicin quinone as the main product of the enzymatic oxidation. Growth of Escherichia coli strains in the presence of final degradation products (FP) and minimum inhibitory concentration (MIC) measurements confirmed that FP are non-anti-microbial compounds, and bioassays suggest that FP is not toxic to eukaryotic organisms. Moreover, competitive fitness assays between susceptible and RIF-resistant bacteria show that susceptible bacteria is strongly favoured in the presence of FP. Our results show that we have developed a robust and environmentally friendly process to effectively remediate rifampicin from antibiotic contaminated environments.

Reprogramming Filamentous fd Viruses to Capture Copper Ions.

C-terminal truncated variants (A, VA, NVA, ANVA, FANVA and GFANVA) of our recently identified Cu(II) specific peptide "HGFANVA" were displayed on filamentous fd phages. Wild type fd-tet and engineered virus variants were treated with 100 mM Cu(II) solution at a final phage concentration of 1011 vir/ml and 1012 vir/ml. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) imaging before Cu(II) exposure showed ≈6-8 nm thick filamentous virus layer formation. Cu(II) treatment resulted in aggregated bundle-like assemblies with mineral deposition. HGFANVA phage formed aggregates with an excessive mineral coverage. As the virus concentration was 10-fold decreased, nanowire-like assemblies were observed for shorter peptide variants A, NVA and ANVA. Wild type fd phages did not show any mineral formation. Energy dispersive X-ray spectroscopy (EDX) analyses revealed the presence of C and N peaks on phage organic material. Cu peak was only detected for engineered viruses. Metal ion binding of viruses was next investigated by enzyme-linked immunosorbent assay (ELISA) analyses. Engineered viruses were able to bind Cu(II) forming mineralized intertwined structures although no His (H) unit was displayed. Such genetically reprogrammed virus based biological materials can be further applied for bioremediation studies to achieve a circular economy.

From mec cassette to rdhA: a key Dehalobacter genomic neighborhood in a chloroform and dichloromethane-transforming microbial consortium.

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, which transforms CF via DCM to CO2. This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO2. Here, we use metagenomic and metaproteomic analyses to identify the functional genes involved in each of these transformations. Though 91 metagenome-assembled genomes were assembled, the genes for an RDase-named acdA-and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter. AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM and subsequent mineralization of DCM using the mec cassette. IMPORTANCE: Chloroform (CF) and dichloromethane (DCM) are regulated groundwater contaminants. A cost-effective approach to remove these pollutants from contaminated groundwater is to employ microbes that transform CF and DCM as part of their metabolism, thus depleting the contamination as the microbes continue to grow. In this work, we investigate bioaugmentation culture SC05, a mixed microbial consortium that effectively and simultaneously degrades both CF and DCM coupled to the growth of Dehalobacter. We identified the functional genes responsible for the transformation of CF and DCM in SC05. These genetic biomarkers provide a means to monitor the remediation process in the field.

The critical roles of propanethiol oxidoreductase and sulfide-quinone oxidoreductase in the propanethiol catabolism pathway in Pseudomonas putida S-1.

Propanethiol (PT) is a hazardous pollutant that poses risks to both the environment and human well-being. Pseudomonas putida S-1 has been identified as a microorganism capable of utilizing PT as its sole carbon source. However, the metabolic pathway responsible for PT degradation in P. putida S-1 has remained poorly understood, impeding its optimization and practical application. In this study, we investigated the catabolic network involved in PT desulfurization with P. putida S-1 and identified key gene modules crucial to this process. Notably, propanethiol oxidoreductase (PTO) catalyzes the initial degradation of PT, a pivotal step for P. putida S-1's survival on PT. PTO facilitates the oxidation of PT, resulting H2S, H2O2, and propionaldehyde (PA). Catalase-peroxidase catalyzes the conversion of H2O2 to oxygen and water, while PA undergoes gradual conversion to Succinyl-CoA, which is subsequently utilized in the tricarboxylic acid cycle. H2S is digested in a comprehensive desulfurization network where sulfide-quinone oxidoreductase (SQOR) predominantly converts it to sulfane sulfur. The transcriptome analysis suggests that sulfur can be finally converted to sulfite or sulfate and exported out of the cell. The PT degradation capacity of P. putida S-1 was enhanced by increasing the transcription level of PTO and SQOR genes in vivo.IMPORTANCEThis work investigated the PT catabolism pathway in Pseudomonas putida S-1, a microorganism capable of utilizing PT as the sole carbon source. Critical genes that control the initiation of PT degradation were identified and characterized, such as pto and sqor. By increasing the transcription level of pto and sqor genes in vivo, we have successfully enhanced the PT degradation efficiency and growth rate of P. putida S-1. This work does not only reveal a unique PT degradation pathway but also highlights the potential of enhancing the microbial desulfurization process in the bioremediation of thiol-contaminated environment.

Continuous planting of euhalophyte Suaeda salsa enhances microbial diversity and multifunctionality of saline soil.

Halophyte-based remediation emerges as a novel strategy for ameliorating saline soils, offering a sustainable alternative to conventional leaching methods. While bioremediation is recognized for its ability to energize soil fertility and structure, the complex interplays among plant traits, soil functions, and soil microbial diversity remain greatly unknown. Here, we conducted a 5-year field experiment involving the continuous cultivation of the annual halophyte Suaeda salsa in saline soils to explore soil microbial diversity and their relationships with plant traits and soil functions. Our findings demonstrate that a decline in soil salinity corresponded with increases in the biomass and seed yield of S. salsa, which sustained a consistent seed oil content of approximately 22% across various salinity levels. Significantly, prolonged cultivation of halophytes substantially augmented soil microbial diversity, particularly from the third year of cultivation. Moreover, we identified positive associations between soil multifunctionality, seed yield, and taxonomic richness within a pivotal microbial network module. Soils enriched with taxa from this module showed enhanced multifunctionality and greater seed yields, correlating with the presence of functional genes implicated in nitrogen fixation and nitrification. Genomic analysis suggests that these taxa have elevated gene copy numbers of crucial functional genes related to nutrient cycling. Overall, our study emphasizes that the continuous cultivation of S. salsa enhances soil microbial diversity and recovers soil multifunctionality, expanding the understanding of plant-soil-microbe feedback in bioremediation.IMPORTANCEThe restoration of saline soils utilizing euhalophytes offers a viable alternative to conventional irrigation techniques for salt abatement and soil quality enhancement. The ongoing cultivation of the annual Suaeda salsa and its associated plant traits, soil microbial diversity, and functionalities are, however, largely underexplored. Our investigation sheds light on these dynamics, revealing that cultivation of S. salsa sustains robust plant productivity while fostering soil microbial diversity and multifunctionality. Notably, the links between enhanced soil multifunctionality, increased seed yield, and network-dependent taxa were found, emphasizing the importance of key microbial taxa linked with functional genes vital to nitrogen fixation and nitrification. These findings introduce a novel understanding of the role of soil microbes in bioremediation and advance our knowledge of the ecological processes that are vital for the rehabilitation of saline environments.

Design of a two functional permeable reactive barrier for synergistic enzymatic and microbial bioremediation of phenol-contaminated waters: laboratory column evaluation : Enzymatic and microbial bioremediation of phenol in a bilayer permeable reactive barrier.

The present study aimed to develop a system using a combination of enzymatic and microbial degradation techniques for removing phenol from contaminated water. In our prior research, the HRP enzyme extracted from horseradish roots was utilized within a core-shell microcapsule to reduce phenolic shock, serving as a monolayer column. To complete the phenol removal process, a second column containing degrading microorganisms was added to the last column in this research. Phenol-degrading bacteria were isolated from different microbial sources on a phenolic base medium. Additionally, encapsulated calcium peroxide nanoparticles were used to provide dissolved oxygen for the microbial population. Results showed that the both isolated strains, WC1 and CC1, were able to completely remove phenol from the contaminated influent water the range within 5 to 7 days, respectively. Molecular identification showed 99.8% similarity for WC1 isolate to Stenotrophomonas rizophila strain e-p10 and 99.9% similarity for CC1 isolate to Bacillus cereus strain IAM 12,605. The results also indicated that columns using activated sludge as a microbial source had the highest removal rate, with the microbial biofilm completely removing 100% of the 100 mg/L phenol concentration in contaminated influent water after 40 days. Finally, the concurrent use of core-shell microcapsules containing enzymes and capsules containing Stenotrophomonas sp. WC1 strain in two continuous column reactors was able to completely remove phenol from polluted water with a concentration of 500 mg/L for a period of 20 days. The results suggest that a combination of enzymatic and microbial degrading systems can be used as a new system to remove phenol from polluted streams with higher concentrations of phenol by eliminating the shock of phenol on the microbial population.

Metagenomic 16S rRNA analysis and predictive functional profiling revealed intrinsic organohalides respiration and bioremediation potential in mangrove sediment.

BACKGROUND: Mangrove sediment microbes are increasingly attracting scientific attention due to their demonstrated capacity for diverse bioremediation activities, encompassing a wide range of environmental contaminants. MATERIALS AND METHODS: The microbial communities of five Avicennia marina mangrove sediment samples collected from Al Rayyis White Head, Red Sea (KSA), were characterized using Illumina amplicon sequencing of the 16S rRNA genes. RESULTS: Our study investigated the microbial composition and potential for organohalide bioremediation in five mangrove sediments from the Red Sea. While Proteobacteria dominated four microbiomes, Bacteroidetes dominated the fifth. Given the environmental concerns surrounding organohalides, their bioremediation is crucial. Encouragingly, we identified phylogenetically diverse organohalide-respiring bacteria (OHRB) across all samples, including Dehalogenimonas, Dehalococcoides, Anaeromyxobacter, Desulfuromonas, Geobacter, Desulfomonile, Desulfovibrio, Shewanella and Desulfitobacterium. These bacteria are known for their ability to dechlorinate organohalides through reductive dehalogenation. PICRUSt analysis further supported this potential, predicting the presence of functional biomarkers for organohalide respiration (OHR), including reductive dehalogenases targeting tetrachloroethene (PCE) and 3-chloro-4-hydroxyphenylacetate in most sediments. Enrichment cultures studies confirmed this prediction, demonstrating PCE dechlorination by the resident microbial community. PICRUSt also revealed a dominance of anaerobic metabolic processes, suggesting the microbiome's adaptation to the oxygen-limited environment of the sediments. CONCLUSION: This study provided insights into the bacterial community composition of five mangrove sediments from the Red Sea. Notably, diverse OHRB were detected across all samples, which possess the metabolic potential for organohalide bioremediation through reductive dehalogenation pathways. Furthermore, PICRUSt analysis predicted the presence of functional biomarkers for OHR in most sediments, suggesting potential intrinsic OHR activity by the enclosed microbial community.

How to deal with xenobiotic compounds through environment friendly approach?

Every year, a huge amount of lethal compounds, such as synthetic dyes, pesticides, pharmaceuticals, hydrocarbons, etc. are mass produced worldwide, which negatively affect soil, air, and water quality. At present, pesticides are used very frequently to meet the requirements of modernized agriculture. The Food and Agriculture Organization of the United Nations (FAO) estimates that food production will increase by 80% by 2050 to keep up with the growing population, consequently pesticides will continue to play a role in agriculture. However, improper handling of these highly persistent chemicals leads to pollution of the environment and accumulation in food chain. These effects necessitate the development of technologies to eliminate or degrade these pollutants. Degradation of these compounds by physical and chemical processes is expensive and usually results in secondary compounds with higher toxicity. The biological strategies proposed for the degradation of these compounds are both cost-effective and eco-friendly. Microbes play an imperative role in the degradation of xenobiotic compounds that have toxic effects on the environment. This review on the fate of xenobiotic compounds in the environment presents cutting-edge insights and novel contributions in different fields. Microbial community dynamics in water bodies, genetic modification for enhanced pesticide degradation and the use of fungi for pharmaceutical removal, white-rot fungi's versatile ligninolytic enzymes and biodegradation potential are highlighted. Here we emphasize the factors influencing bioremediation, such as microbial interactions and carbon catabolism repression, along with a nuanced view of challenges and limitations. Overall, this review provides a comprehensive perspective on the bioremediation strategies.

Ecotoxicity characterization assisted performance assessment of electro-bioremediation reactors for nitrate and arsenite elimination.

The performance of combined reduction of nitrate (NO3 - ) to dinitrogen gas (N2 ) and oxidation of arsenite (As[III]) to arsenate (As[V]) by a bioelectrochemical system was assessed, supported by ecotoxicity characterization. For the comprehensive toxicity characterization of the untreated model groundwater and the treated reactor effluents, a problem-specific ecotoxicity test battery was established. The performance of the applied technology in terms of toxicity and target pollutant elimination was compared and analyzed. The highest toxicity attenuation was achieved under continuous flow mode with hydraulic retention time (HRT) = 7.5 h, with 95%, nitrate removal rate and complete oxidation of arsenite to arsenate. Daphnia magna proved to be the most sensitive test organism. The results of the D. magna lethality test supported the choice of the ideal operational conditions based on chemical data analysis. The outcomes of the study demonstrated that the applied technology was able to improve the groundwater quality in terms of both chemical and ecotoxicological characteristics. The importance of ecotoxicity evaluation was also highlighted, given that significant target contaminant elimination did not necessarily lower the environmental impact of the initial, untreated medium, in addition, anomalies might occur during the technology operational process which in some instances, could result in elevated toxicity levels.

Extremophiles and their expanding biotechnological applications.

Microbial life is not restricted to any particular setting. Over the past several decades, it has been evident that microbial populations can exist in a wide range of environments, including those with extremes in temperature, pressure, salinity, and pH. Bacteria and Archaea are the two most reported types of microbes that can sustain in extreme environments, such as hot springs, ice caves, acid drainage, and salt marshes. Some can even grow in toxic waste, organic solvents, and heavy metals. These microbes are called extremophiles. There exist certain microorganisms that are found capable of thriving in two or more extreme physiological conditions simultaneously, and are regarded as polyextremophiles. Extremophiles possess several physiological and molecular adaptations including production of extremolytes, ice nucleating proteins, pigments, extremozymes and exopolysaccharides. These metabolites are used in many biotechnological industries for making biofuels, developing new medicines, food additives, cryoprotective agents etc. Further, the study of extremophiles holds great significance in astrobiology. The current review summarizes the diversity of microorganisms inhabiting challenging environments and the biotechnological and therapeutic applications of the active metabolites obtained as a response to stress conditions. Bioprospection of extremophiles provides a progressive direction with significant enhancement in economy. Moreover, the introduction to omics approach including whole genome sequencing, single cell genomics, proteomics, metagenomics etc., has made it possible to find many unique microbial communities that could be otherwise difficult to cultivate using traditional methods. These findings might be capable enough to state that discovery of extremophiles can bring evolution to biotechnology.

Performance evaluation of rhamnolipid biosurfactant produced by Pseudomonas aeruginosa and its effect on marine oil-spill remediation.

This study aimed to reveal that the effect of biosurfactant on the dispersion and degradation of crude oil. Whole genome analysis showed that Pseudomonas aeruginosa GB-3 contained abundant genes involved in biosurfactant synthesis and metabolic processes and had the potential to degrade oil. The biosurfactant produced by strain GB-3 was screened by various methods. The results showed that the surface tension reduction activity was 28.6 mN·m-1 and emulsification stability was exhibited at different pH, salinity and temperature. The biosurfactant was identified as rhamnolipid by LC-MS and FTIR. The fermentation conditions of strain GB-3 were optimized by response surface methodology, finally the optimal system (carbon source: glucose, nitrogen source: ammonium sulfate, C/N ratio:16:1, pH: 7, temperature: 30-35 °C) was determined. Compared with the initial fermentation, the yield of biosurfactant increased by 4.4 times after optimization. In addition, rhamnolipid biosurfactant as a dispersant could make the dispersion of crude oil reach 38% within seven days, which enhanced the bioavailability of crude oil. As a biostimulant, it could also improve the activity of indigenous microorganism and increase the degradation rate of crude oil by 10-15%. This study suggested that rhamnolipid biosurfactant had application prospect in bioremediation of marine oil-spill.