Single Carbon Substrates Research Articles

Development of a Recombineering System for the Acetogen Eubacterium limosum with Cas9 Counterselection for Markerless Genome Engineering.

Eubacterium limosum is a Clostridial acetogen that efficiently utilizes a wide range of single-carbon substrates and contributes to metabolism of health-associated compounds in the human gut microbiota. These traits have led to interest in developing it as a platform for sustainable CO2-based biofuel production to combat carbon emissions, and for exploring the importance of the microbiota in human health. However, synthetic biology and metabolic engineering in E. limosum have been hindered by the inability to rapidly make precise genomic modifications. Here, we screened a diverse library of recombinase proteins to develop a highly efficient oligonucleotide-based recombineering system based on the viral recombinase RecT. Following optimization, the system is capable of catalyzing ssDNA recombination at an efficiency of up to 2%. Addition of a Cas9 counterselection system eliminated unrecombined cells, with up to 100% of viable cells encoding the desired mutation, enabling creation of genomic point mutations in a scarless and markerless manner. We deployed this system to create a clean knockout of the extracellular polymeric substance (EPS) gene cluster, generating a strain incapable of biofilm formation. This approach is rapid and simple, not requiring laborious homology arm cloning, and can readily be retargeted to almost any genomic locus. This work overcomes a major bottleneck in E. limosum genetic engineering by enabling precise genomic modifications, and provides both a roadmap and associated recombinase plasmid library for developing similar systems in other Clostridia of interest.

Efficient production of itaconic acid from the single-carbon substrate methanol with engineered Komagataella phaffii

BackgroundAmidst the escalating carbon dioxide levels resulting from fossil fuel consumption, there is a pressing need for sustainable, bio-based alternatives to underpin future global economies. Single-carbon feedstocks, derived from CO2, represent promising substrates for biotechnological applications. Especially, methanol is gaining prominence for bio-production of commodity chemicals.ResultsIn this study, we show the potential of Komagataella phaffii as a production platform for itaconic acid using methanol as the carbon source. Successful integration of heterologous genes from Aspergillus terreus (cadA, mttA and mfsA) alongside fine-tuning of the mfsA gene expression, led to promising initial itaconic acid titers of 28 g·L−1 after 5 days of fed-batch cultivation. Through the combined efforts of process optimization and strain engineering strategies, we further boosted the itaconic acid production reaching titers of 55 g·L−1 after less than 5 days of methanol feed, while increasing the product yield on methanol from 0.06 g·g−1 to 0.24 g·g−1.ConclusionOur results highlight the potential of K. phaffii as a methanol-based platform organism for sustainable biochemical production.

Natural Polyhydroxyalkanoates—An Overview of Bacterial Production Methods

Polyhydroxyalkanoates (PHAs) are intracellular biopolymers that microorganisms use for energy and carbon storage. They are mechanically similar to petrochemical plastics when chemically extracted, but are completely biodegradable. While they have potential as a replacement for petrochemical plastics, their high production cost using traditional carbon sources remains a significant challenge. One potential solution is to modify heterotrophic PHA-producing strains to utilize alternative carbon sources. An alternative approach is to utilize methylotrophic or autotrophic strains. This article provides an overview of bacterial strains employed for PHA production, with a particular focus on those exhibiting the highest PHA content in dry cell mass. The strains are organized according to their carbon source utilization, encompassing autotrophy (utilizing CO2, CO) and methylotrophy (utilizing reduced single-carbon substrates) to heterotrophy (utilizing more traditional and alternative substrates).

Characterization of biosurfactant produced through co-utilization of substrates by the novel strain Pseudomonas aeruginosa NG4

Biosurfactants are extremely valuable and promising compounds that have a wide range of applications across various industries and aspects of human life. Thus, there is a great need to enhance their production yield, by developing new and more efficient fermentation methods. In pursuit of this objective, batch fermentation was carried out with different feed concentrations and addition. The results were compared in relation to their effectiveness in producing biosurfactants using Pseudomonas aeruginosa. Batch fermentation experiments were conducted using two different substrates feeding strategies, such as single substrate utilization and co-utilization of substrates. The comparison of these substrates utilization processes revealed that the co-utilization of glucose and crude glycerol was a superior cultivation strategy compared to the single substrate utilization for biosurfactant production. The biosurfactant production by P. aeruginosa from co-utilization of glucose and crude glycerol reached to a yield of 2.5 gL-1. There was about 2.2 fold enhancement in the yield compared to single carbon substrates. Such co-utilization of substrates is not reported for biosurfactant production. The biosurfactant was characterized and the presence of mono-rhamnolipid and di-rhamnolipid compounds was confirmed through FT-IR and LC-MS.

Electrical-biological hybrid system for carbon efficient isobutanol production

We have developed an electrical-biological hybrid system wherein an engineered microorganism consumes electrocatalytically produced formate from CO2 to supplement the bioproduction of isobutanol, a valuable fuel chemical. Biological CO2 sequestration is notoriously slow compared to electrochemical CO2 reduction, while electrochemical methods struggle to generate carbon-carbon bonds which readily form in biological systems. A hybrid system provides a promising method for combining the benefits of both biology and electrochemistry. Previously, Escherichia coli was engineered to assimilate formate and CO2 in central metabolism using the reductive glycine pathway. In this work, we have shown that chemical production in E. coli can benefit from single carbon substrates when equipped with the RGP. By installing the RGP and the isobutanol biosynthetic pathway into E. coli and by further genetic modifications, we have generated a strain of E. coli that can consume formate and produce isobutanol at a yield of >100% of theoretical maximum from glucose. Our results demonstrate that carbon produced from electrocatalytically reduced CO2 can bolster chemical production in E. coli. This study shows that E. coli can be engineered towards carbon efficient methods of chemical production.

FreeFlux: A Python Package for Time-Efficient Isotopically Nonstationary Metabolic Flux Analysis.

13C metabolic flux analysis is a powerful tool for metabolism characterization in metabolic engineering and synthetic biology. However, the widespread adoption of this tool is hindered by limited software availability and computational efficiency. Currently, the most widely accepted 13C-flux tools, such as INCA and 13CFLUX2, are developed in a closed-source environment. While several open-source packages or software are available, they are either computationally inefficient or only suitable for flux estimation at isotopic steady state. To address the need for a time-efficient computational tool for the more complicated flux analysis at an isotopically nonstationary state, especially for understanding the single-carbon substrate metabolism, we present FreeFlux. FreeFlux is an open-source Python package that performs labeling pattern simulation and flux analysis at both isotopic steady state and transient state, enabling a more comprehensive analysis of cellular metabolism. FreeFlux provides a set of interfaces to manipulate the objects abstracted from a labeling experiment and computational process, making it easy to integrate into other programs or pipelines. The flux estimation by FreeFlux is fast and reliable, and its validity has been confirmed by comparison with results from other computational tools using both synthetic and experimental data. FreeFlux is freely available at https://github.com/Chaowu88/freeflux with a detailed online tutorial and documentation provided at https://freeflux.readthedocs.io/en/latest/index.html.

Deletion of biofilm synthesis in Eubacterium limosum ATCC 8486 improves handling and transformation efficiency.

Eubacterium limosum is an acetogenic bacterium of potential industrial relevance for its ability to efficiently metabolize a range of single carbon compounds. However, extracellular polymeric substance (EPS) produced by the type strain ATCC 8486 is a serious impediment to bioprocessing and genetic engineering. To remove these barriers, here we bioinformatically identified genes involved in EPS biosynthesis, and targeted several of the most promising candidates for inactivation, using a homologous recombination-based approach. Deletion of a single genomic region encoding homologues for epsABC, ptkA, and tmkA resulted in a strain incapable of producing EPS. This strain is significantly easier to handle by pipetting and centrifugation, and retains important wild-type phenotypes including the ability to grow on methanol and carbon dioxide and limited oxygen tolerance. Additionally, this strain is also more genetically tractable with a 2-fold increase in transformation efficiency compared to the highest previous reports. This work advances a simple, rapid protocol for gene knockouts in E. limosum using only the native homologous recombination machinery. These results will hasten the development of this organism as a workhorse for valorization of single carbon substrates, as well as facilitate exploration of its role in the human gut microbiota.

Quorum Sensing and Antimicrobial Production Orchestrate Biofilm Dynamics in Multispecies Bacterial Communities.

Microbial interactions are often mediated by diffusible small molecules, including secondary metabolites, that play roles in cell-to-cell signaling and inhibition of competitors. Biofilms are often "hot spots" for high concentrations of bacteria and their secondary metabolites, which make them ideal systems for the study of small-molecule contributions to microbial interactions. Here, we use a five-member synthetic community consisting of Roseobacteraceae representatives to investigate the role of secondary metabolites on microbial biofilm dynamics. One synthetic community member, Rhodobacterales strain Y4I, possesses two acylated homoserine lactone (AHL)-based cell-to-cell signaling systems (pgaRI and phaRI) as well as a nonribosomal peptide synthase gene (igi) cluster that encodes the antimicrobial indigoidine. Through serial substitution of Y4I with mutants deficient in single signaling molecule pathways, the contribution of these small-molecule systems could be assessed. As secondary metabolite production is dependent upon central metabolites, the influence of growth substrate (i.e., complex medium versus defined medium with a single carbon substrate) on these dynamics was also considered. Depending on the Y4I mutant genotype included, community dynamics ranged from competitive to cooperative. The observed interactions were mostly competitive in nature. However, the community harboring a Y4I variant that was both impaired in quorum sensing (QS) pathways and unable to produce indigoidine (pgaR variant) shifted toward more cooperative interactions over time. These cooperative interactions were enhanced in the defined growth medium. The results presented provide a framework for deciphering complex, small-molecule-mediated interactions that have broad application to microbial biology. IMPORTANCE Microbial biofilms play critical roles in marine ecosystems and are hot spots for microbial interactions that play a role in the development and function of these communities. Roseobacteraceae are an abundant and active family of marine heterotrophic bacteria forming close associations with phytoplankton and carrying out key transformations in biogeochemical cycles. Group members are aggressive primary colonizers of surfaces, where they set the stage for the development of multispecies biofilm communities. Few studies have examined the impact of secondary metabolites, such as cell-to-cell signaling and antimicrobial production, on marine microbial biofilm community structure. Here, we assessed the impact of secondary metabolites on microbial interactions using a synthetic, five-member Roseobacteraceae community by measuring species composition and biomass production during biofilm growth. We present evidence that secondary metabolites influence social behaviors within these multispecies microbial biofilms, thereby improving understanding of bacterial secondary metabolite production influence on social behaviors within marine microbial biofilm communities.

Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum.

Acetogenic bacteria are an increasingly popular choice for producing fuels and chemicals from single carbon (C1) substrates. Eubacterium limosum is a promising acetogen with several native advantages, including the ability to catabolize a wide repertoire of C1 feedstocks and the ability to grow well on agar plates. However, despite its promise as a strain for synthetic biology and metabolic engineering, there are insufficient engineering tools and molecular biology knowledge to leverage its native strengths for these applications. To capitalize on the natural advantages of this organism, here we extended its limited engineering toolbox. We evaluated the copy number of three common plasmid origins of replication and devised a method of controlling copy number and heterologous gene expression level by modulating antibiotic concentration. We further quantitatively assessed the strength and regulatory tightness of a panel of promoters, developing a series of well-characterized vectors for gene expression at varying levels. In addition, we developed a black/white colorimetric genetic reporter assay and leveraged the high oxygen tolerance of E. limosum to develop a simple and rapid transformation protocol that enables benchtop transformation. Finally, we developed two new antibiotic selection markers—doubling the number available for this organism. These developments will enable enhanced metabolic engineering and synthetic biology work with E. limosum.

Seemingly trivial secondary factors may determine microbial competition: a cautionary tale on the impact of iron supplementation through corrosion.

ABSTRACTMicrobial community engineering aims for enrichment of a specific microbial trait by imposing specific cultivation conditions. This work demonstrates that things may be more complicated than typically presumed and that microbial competition can be affected by seemingly insignificant variables, like in this case the type of acid used for pH control. Aerobic bioreactors pulse fed with acetate operated with hydrochloric acid resulted in the enrichment of Plasticicumulans acidivorans, and changing the pH controlling agent to sulfuric acid shifted the community towards Zoogloea sp. Further research demonstrated that the change in community structure was not directly caused by the change in acid used for pH control, but resulted from the difference in corrosive strength of both acids and the related iron leaching from the bioreactor piping. Neither system was iron deficient, suggesting that the biological availability of iron is affected by the leaching process. Our results demonstrate that microbial competition and process development can be affected dramatically by secondary factors related to nutrient supply and bioavailability, and is way more complex than generally assumed in a single carbon substrate limited process.

Extracellular electron transfer of Methylophilus methylotrophs

Methylophilus methylotrophs, as representatives of C1 microbes, widely exist in the interface of hyperoxia–hypoxia environment and plays a key role in global carbon cycle. Most studies of Methylophilus were carried out under hyperoxic conditions, while it remains unclear how Methylophilus survive under oxygen-limited conditions. In this study, we report that ferrihydrite could serve as an alternative electron acceptor for Methylophilus strain with reduction rate of 53.6 μMFe(II) d−1, when cells were deprived of oxygen. Genomic analysis and electrochemical measurements suggested that two strategies, cytochrome c and riboflavin, were involved in the extracellular mineral respiration. Respiration of extracellular electron acceptor by a Methylophilus strain demonstrated in this study broadens our understanding of energy metabolism by Methylophilus and suggests as a survival strategy for these microbes under oxygen-limited conditions. The results also demonstrate Methylophilus spp. as novel type of electrochemical active microbes that could be applied in bioelectrochemical systems with single-carbon substrate.

High temperature shockwave stabilized single atoms.

The stability of single-atom catalysts is critical for their practical applications. Although a high temperature can promote the bond formation between metal atoms and the substrate with an enhanced stability, it often causes atom agglomeration and is incompatible with many temperature-sensitive substrates. Here, we report using controllable high-temperature shockwaves to synthesize and stabilize single atoms at very high temperatures (1,500-2,000 K), achieved by a periodic on-off heating that features a short on state (55 ms) and a ten-times longer off state. The high temperature provides the activation energy for atom dispersion by forming thermodynamically favourable metal-defect bonds and the off-state critically ensures the overall stability, especially for the substrate. The resultant high-temperature single atoms exhibit a superior thermal stability as durable catalysts. The reported shockwave method is facile, ultrafast and universal (for example, Pt, Ru and Co single atoms, and carbon, C3N4 and TiO2 substrates), which opens a general route for single-atom manufacturing that is conventionally challenging.

Bioconversion of Methanol into Value-added Chemicals in Native and Synthetic Methylotrophs.

Methanol, commercially generated from methane, is a renewable chemical feedstock that is highly soluble, relatively inexpensive, and easy to handle. The concept of native methylotrophic bacteria serving as whole cell catalysts for production of chemicals and materials using methanol as a feedstock is highly attractive. In recent years, the available omics data for methylotrophic bacteria, especially for Methylobacterium extorquens, the most well-characterized model methylotroph, have provided a solid platform for rational engineering of methylotrophic bacteria for industrial production. In addition, there is a strong interest in converting the more traditional heterotrophic production platforms toward the use of single carbon substrates, including methanol, through metabolic engineering. In this chapter, we review the recent progress toward achieving the desired growth and production yields from methanol, by genetically engineered native methylotrophic strains and by the engineered synthetic methylotrophs.

Basics of genome-scale metabolic modeling and applications on C1-utilization.

It is fundamental to understand the relationship between genotype and phenotype in biology. This requires comprehensive knowledge of metabolic pathways, genetic information and well-defined mathematic modeling. Integration of knowledge on metabolism with mathematical modeling results in genome-scale metabolic models which have proven useful to investigate bacterial metabolism and to engineer bacterial strains capable of producing value-added biochemical. Single carbon substrates such as methane and carbon monoxide have drawn interests and they assumed one of next-generation feedstocks because of their high abundance and low price. The methylotroph and acetogen-based biorefineries hold promises for bioconversion of C1 substrates into biofuels and high value compounds. As an effort on expanding our knowledge on C1 utilization approaches, in silico computational framework of C1-metabolism in methylotrophic and acetogenic bacteria has been developed. In this review, genome-scale metabolic models for C1-utilizing bacteria and well-established analysis tools are presented for potential uses for study of C1 metabolism at the genome scale and its application in metabolic engineering.

Natural Selection in Synthetic Communities Highlights the Roles of Methylococcaceae and Methylophilaceae and Suggests Differential Roles for Alternative Methanol Dehydrogenases in Methane Consumption

We describe experiments that follow species dynamics and gene expression patterns in synthetic bacterial communities including species that compete for the single carbon substrate supplied, methane, and species unable to consume methane, which could only succeed through cooperative interactions. We demonstrate that these communities mostly select for two functional guilds, methanotrophs of the family Methylococcaceae and non-methanotrophic methylotrophs of the family Methylophilaceae, these taxonomic guilds outcompeting all other species included in the synthetic mix. The metatranscriptomics analysis uncovered that in both Methylococcaceae and Methylophilaceae, some of the most highly transcribed genes were the ones encoding methanol dehydrogenases (MDH). Remarkably, expression of alternative MDH genes (mxaFI versus xoxF), previously shown to be subjects to the rare Earth element switch, was found to depend on environmental conditions such as nitrogen source and methane and O2 partial pressures, and also to be species-specific. Along with the xoxF genes, genes encoding divergent cytochromes were highly expressed in both Methylophilaceae and Methylococcaceae, suggesting their function in methanol metabolism, likely encoding proteins serving as electron acceptors from XoxF enzymes. The research presented tested a synthetic community model that is much simplified compared to natural communities consuming methane, but more complex than the previously utilized two-species model. The performance of this model identifies prominent species for future synthetic ecology experiments and highlights both advantages of this approach and the challenges that it presents.

Strategies for Efficient Microbial Fuel Cell Operation

Microbial fuel cells (MFCs) are bioelectrochemical systems that exploit microbial respiration for the conversion of organic compounds into electrical energy. Within a typical system, electrochemically active microorganisms extracellularly transfer electrons released during the oxidation of organic compounds to the anode surface. The electrons travel from the anode, across an external load to the cathode, where they react with the terminal electron acceptor (e.g. oxygen from air). MFCs are a promising means for wastewater treatment and as a source of renewable energy. The operational mode of the MFC can directly control microbial metabolism and respiration and thus define the wastewater treatment rate, energy recovery and final products from wastewater treatment. Despite the advantages, MFCs are not yet a viable solution for large-scale wastewater treatment and energy generation. Extensive research must still be conducted to optimize the system including MFC start-up time, stability and treatment rates. MFC start-up time is widely variable depending on external conditions, inoculum source and substrate. There are challenges associated with stability and reproducibility of the system. Finally, wastewater treatment rates using MFCs still are not comparable to conventional wastewater treatment methods, and the magnitude of energy recovery is not at a meaningful scale. In this study, we evaluated different enrichment and operational strategies to optimize MFC performance. At the beginning of the experiment, during the enrichment phase, the anode potential was modulated either by an external resistor or an applied voltage. It has been previously shown that the anodic community composition and biomass formation changes according to the external resistor or potential applied [1,2]. Under such operational conditions, a faster start up time can also be achieved [3]. In addition, start-up time and overall performance will also be impacted by inoculum source and substrate selection. Previous reports addressed MFC performance as a function of operation and startup time using either a defined media and single carbon substrate [2] or a single enrichment strategy with primary clarifier effluent as the single inoculum and substrate [1]. Therefore our evaluations addressed a highly diverse inoculum source including lagoon sediment and swine waste and different methodologies for enrichment to expand the knowledge base about what enrichment strategies may be best for a given wastewater treatment application. Varying ranges of external resistors and applied voltages were tested. A periodically induced open circuit condition was also evaluated to study its effect on MFC output. With these experimental conditions, we are aiming to identify an optimal strategy for MFC start-up and operation, and evaluate the long-term effects on MFC performance including the electrochemical performance, community taxonomic dynamics, biomass formation, and wastewater treatment rate.

Triosephosphate isomerase is dispensable in vitro yet essential for Mycobacterium tuberculosis to establish infection.

ABSTRACTTriosephosphate isomerase (TPI) catalyzes the interconversion of dihydroxyacetone phosphate (DHAP) and glyceraldehyde-3-phosphate (G3P). This reaction is required for glycolysis and gluconeogenesis, and tpi has been predicted to be essential for growth of Mycobacterium tuberculosis. However, when studying a conditionally regulated tpi knockdown mutant, we noticed that depletion of TPI reduced growth of M. tuberculosis in media containing a single carbon source but not in media that contained both a glycolytic and a gluconeogenic carbon source. We used such two-carbon-source media to isolate a tpi deletion (Δtpi) mutant. The Δtpi mutant did not survive with single carbon substrates but grew like wild-type (WT) M. tuberculosis in the presence of both a glycolytic and a gluconeogenic carbon source. 13C metabolite tracing revealed the accumulation of TPI substrates in Δtpi and the absence of alternative triosephosphate isomerases and metabolic bypass reactions, which confirmed the requirement of TPI for glycolysis and gluconeogenesis in M. tuberculosis. The Δtpi strain was furthermore severely attenuated in the mouse model of tuberculosis, suggesting that M. tuberculosis cannot simultaneously access sufficient quantities of glycolytic and gluconeogenic carbon substrates to establish infection in mice.

Elucidation of intrinsic biosynthesis yields using 13C-based metabolism analysis

This paper discusses the use of 13C-based metabolism analysis for the assessment of intrinsic product yields — the actual carbon contribution from a single carbon substrate to the final product via a specific biosynthesis route — in the following four cases. First, undefined nutrients (such as yeast extract) in fermentation may contribute significantly to product synthesis, which can be quantified through an isotopic dilution method. Second, product and biomass synthesis may be dependent on the co-metabolism of multiple-carbon sources. 13C labeling experiments can track the fate of each carbon substrate in the cell metabolism and identify which substrate plays a main role in product synthesis. Third, 13C labeling can validate and quantify the contribution of the engineered pathway (versus the native pathway) to the product synthesis. Fourth, the loss of catabolic energy due to cell maintenance (energy used for functions other than production of new cell components) and low P/O ratio (Phosphate/Oxygen Ratio) significantly reduces product yields. Therefore, 13C-metabolic flux analysis is needed to assess the influence of suboptimal energy metabolism on microbial productivity, and determine how ATP/NAD(P)H are partitioned among various cellular functions. Since product yield is a major determining factor in the commercialization of a microbial cell factory, we foresee that 13C-isotopic labeling experiments, even without performing extensive flux calculations, can play a valuable role in the development and verification of microbial cell factories.

Comparison of the Biodegradation Capacity of Two Fungal Strains Toward a Mixture of Phenol and Cresol by Mathematical Modeling

ABSTRACTThe examined strains of Aspergillus awamori NRRL 3112 and Trametes versicolor 1 were cultured in a culture medium containing phenol, p-cresol and a mixture of them as single carbon substrates. Both strains were able to utilize both examined compounds. The results showed that the strain of Trametes versicolor 1 degraded p-cresol much faster than the strain Aspergillus awamori NRRL 3112. In the experiments on degradation of the mixture of phenol and p-cresol by both strains, complete carbon utilization was also observed. The influence of the mixed substrate on the growth of microbial cultures was studied. A positive influence of phenol on the degradation of cresol by Aspergillus awamori NRRL 3112 and Trametes versicolor 1 was observed. SKIP models (Sum kinetics with interaction parameters) expressing the total kinetics of the degradation of a mixture of phenol/p-cresol in Aspergillus awamori NRRL 3112 and Trametes versicolor 1 were created. The obtained values of the interaction coefficients in the model (Iph/cr and Icr/ph) corresponded to the influence exerted by each of the investigated mixed compounds on the development and degradation capacities of both fungal strains.

Degradation of Mixed Aromatic Pollutants by Trametes Versicolor Strain 1

ABSTRACTOne of the most distributed groups of industrial pollutants consists of aromatic hydrocarbons. Because of the complex nature of such wastes the obtaining of new strains able to degrade different toxic aromatic compounds in mixtures is a challenge to many scientists exploring the area of environmental microbiology.In this study the strain of white rot fungus Trametes versicolor was cultivated in a medium comprising a mixture of phenol (0.2 g/l), resorcinol (0.1 g/l), p-cresol (0.1 g/l), and o-nitrophenol (0.14 g/l). Phenol, resorcinol and p-cresol were degraded in 24 hours and then a slow o-nitrophenol degradation was observed. There was about 50% decrease of o-nitrophenol concentration in 144 hours.The degradation of the tested concentrations of phenol, resorcinol and p-cresol in a mixture showed similar characteristics as in the experiments with each of them used as a single carbon substrate. The data showed that the presence of o-nitrophenol does not influence the biodegradation rate of other compounds included in the mixture. The capability of the investigated strain demonstrated its potential for future application in bioremediation technologies oriented to cleaning and preservation of industrially polluted water.