Expression of filaments of the Geobacter extracellular cytochrome OmcS in Shewanella oneidensis.

The physiological role of Geobacter sulfurreducens extracellular cytochrome filaments is a matter of debate and the development of proposed electronic device applications of cytochrome filaments awaits methods for large-scale cytochrome nanowire production. Functional studies in G. sulfurreducens are stymied by the broad diversity of redox-active proteins on the outer cell surface and the redundancy and plasticity of extracellular electron transport routes. G. sulfurreducens is a poor chassis for producing cytochrome nanowires for electronics because of its slow, low-yield, anaerobic growth. Here we report that filaments of the G. sulfurreducens cytochrome OmcS can be heterologously expressed in Shewanella oneidensis. Multiple lines of evidence demonstrated that a strain of S. oneidensis, expressing the G. sulfurreducens OmcS gene on a plasmid, localized OmcS on the outer cell surface. Atomic force microscopy revealed filaments with the unique morphology of OmcS filaments emanating from cells. Electron transfer to OmcS appeared to require a functional outer-membrane porin-cytochrome conduit. The results suggest that S. oneidensis, which grows rapidly to high culture densities under aerobic conditions, may be suitable for the development of a chassis for producing cytochrome nanowires for electronics applications and may also be a good model microbe for elucidating cytochrome filament function in anaerobic extracellular electron transfer.

Redox Concomitant Formation Method for Fabrication of Cu(I)-MOF/Polymer Composites with Antifouling Properties.

Marine biofouling, which is one of the technical challenges hindering the growth of the marine economy, has been controlled using cuprous oxide (Cu2O) nanoparticles due to the exceptional antifouling properties of Cu(I) ions. However, Cu2O nanoparticles have encountered bottlenecks due to explosive releases of Cu+ ions, high toxicity at elevated doses, and long-term instability. Here, we present a novel method called Redox Concomitant Formation (RCF) for fabricating a hierarchical Cu(I) metal-organic framework polypyrrole (Cu(I)-MOF/PPy) composite. This method enables in-situ phase transition via successive redox reactions that change the chemical valence state and coordination mode of Cu(II)-MOF, resulting in a new structure of Cu(I)-MOF while creating a PPy layer surrounded by the hierarchical structure. Owing to the steady release of Cu+ ions from the Cu(I) sites and photothermal properties of PPy, Cu(I)-MOF/PPy exhibits superior and broad-spectrum resistance to marine bacteria, algae, and surface-adhered biofilms in complex biological environments, as well as long-term stability, resulting in 100% eradication efficiency under solar-driven heating. Mechanistic insights into successive structural redox reactions and formation using the RCF method are provided in detail, enabling the fabrication of novel MOFs with the desired composition and structure for a wide range of potential applications.

Accelerated Microbial Corrosion by Magnetite and Electrically Conductive Pili through Direct Fe0 -to-Microbe Electron Transfer.

Electrobiocorrosion, the process in which microbes extract electrons from metallic iron (Fe0 ) through direct Fe0 -microbe electrical connections, is thought to contribute to the costly corrosion of iron-containing metals that impacts many industries. However, electrobiocorrosion mechanisms are poorly understood. We report here that electrically conductive pili (e-pili) and the conductive mineral magnetite play an important role in the electron transfer between Fe0 and Geobacter sulfurreducens, the first microbe in which electrobiocorrosion has been rigorously documented. Genetic modification to express poorly conductive pili substantially diminished corrosive pitting and rates of Fe0 -to-microbe electron flux. Magnetite reduced resistance to electron transfer, increasing corrosion currents and intensifying pitting. Studies with mutants suggested that the magnetite promoted electron transfer in a manner similar to the outer-surface c-type cytochrome OmcS. These findings, and the fact that magnetite is a common product of iron corrosion, suggest a potential positive feedback loop of magnetite produced during corrosion further accelerating electrobiocorrosion. The interactions of e-pili, cytochromes, and magnetite demonstrate mechanistic complexities of electrobiocorrosion, but also provide insights into detecting and possibly mitigating this economically damaging process.

Enhanced antibacterial behavior of a novel Cu-bearing high-entropy alloy.

Contact infection of bacteria and viruses has been a critical threat to human health. The worldwide outbreak of COVID-19 put forward urgent requirements for the research and development of the self-antibacterial materials, especially the antibacterial alloys. Based on the concept of high-entropy alloys, the present work designed and prepared a novel Co0.4FeCr0.9Cu0.3 antibacterial high-entropy alloy with superior antibacterial properties without intricate or rigorous annealing processes, which outperform the antibacterial stainless steels. The antibacterial tests presented a 99.97% antibacterial rate against Escherichia coli and a 99.96% antibacterial rate against Staphylococcus aureus after 24 h. In contrast, the classic antibacterial copper-bearing stainless steel only performed the 71.50% and 80.84% antibacterial rate, respectively. The results of the reactive oxygen species analysis indicated that the copper ion release and the immediate contact with copper-rich phase had a synergistic effect in enhancing antibacterial properties. Moreover, this alloy exhibited excellent corrosion resistance when compared with the classic antibacterial stainless steels, and the compression test indicated the yield strength of the alloy was 1015 MPa. These findings generate fresh insights into guiding the designs of structure-function-integrated antibacterial alloys.

Antimicrobial Activity of Nano-Magnesium Hydroxide Against Oral Bacteria and Application in Root Canal Sealer.

BACKGROUND The goal of the present work was to assess the antibacterial activity of nano-magnesium hydroxide (NMH) against Streptococcus mutans (S. mutans) and to explore the antimicrobial function of AH Plus™ sealer incorporating NMH. MATERIAL AND METHODS The antimicrobial behavior of NMH against S. mutans was evaluated with bactericidal tests. A modified direct contact test was used to assess the antimicrobial activity of unset AH Plus containing NMH after 5 minutes, 20 minutes, and 60 minutes of contact with bacteria. The antimicrobial effects and the amount of surface-adhering bacteria of the solidified materials were explored by SEM and confocal laser scanning microscopy, respectively. RESULTS NMH powder presented excellent antimicrobial activity against S. mutans. Mg²âº and OH⁻ were not the main factors resulting in bacterial death. Approximately 93.1% and 98% of the S. mutans were killed in the AH Plus+7% NMH group after incubation for 5 minutes and 20 minutes, respectively. AH Plus with 5% or 7% NMH were more potent against S. mutans compared with AH Plus alone (P<0.05). Moreover, the antibacterial function of AH Plus was lost after setting. NMH enabled the solidified AH Plus to still have antibacterial properties on the seventh day. CONCLUSIONS NMH can be used to modify AH Plus sealer to eradicate residual bacteria and prevent reinfection.

Mitigation of the corrosion-causing Desulfovibrio desulfuricans biofilm using an organic silicon quaternary ammonium salt in alkaline media simulated concrete pore solutions.

Organic silicon quaternary ammonium salt (OSA), an environmentally friendly naturally occurring chemical, was used as a bacteriostatic agent against sulphate-reducing bacteria (SRB) on a 20SiMn steel surface in simulated concrete pore solutions (SCP). Four different media were used: No SRB (NSRB), No SRB and OSA (NSRB + OSA), With SRB (WSRB), With SRB and OSA (WSRB + OSA). After biofilm growth for 28 days, optimized sessile SRB cells survived at the high pH of 11.35 and as a result these cells caused the breakdown of the passive film due to the metabolic activities of the SRB. Corrosion prevention results showed that the OSA was effective in mitigating the growth of the sessile SRB cells and reduced corrosion in the SCP. These results were further confirmed by scanning electron microscope images, energy dispersive X-ray analysis, confocal-laser scanning microscopy, X-ray photoelectron spectroscopy and corrosion testing using electrochemical analysis.

[Efficacy and Safety Evaluation of Liujin Runzao Concentrated Decoction in Treating Primary Sjögren's Syndrome].

Objective To evaluate the efficacy and safety of Liujin Runzao Concentrated Decoction (LRCD) for the treatment of primary Sjögren's syndrome (pSS). Methods Forty pSS patients with fluid depletion and distribution obstacles syndrome (FDDOS) were randomly assigned to the experimen- tal group and the control group according to 1:1 proportion. All patients received standard therapy: Radix Paeoniae alba total glycosides 600 mg, twice per day. Patients in the experimental group additionally took LRCD, 30 mL each time, twice per day. The therapeutic course for all was 4 weeks, and two courses for all. The improvement of dry mouth and dry eyes were comprehensively evaluated. Each outcome of composite index constitutions (integrals of dry eyes and dry mouth, salivary flow rate, Schirmer test) was respectively reported. Schirmer test and salivary flow rate were determined as well. Score of TCM syndrome, blood sedimentation,'immunoglobulin, and adverse drug reactions were observed. Results The effective rate of comprehensive effect for dry eyes and dry mouth improvement at the end of 8 weeks was 80% in the experimental group and 35% in the control group, with statistical difference (X² =8. 286, P <0. 05). As for the composition of comprehensive effect for dry eyes and dry mouth improvement: The score for dry eyes and dry mouth decreased in the two groups more after treatment than before treatment. The difference in pre-post treatment score for dry eyes and dry mouth at week 8 was higher in the experimental group than in the control group. The difference in pre-post treatment score at week 8 was 1. 71 (95% Cl: -0. 37 -3. 78) between the two groups (P <0. 05). The difference in pre-post treatment Schirmer test and salivary flow rate at week 8 was higher in the experimental group than in the control group, but with on statistical difference (P >0. 05). The difference in pre-post treatment Schirmer test and salivary flow rate at week 8 was 2. 74 mL/15 min (95% Cl: 0. 49 -4.98) and 0. 13 mm/5 min (95% Cl: 0. 92 -1. 23) between the two groups (P <0. 05). The score of TCM syndrome decreased more in the two groups, as compared with before treatment. The difference in pre-post treatment score of TCM syn- drome at week 8 was 1. 71 (95% CI: -1. 40 -4. 81) between the two groups (P >0. 05). One case of uri- nary tract infections occurred in the control group, while no obvious adverse event occurred in the exper- imental group. Conclusion Standard treatment combined LRCD showed better comprehensive effect for dry eyes and dry mouth in pSS patients with FDDOS, and was more safe.

Laboratory investigation of the microbiologically influenced corrosion (MIC) resistance of a novel Cu-bearing 2205 duplex stainless steel in the presence of an aerobic marine Pseudomonas aeruginosa biofilm.

The microbiologically influenced corrosion (MIC) resistance of a novel Cu-bearing 2205 duplex stainless steel (2205 Cu-DSS) against an aerobic marine Pseudomonas aeruginosa biofilm was investigated. The electrochemical test results showed that Rp increased and icorr decreased sharply after long-term immersion in the inoculation medium, suggesting that 2205 Cu-DSS possessed excellent MIC resistance to the P. aeruginosa biofilm. Fluorescence microscope images showed that 2205 Cu-DSS possessed a strong antibacterial ability, and its antibacterial efficiency after one and seven days was 7.75% and 96.92%, respectively. The pit morphology comparison after 14 days between 2205 DSS and 2205 Cu-DSS demonstrated that the latter showed a considerably reduced maximum MIC pit depth compared with the former (1.44 µm vs 9.50 µm). The experimental results suggest that inhibition of the biofilm was caused by the copper ions released from the 2205 Cu-DSS, leading to its effective mitigation of MIC by P. aeruginosa.

Harnessing active biofilm for microbial corrosion protection of carbon steel against Geobacter sulfurreducens.

Microbiologically influenced corrosion (MIC) caused by corrosive microorganisms poses significant economic losses and safety hazards. Conventional corrosion prevention methods have limitations, so it is necessary to develop the eco-friendly and long-term effective strategies to mitigate MIC. This study investigated the inhibition of Vibrio sp. EF187016 biofilm on Geobacter sulfurreducens on carbon steel. Vibrio sp. EF187016 biofilm reduced the corrosion current density and impeded pitting corrosion. A thick and uniform Vibrio sp. EF187016 biofilm formed on the coupon surfaces, acting as a protective layer against corrosive ions and electron acquisition by G. sulfurreducens. The pre-grown mature Vibrio sp. EF187016 biofilms, provided enhanced protection against G. sulfurreducens corrosion. Additionally, the extracellular polymeric substances from Vibrio sp. EF187016 was confirmed to act as a green corrosion inhibitor to mitigate microbial corrosion. This study highlights the potential of active biofilms for eco-friendly corrosion protection, offering a novel perspective on material preservation against microbial corrosion.

Elucidating microbial iron corrosion mechanisms with a hydrogenase-deficient strain of Desulfovibrio vulgaris.

Sulfate-reducing microorganisms extensively contribute to the corrosion of ferrous metal infrastructure. There is substantial debate over their corrosion mechanisms. We investigated Fe0 corrosion with Desulfovibrio vulgaris, the sulfate reducer most often employed in corrosion studies. Cultures were grown with both lactate and Fe0 as potential electron donors to replicate the common environmental condition in which organic substrates help fuel the growth of corrosive microbes. Fe0 was corroded in cultures of a D. vulgaris hydrogenase-deficient mutant with the 1:1 correspondence between Fe0 loss and H2 accumulation expected for Fe0 oxidation coupled to H+ reduction to H2. This result and the extent of sulfate reduction indicated that D. vulgaris was not capable of direct Fe0-to-microbe electron transfer even though it was provided with a supplementary energy source in the presence of abundant ferrous sulfide. Corrosion in the hydrogenase-deficient mutant cultures was greater than in sterile controls, demonstrating that H2 removal was not necessary for the enhanced corrosion observed in the presence of microbes. The parental H2-consuming strain corroded more Fe0 than the mutant strain, which could be attributed to H2 oxidation coupled to sulfate reduction, producing sulfide that further stimulated Fe0 oxidation. The results suggest that H2 consumption is not necessary for microbially enhanced corrosion, but H2 oxidation can indirectly promote corrosion by increasing sulfide generation from sulfate reduction. The finding that D. vulgaris was incapable of direct electron uptake from Fe0 reaffirms that direct metal-to-microbe electron transfer has yet to be rigorously described in sulfate-reducing microbes.

Insight into a single-chamber air-cathode microbial fuel cell for nitrate removal and ecological roles.

Bioelectrochemical systems are sustainable and potential technology systems in wastewater treatment for nitrogen removal. The present study fabricated an air-cathode denitrifying microbial fuel cell (DNMFC) with a revisable modular design and investigated metabolic processes using nutrients together with the spatiotemporal distribution characteristics of dominated microorganisms. Based on the detection of organics and solvable nitrogen concentrations as well as electron generations in DNMFCs under different conditions, the distribution pattern of nutrients could be quantified. By calculation, it was found that heterotrophic denitrification performed in DNMFCs using 56.6% COD decreased the Coulombic efficiency from 38.0% to 16.5% at a COD/NO3 --N ratio of 7. Furthermore, biological denitrification removed 92.3% of the nitrate, while the residual was reduced via electrochemical denitrification in the cathode. Correspondingly, nitrate as the electron acceptor consumed 16.7% of all the generated electrons, and the residual electrons were accepted by oxygen. Microbial community analysis revealed that bifunctional bacteria of electroactive denitrifying bacteria distributed all over the reactor determined the DNMFC performance; meanwhile, electroactive bacteria were mainly distributed in the anode biofilm, anaerobic denitrifying bacteria adhered to the wall, and facultative anaerobic denitrifying bacteria were distributed in the wall and cathode. Characterizing the contribution of specific microorganisms in DNMFCs comprehensively revealed the significant role of electroactive denitrifying bacteria and their cooperative relationship with other functional bacteria.

Bioengineering approaches for the endometrial research and application.

The endometrium undergoes a series of precise monthly changes under the regulation of dynamic levels of ovarian hormones that are characterized by repeated shedding and subsequent regeneration without scarring. This provides the potential for wound healing during endometrial injuries. Bioengineering materials highlight the faithful replication of constitutive cells and the extracellular matrix that simulates the physical and biomechanical properties of the endometrium to a larger extent. Significant progress has been made in this field, and functional endometrial tissue bioengineering allows an in-depth investigation of regulatory factors for endometrial and myometrial defects in vitro and provides highly therapeutic methods to alleviate obstetric and gynecological complications. However, much remains to be learned about the latest progress in the application of bioengineering technologies to the human endometrium. Here, we summarize the existing developments in biomaterials and bioengineering models for endometrial regeneration and improving the female reproductive potential.

The identification of arsenic species produced in the dissolution of crystalline orpiment under anoxic conditions: XAS evidence.

The orpiment (As2S3) is an important secondary mineral in the geochemical process of arsenic (As) in the environment. The dissolution of orpiment has a close relationship with the migration and transformation of As. The dissolved species of As2S3 is closely related to sulfide (S-II) in the anoxic and sulfidic environment. This paper focuses on the various As species formed when As2S3 dissolved in the presence and absence of excess S-II under anoxic conditions with simulation tests via X-ray absorption spectroscopy (XAS), liquid chromatography with (hydride generation) atomic fluorescence spectrophotometry, and Raman spectroscopy. The results showed that the As produced when As2S3 dissolved in the excess S-II contained a mixture of arsenite and thioarsenite (ThioAsIII). Based on the linear combination fitting, ThioAsIII is the dominant As species (88.2 %) with arsenite as the leftover component. However, the percentage of ThioAsIII decreased to 43.7 % if As2S3 dissolved in the absence of excess S-II, indicting ThioAsIII favored under sulfidic conditions. The findings may give further insights about the role and formation mechanism of ThioAsIII in the dissolution process of As2S3. ENVIRONMENTAL IMPLICATION: The dissolution of crystallization orpiment has a close relationship with the transport of As in the environment. Qualitatively and quantitatively identification of the dissolved species of As2S3 in the presence and absence of excess S-II may be helpful for a better understanding and predicting the fate of As. The formed trithioarsenite was the dominant dissolved species compared to arsenite in the sulfidic system. It has higher mobility than AsV and AsIII, and has been found in many As-related adsorption/desorption and redox reactions. Therefore, great cautions should be given when choosing technologies to remediate the As contaminated soils and waters.

Design and fabrication of intermetallic NiCo electrocatalysts for the alkaline HER.

The design and fabrication of highly efficient electrocatalysts are crucial for reducing energy consumption, improving hydrogen production rates, and prolonging the service life of alkaline electrolyzers. In this study, intermetallic L10-NiCo electrocatalysts were designed using DFT calculations and fabricated through a one-step solid-state reaction method. The DFT calculations indicated that L10-NiCo presented a lower H adsorption Gibbs free energy and a moderate H2O dissociation barrier compared to the commonly used Ni catalyst and disordered NiCo alloy. Increasing the solid-state reaction temperature facilitated the formation of intermetallic L10-NiCo. Electrocatalytic tests for the alkaline HER demonstrated that the ECSA of L10-NiCo nanoparticles increased to 2.3 times, the overpotential decreased by 19%, the electrocatalytic activity increased to 1.5 times, and the stability improved to 2.2 times compared to those of the Ni nanoparticles. This research provides insights into the design and fabrication of highly efficient catalytic electrodes for alkaline electrolyzers.

Enhanced corrosion of 2205 duplex stainless steel by Acetobacter aceti through synergistic electron transfer and organic acids acceleration.

Acetobacter aceti is a microbe that produces corrosive organic acids, causing severe corrosion of industrial equipment. Previous studies have focused on the organic acid corrosion of A. aceti, but neglected the possibility that it has electron transfer corrosion. This study found that electron transfer and organic acids can synergistically promote the corrosion of 2205 duplex stainless steel (DSS). Electrochemical measurement results showed that corrosion of 2205 DSS was more severe in the presence of A. aceti. Surface analysis indicated a thick biofilm formed on the steel surface, with low pH and dissolved oxygen concentrations under the biofilm. Corrosion intensified when A. aceti lacked a carbon source, suggesting that A. aceti can corrode metals by using metallic substrates as electron donors, in addition to its acidic by-products.

Corrosive Pseudomonas aeruginosa detection by measuring pyocyanin with a lab-on-fiber optical surface plasmon resonance biosensor in aquatic environments.

Oceanic facilities and equipment corrosion present considerable economic and safety concerns, predominantly due to microbial corrosion. Early detection of corrosive microbes is pivotal for effective monitoring and prevention. Yet, traditional detection methods often lack specificity, require extensive processing time, and yield inaccurate results. Hence, the need for an efficient real-time corrosive microbe monitoring technology is evident. Pseudomonas aeruginosa, a widely distributed microorganism in aquatic environments, utilizes its production of quinone-like compounds, specifically pyocyanin (PYO), to corrode metals. Here, we report a novel fiber optic surface plasmon resonance (SPR) sensor modified by the C-terminal of BrlR protein (BrlR-C), which is a specific receptor of PYO molecule, to detect P. aeruginosa in aquatic environments. The results showed that the sensor had a good ability to recognize PYO in the concentration range of 0-1 µg/mL, and showed excellent sensing performance in real-time monitoring the growth status of P. aeruginosa. With a strong selectivity of PYO, the sensor could clearly detect P. aeruginosa against other bacteria in seawater environment, and exhibited excellent anti-interference ability against variations in pH, temperature and pressure and other interfering substances. This study provides a useful tool for monitoring corrosive P. aeruginosa biofilm in aquatic environments, which is a first of its kind example that serves as a laboratory model for the application of fiber optic technology in real-world scenarios to monitoring biofilms in microbial corrosion and biofouling.

Robust Chiral Metal-Organic Framework Coatings for Self-Activating and Sustainable Biofouling Mitigation.

Surface coatings are designed to mitigate pervasive biofouling herald, a new era of surface protection in complex biological environments. However, existing strategies are plagued by persistent and recurrent biofilm attachment, despite the use of bactericidal agents. Herein, a chiral metal-organic framework (MOF)-based coating with conformal microstructures to enable a new anti-biofouling mode that involves spontaneous biofilm disassembly followed by bacterial eradication is developed. A facile and universal metal-polyphenol network (MPN) is designed to robustly anchor the MOF nanoarmor of biocidal Cu2+ ions and anti-biofilm d-amino acid ligands to a variety of substrates across different material categories and surface topologies. Incorporating a diverse array of chiral amino acids endows the resultant coatings with widespread signals for biofilm dispersal, facilitating copper-catalyzed chemodynamic reactions and inherent mechano-bactericidal activities. This synergistic mechanism yields unprecedented anti-biofouling efficacy elucidated by RNA-sequencing transcriptomics analysis, enhancing broad-spectrum antibacterial activities, preventing biofilm formation, and destroying mature biofilms. Additionally, the chelation-directed amorphous/crystalline coatings can activate photoluminescent properties to inhibit the settlement of microalgae biofilms. This study provides a distinctive perspective on chirality-enhanced antimicrobial behaviors and pioneers a rational pathway toward developing next-generation anti-biofouling coatings for diverse applications.

Enhanced antibacterial and corrosion resistance of copper-containing 2205 duplex stainless steel against the corrosive bacterium Shewanella algae.

2205 DSS is an excellent corrosion-resistant engineering metal material, but it is still threatened by microbiological corrosion. The addition of copper elements is a new approach to improving the resistance of 2205 DSS to microbiological corrosion. In this study, 2205-Cu DSS was compared with 2205 DSS to study its antimicrobial properties and resistance to microbiological corrosion in the presence of the electroactive bacterium Shewanella algae. The results showed that compared to 2205 DSS, the biofilm thickness and the number of live bacteria on the surface of 2205-Cu DSS were significantly reduced, demonstrating excellent antimicrobial properties against S. algae. Electrochemical tests and surface morphology characterization results showed that the corrosion rate and pitting of 2205-Cu DSS by S. algae were lower than that of 2205 DSS, indicating better resistance to microbiological corrosion. The good antimicrobial properties and resistance to microbiological corrosion exhibited by 2205-Cu DSS are attributed to the contact antimicrobial properties of copper elements in the 2205-Cu DSS matrix and the release of copper ions for antimicrobial effects. This study provides a new strategy for combating microbiological corrosion.

The long-term transformation of FeIII-AsV coprecipitates at room temperature under oxic conditions: New insights for the fate and the speciation of As.

The long-term stability of FeIII-AsV coprecipitates, a typically hydrometallurgical or naturally produced As-bearing wastes in tailings or in other environments, is critical to evaluating the As risk caused by them. A wide pH range, different Fe/As molar ratios, reaction media, and neutralization reagents were considered in order to find the mechanisms controlling the fate of As during the 1640 days of transformation at 25 °C. The results indicated that at pH 4 and 12, As continuously released from the solid phase. The components and their proportions determined the fate of As at pH 4. However, at pH 12, crystalline calcium carbonates (CCA) formed due to the CO2 in the air and this combined with the adsorption capacity of As on the 2-line ferrihydrite controlling the fate of As. If pH changed to 8 and 10, yukonite formed after the release of As. The CCA also appeared in the presence of Ca. Therefore, these two processes controlled the fate of As at this pH range. These findings are important for understanding and predicting the transport of As under various environmental conditions. The technology chosen for As remediation in soils and As removal from waste waters will also be benefit from these results.