Dynamics of the role of LacMeta laccase in the complete degradation and detoxification of malachite green.

Laccases highlight for xenobiotic bioremediation, as well as application in the fine chemical, textile, biofuel and food industries. In a previous work, we described the preliminary characterization of laccase LacMeta, a promising enzyme for the bioremediation of dyes, able to decolorization malachite green (MG), trypan blue, methylene blue. Here we demonstrate that LacMeta is indeed suitable for the complete degradation and detoxification of MG dye, not just for its discoloration, since some works show false positives due to the formation of colorless intermediates such as leucomalachite. The optimal pH and temperature parameters of LacMeta were 5.0 and 50 °C, respectively (MG as substrate). LacMeta was tolerant of up to 10 mmol L- 1 EDTA (82%) and up to 5% (V/V) acetone (91%) and methanol (71%), while SDS promoted severe inhibition. For ions, a high tolerance to cobalt, zinc, manganese, and calcium (10 mmol L- 1) was also observed (> 90%). Even under high-salinity conditions (1 mol L- 1 NaCl), the residual bleaching activity of the dye remained at 61%. Furthermore, the bleaching product of MG did not inhibit the germination of sorghum and tomato seeds and was inert to the vegetative structures of the germinated seedlings. Additionally, this treatment effectively reduced the cytotoxic effect of the dye on microorganisms (Escherichia coli and Azospirillum brasilense), which can be explained by H-NMR spectral analysis results since LacMeta completely degraded the peak signals corresponding to the aromatic rings in the dye, demonstrating extreme efficiency in the bioremediation of the xenobiotic at high concentrations (50 mg L- 1).

3-D Time-Lapse Imaging of Cell Wall Dynamics Using Calcofluor in the Moss Physcomitrium patens.

Time-lapse imaging with fluorescence microscopy allows observation of the dynamic changes of growth and development at cellular and subcellular levels. In general, for observations over a long period, the technique requires transformation of a fluorescent protein; however, for most systems, genetic transformation is either time-consuming or technically unavailable. This manuscript presents a protocol for 3-D time-lapse imaging of cell wall dynamics over a 3 day period using calcofluor dye (which stains cellulose in the plant cell wall), developed in the moss Physcomitrium patens. The calcofluor dye signal from the cell wall is stable and can last for 1 week without obvious decay. Using this method, it has been shown that the detachment of cells in ggb mutants (in which the protein geranylgeranyltransferase-I beta subunit is knocked out) is caused by unregulated cell expansion and cell wall integrity defects. Moreover, the patterns of calcofluor staining change over time; less intensely stained regions correlate with the future cell expansion/branching sites in the wild type. This method can be applied to many other systems that contain cell walls and that can be stained by calcofluor.

Nonreducing terminal chimeric isomaltomegalosaccharide and its integration with azoreductase for the remediation of soil-contaminated lipophilic azo dyes.

Lipophilic azo dyes are practically water-insoluble, and their dissolution by organic solvents and surfactants is harmful to biological treatment with living cells and enzymes. This study aimed to evaluate the feasibility of a newly synthesized nonreducing terminal chimeric isomaltomegalosaccharide (N-IMS) as a nontoxic solubilizer of four simulated lipophilic azo dye wastes for enzymatic degradation. N-IMS bearing a helical α-(1 â†’ 4)-glucosidic segment derived from a donor substrate α-cyclodextrin was produced by a coupling reaction of cyclodextrin glucanotransferase. Inclusion complexing by N-IMS overcame the solubility issue with equilibrium constants of 1786-242 M-1 (methyl yellow > ethyl red > methyl red > azo violet). Circular dichroism spectra revealed the axial alignment of the aromatic rings in the N-IMS cavity, while UV-visible absorption quenching revealed that the azo bond of methyl yellow was particularly induced. Desorption of the dyes from acidic and neutral soils was specific to aqueous organic over alkali extraction. The dissolution kinetics of the incorporated dyes followed a sigmoid pattern facilitating the subsequent decolorization process with azoreductase. It was demonstrated that after soil extraction, the solid dyes dissolved with N-IMS assistance and spontaneously digested by coupled azoreductase/glucose dehydrogenase (for a cofactor regeneration system) with the liberation of the corresponding aromatic amine.

Bioprospecting of novel ligninolytic bacteria for effective bioremediation of agricultural by-product and synthetic pollutant dyes.

Lignin is a significant renewable carbon source that needs to be exploited to manufacture bio-ethanol and chemical feedstocks. Lignin mimicking methylene blue (MB) dye is widely used in industries and causes water pollution. Using kraft lignin, methylene blue, and guaiacol as a full carbon source, 27 lignin-degrading bacteria (LDB) were isolated from 12 distinct traditional organic manures for the current investigation. The ligninolytic potential of 27 lignin-degrading bacteria was assessed by qualitative and quantitative assay. In a qualitative plate assay, the LDB-25 strain produced the largest zone, measuring 6.32 ± 0.297, on MSM-L-kraft lignin plates, while the LDB-23 strain produced the largest zone, measuring 3.44 ± 0.413, on MSM-L-Guaiacol plates. The LDB-9 strain in MSM-L-kraft lignin broth was able to decolorize lignin to a maximum of 38.327 ± 0.011% in a quantitative lignin degradation assay, which was later verified by FTIR assay. In contrast, LDB-20 produced the highest decolorization (49.633 ± 0.017%) in the MSM-L-Methylene blue broth. The highest manganese peroxidase enzyme activity, measuring 6322.314 ± 0.034 U L-1, was found in the LDB-25 strain, while the highest laccase enzyme activity, measuring 1.5105 ± 0.017 U L-1, was found in the LDB-23 strain. A preliminary examination into the biodegradation of rice straw using effective LDB was carried out, and efficient lignin-degrading bacteria were identified using 16SrDNA sequencing. SEM investigations also supported lignin degradation. LDB-8 strain had the highest percentage of lignin degradation (52.86%), followed by LDB-25, LDB-20, and LDB-9. These lignin-degrading bacteria have the ability to significantly reduce lignin and lignin-analog environmental contaminants, therefore they can be further researched for effective bio-waste management mediated breakdown.

A bacterial cold-active dye-decolorizing peroxidase from an Antarctic Pseudomonas strain.

DyP (dye-decolorizing peroxidase) enzymes are hemeproteins that catalyze the H2O2-dependent oxidation of various molecules and also carry out lignin degradation, albeit with low activity. We identified a dyp gene in the genome of an Antarctic cold-tolerant microbe (Pseudomonas sp. AU10) that codes for a class B DyP. The recombinant protein (rDyP-AU10) was produced using Escherichia coli as a host and purified. We found that rDyP-AU10 is mainly produced as a dimer and has characteristics that resemble psychrophilic enzymes, such as high activity at low temperatures (20 °C) when using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and H2O2 as substrates, thermo-instability, low content of arginine, and a catalytic pocket surface larger than the DyPs from some mesophilic and thermophilic microbes. We also report the steady-state kinetic parameters of rDyP-AU10 for ABTS, hydroquinone, and ascorbate. Stopped-flow kinetics revealed that Compound I is formed with a rate constant of (2.07 ± 0.09) × 106 M-1 s-1 at pH 5 and that this is the predominant species during turnover. The enzyme decolors dyes and modifies kraft lignin, suggesting that this enzyme may have potential use in bioremediation and in the cellulose and biofuel industries. KEY POINTS: • An Antarctic Pseudomonas strain produces a dye-decolorizing peroxidase. • The recombinant enzyme (rDyP-AU10) was produced in E. coli and purified. • rDyP-AU10 showed high activity at low temperatures. • rDyP-AU10 is potentially useful for biotechnological applications.

Quantification of a Fluorescent Lipid DOPE-NBD by an HPLC Method in Biological Tissue: Application to Study Liposomes' Uptake by Human Placenta.

Nanomedicine offers the possibility of modifying the distribution of encapsulated drugs and biomolecules. Nanomedicine could limit the transplacental passage and/or enhance the concentration of drugs in placental tissue; this approach could be exploited for the treatment of pregnancy disorders. In the context of pregnancy, tackling the biological fate of both the nanocarrier and the drug has high importance in ensuring both the mother's and the fetus' safety.In this study, we propose a method for quantifying the uptake of liposomes inside placental tissue using covalently labeled liposomes and adapting a high-performance liquid chromatography (HPLC) method using a fluorescent detector. An optimized protocol for liquid-liquid extraction of fluorescent lipids from placental tissue extracts, followed by HPLC analysis, is detailed in this chapter. The HPLC method allows the quantification of fluorescent lipids using a calibration curve, including the biological matrix and extraction procedures. The internalization rate of fluorescent liposomes within human villous placental explants was quantitatively assessed, thanks to the HPLC developed method and suitable analytical tools.

Actin: Static and Dynamic Studies.

The actin cytoskeleton is a highly dynamic network in plant cells, which is precisely regulated by numerous actin-binding proteins. Hence, characterizing the biochemical activities of actin-binding proteins is of great importance. Here we describe methods for determining the binding and bundling of microfilaments as well as methods for visualizing microfilaments using fluorescent phalloidin and single-molecule TIRF imaging.

Enhanced textile dye removal from wastewater using natural biosorbent and Shewanella algae B29: Application of Box Behnken design and genomic approach.

In this study, Box-Behnken design combining seven factors at three levels were used to optimize the elimination of CI Reactive Red 66 in artificial seawater, by the combination of eco-friendly bio-sorbents and acclimated halotolerant microbial strain. Results showed that macro-algae and cuttlebone (2 %) were the best natural bio-sorbent. Additionally, the selected halotolerant strain able to rapidly remove dye was identified as Shewanella algae B29. The optimization process revealed that decolourization of CI Reactive Red 66 yields reached 91.04 % under the following variable values: dyes concentration (100 mg/l), salinity (30 g/l), peptone (2 %), pH (5), algae C (3 %), cuttlebone (1.5 %) and agitation (150 rpm). The whole genome analysis of S. algae B29 demonstrated the presence of several genes coding for valuable enzymes involved in textile dyes biotransformation, adaptation to stress as well as biofilm formation implying its potential use in biological textile wastewater treatment.

Optimization of biodegradation yield of reactive blue 49: An integrated approach using response surface methodology based marine predators algorithm.

Biodegradation involving the use of biological systems are proving to be more cost-effective, energy efficient and environmentally friendly method for treatment with dye. There are also some advantages of enzymatic degradation to remove pollutants from textile effluents. They are specific and selectable for the substrates. Moreover, the usage of enzymatic degradation with laccase has environmental and social dimensions of sustainable bio-economy when compared with commercial solutions. In this study, besides the conventional RSM (Response Surface Methodology) approaches, the performance of a new metaheuristic, MPA (Marine Predators Algorithm) integrated with RSM has also been tested. The effect of four independent variables time, enzyme amount, agitation speed, and initial dye concentration have been studied to obtain maximum decolorization. Two major techniques of RSM, namely Box Behnken Design (BBD) and Central Composite Design (CCD) have been used to determine optimal levels of parameters. The highest decolorization efficiency (%) of Reactive Blue 49 (RB49) has been achieved as 92.82% with BBD and 90.56% with CCD. Maximum efficiency for BBD based MPA as being 94.69% has been obtained at 70.96 min, 1.5 mL, 74.63 rpm, and 99.09 ppm. For CCD based MPA, the level of efficiency has been obtained as 93.1% at 39.69 min, 1 mL, 72.07 rpm, and 74.41 ppm. The results of MPA indicate that using metaheuristics combined with RSM is a suitable and sustainable way to optimize parameters of RB49% for decolorization.

Biosynthesis of Silver Nanoparticles Utilizing Leaf Extract of Trigonella foenum-graecum L. for Catalytic Dyes Degradation and Colorimetric Sensing of Fe3+/Hg2.

The aqueous Trigonella foenum-graecum L. leaf extract belonging to variety HM 444 was used as reducing agent for silver nanoparticles (AgNPs) synthesis. UV-Visible spectroscopy, Particle size analyser (PSA), Field emission scanning electron microscopy coupled to energy dispersive X-ray spectroscopy (FESEM-EDX) and High-resolution transmission electron microscopy (HRTEM) were used to characterize AgNPs. Selected area electron diffraction (SAED) confirmed the formation of metallic Ag. Fourier Transform Infrared Spectroscopy (FTIR) was done to find out the possible phytochemicals responsible for stabilization and capping of the AgNPs. The produced AgNPs had an average particle size of 21 nm, were spherical in shape, and monodispersed. It showed catalytic degradation of Methylene blue (96.57%, 0.1665 ± 0.03 min-1), Methyl orange (71.45%, 0.1054 ± 0.002 min-1), and Rhodamine B (92.72%, 0.2004 ± 0.01 min-1). The produced AgNPs were excellent solid bio-based sensors because they were very sensitive to Hg2+ and Fe3+ metal ions with a detection limit of 11.17 µM and 195.24 µM, respectively. From the results obtained, it was suggested that aqueous leaf extract demonstrated a versatile and cost-effective method and should be utilized in future as green technology for the fabrication of nanoparticles.

Impact of textile dyes on human health and bioremediation of textile industry effluent using microorganisms: current status and future prospects.

Environmental contamination brought on by the discharge of wastewater from textile industries is a growing concern on a global scale. Textile industries produce a huge quantity of effluents containing a myriad of chemicals, mostly dyes. The discharge of such effluents into the aquatic environment results in pollution that adversely affects aquatic organisms. Synthetic dyes are complex aromatic chemical structures with carcinogenic and mutagenic properties in addition to high biological oxygen demand (BOD) and chemical oxygen demand (COD). This complex aromatic structure resists degradation by conventional techniques. The bioremediation approach is the biological clean-up of toxic contaminants from industrial effluents. Biological treatment methods produce less or no sludge and are cost-effective, efficient, and eco-friendly. Microorganisms, mostly microalgae and bacteria, and, in some instances, fungi, yeast, and enzymes decolorize textile dye compounds into simple, non-toxic chemical compounds. Following a thorough review of the literature, we are persuaded that microalgae and bacteria might be one of the potential decolorizing agents substituting for most other biological organisms in wastewater treatment. This article presents extensive literature information on textile dyes, their classification, the toxicity of dyes, and the bioremediation of toxic textile industry effluent utilizing microalgae and bacteria. Additionally, it combines data on factors influencing textile dye bioremediation, and a few suggestions for future research are proposed.

A mutant R70V/E166A of short manganese peroxidase showing Mn2+-independent dye decolorization.

Il-MnP1, a short-type manganese peroxidase from Irpex lacteus F17, can oxidize some substrates in the absence of Mn2+, but the catalysis was much lower than in the presence of Mn2+. Here, we report a mutant R70V/E166A of Il-MnP1 with some unique properties, which possessed clearly higher catalysis for the decolorization of anthraquinone and azo dyes in the absence of Mn2+ than that of Il-MnP1. Importantly, the optimum pH of R70V/E166A for decolorization of anthraquinone dyes (Reactive Blue 19, RB19) was 6.5, and the mutant achieved high decolorization activities in the range of pH 4.0-7.0, whereas Il-MnP1 only showed decolorization for RB19 at pH 3.5-4.0. In addition, the optimum H2O2 concentration of R70V/E166A for RB19 decolorization was eight times that of Il-MnP1 and the H2O2 stability has improved 1.4 times compared with Il-MnP1. Furthermore, Mn2+ competitively inhibited the oxidation of RB19 by R70V/E166A, explaining the higher catalytic activity of the mutant R70V/E166A in the absence of Mn2+. Molecular docking results suggested that RB19 binds to the distal side of the heme plane in mutant R70V/E166A, which extended from the heme δ-side to the heme γ-side, and close to the mutated residues of R70V and E166A, whereas RB19 could not access the heme pocket of Il-MnP1 due to the steric hindrance of the side-chain group of Arg 70. Thus, this study constructed a useful mutant R70V/E166A and analyzed its higher Mn2+-independent activity, which is very important for better understanding the Mn2+-independent catalytic mechanism for short manganese peroxidases. KEY POINTS: • The mutant R70V/E166A of atypical MnP1 of I. lacteus F17 shows unique catalytic properties. • At pH 6.5, the R70V/E166A had a strong ability to decolorize anthraquinone dyes in the absence of Mn2+. • The binding sites of Reactive Blue 19 in mutant R70V/E166A were elucidated.

Biotreatment of azo dye containing textile industry effluent by a developed bacterial consortium immobilised on brick pieces in an indigenously designed packed bed biofilm reactor.

This study highlights the development of a lab-scale, indigenously designed; Packed-Bed Biofilm Reactor (PBBR) packed with brick pieces. The developed biofilm in the reactor was used for the decolourisation and biodegradation of the textile industry effluent. The PBBR was continuously operated for 264 days, during which 301 cycles of batch and continuous treatment were operated. In batch mode under optimised conditions, more than 99% dye decolourisation and ≥ 92% COD reduction were achieved in 6 h of contact time upon supplementation of effluent with 0.25 g L-1 glucose, 0.25 g L-1 urea, and 0.1 g L-1 phosphates. A decolourisation rate of 133.94 ADMI units h-1 was achieved in the process. PBBR, when operated in continuous mode, showed ≥ 95% and ≥ 92% reduction in ADMI and COD values. Subsequent aeration and passage through the charcoal reactor assisted in achieving a ≥ 96% reduction in COD and ADMI values. An overall increase of 81% in dye-laden effluent decolourisation rate, from 62 to 262 mg L-1 h-1, was observed upon increasing the flow rate from 18 to 210 mL h-1. Dye biodegradation was determined by UV-Vis and FTIR spectroscopy and toxicity study. SEM analysis showed the morphology of the attached-growth biofilm.

Seeing Neurodegeneration in a New Light Using Genetically Encoded Fluorescent Biosensors and iPSCs.

Neurodegenerative diseases present a progressive loss of neuronal structure and function, leading to cell death and irrecoverable brain atrophy. Most have disease-modifying therapies, in part because the mechanisms of neurodegeneration are yet to be defined, preventing the development of targeted therapies. To overcome this, there is a need for tools that enable a quantitative assessment of how cellular mechanisms and diverse environmental conditions contribute to disease. One such tool is genetically encodable fluorescent biosensors (GEFBs), engineered constructs encoding proteins with novel functions capable of sensing spatiotemporal changes in specific pathways, enzyme functions, or metabolite levels. GEFB technology therefore presents a plethora of unique sensing capabilities that, when coupled with induced pluripotent stem cells (iPSCs), present a powerful tool for exploring disease mechanisms and identifying novel therapeutics. In this review, we discuss different GEFBs relevant to neurodegenerative disease and how they can be used with iPSCs to illuminate unresolved questions about causes and risks for neurodegenerative disease.

Detection of microscopic filamentous fungal biofilms - Choosing the suitable methodology.

Microscopic filamentous fungi are ubiquitous microorganisms that adapt very easily to a variety of environmental conditions. Due to this adaptability, they can colonize a number of various surfaces where they are able to start forming biofilms. Life in the form of biofilms provides them with many benefits (increased resistance to desiccation, UV radiation, antimicrobial compounds, and host immune response). The aim of this study is to find a reliable and reproducible methodology to determine biofilm growth of selected microscopic filamentous fungi strains. Several methods (crystal violet staining, MTT assay, XTT assay, resazurin assay) for the determination of total biofilm biomass and its metabolic activity were tested on four fungi - Alternaria alternata, Aspergillus niger, Fusarium culmorum and Fusarium graminearum, and their biofilm was also imaged by spinning disc confocal microscopy using fluorescent dyes. A reproducible biofilm quantification method is essential for the subsequent testing of the biofilm growth suppression using antifungal agents or physical methods. Crystal violet staining was found to be a suitable method for the determination of total biofilm biomass of selected strains, and the MTT assay for the determination of metabolic activity of the biofilms. Calcofluor white and Nile red fluorescent stains successfully dyed the hyphae of microscopic fungi.

Optimized fluorescent proteins for 4-color and photoconvertible live-cell imaging in Neurospora crassa.

Fungal cells are quite unique among life in their organization and structure, and yet implementation of many tools recently developed for fluorescence imaging in animal systems and yeast has been slow in filamentous fungi. Here we present analysis of properties of fluorescent proteins in Neurospora crassa as well as describing genetic tools for the expression of these proteins that may be useful beyond cell biology applications. The brightness and photostability of ten different fluorescent protein tags were compared in a well-controlled system; six different promoters are described for the assessment of the fluorescent proteins and varying levels of expression, as well as a customizable bidirectional promoter system. We present an array of fluorescent proteins suitable for use across the visible light spectrum to allow for 4-color imaging, in addition to a photoconvertible fluorescent protein that enables a change in the color of a small subset of proteins in the cell. These tools build on the rich history of cell biology research in filamentous fungi and provide new tools to help expand research capabilities.

Application of Salvinia sps. in remediation of reactive mixed azo dyes and Cr (VI) - Its pathway elucidation.

The emerging industrialization has resulted in the rapid growth of textile industries across the globe. The presence of xenobiotic pollutants in textile wastewater threatens the ecosystem. Applying different microbes (bacteria, fungi & algae) has paved the way for phytoremediation - the eco-friendly, cost-effective method. The present study focuses on the phytoremediation of reactive dyes - Reactive red, Reactive Brown & Reactive Black and Cr (VI) in synthetic textile wastewater using Salvinia sps. The mixed azo dyes of each 100 mg/L showed decolourization of 75 ± 0.5% and 82 ± 0.5% of removal of 20 mg/L of Cr (VI) after eight days of incubation in a phytoreactor setup. Chlorophyll analysis revealed the gradual decrease in the photosynthetic pigments during the remediation. The degraded metabolites were analyzed using FT-IR and showed the presence of aromatic amines on day zero, which were converted to aliphatic amines on day four. The GC-MS analysis revealed the disruption of -NN- bond, rupture of -CN- bond, scission of -N-N-bond, and loss of -SO3H from the Reactive Black dye leading to the formation of an intermediate p-Hydroxy phenylhydrazinyl. The rupture of Reactive red dye resulted in the formation of p-Hydrazinyl toluene sulphonic acid, Naphthyl amine -3,6-disulphonic acid and 8-Hydroxy Naphthyl amine -3,6-disulphonic acid. Decarboxylation, desulphonation, deoxygenation and deamination of Reactive Brown dye showed the presence of different metabolites and metabolic pathways were proposed for the reactive azo dyes which were phytoremediated.

Eco-friendly degradation of reactive red 195, reactive blue 214, and reactive yellow 145 by Klebsiella pneumoniae MW815592 isolated from textile waste.

The water is used in many textile manufacturing steps beyond cleaning. The quantity and the significant chemical load of the effluents generated constitute the primary challenge of the textile industry. In order to discover new sustainable methods to overcome this problem, the aim of this research was to study the potential for degradation of Reactive Blue 214, Reactive Red 195, and Reactive Yellow 145 using a dye degrading bacterium. Sequencing analysis reveals it to be Klebsiella pneumoniae MW815592. This strain completely decolorized artificial effluent (200 mg/L) after 42 h at pH 9 and 46 °C. The decolorization rate increased in the presence of glucose and yeast extract (2 g). In addition, our finding revealed that the decolorization is due to biodegradation rather than adsorption on the bacterial surface.

Investigation on the change of spermatozoa flagellar beating forces before and after capacitation.

The swimming forces exerted by mammalian spermatozoa during the pathway to the ovary and during the interaction with the oocyte are thought to play a fundamental role in the fertilization of the egg. In particular, a process named capacitation is of key relevance for its success. Capacitation enables spermatozoa to undergo the acrosome reaction and to exhibit different motility called hyperactivation with a change in the sperm cell tail motion from symmetric to a more asymmetric beating, characterized by wider flagellar bending at lower frequencies. Despite several studies about the mechanism that underlies capacitation, no quantitative information is available about the forces associated with sperm motility. Sperm cell motility has been widely studied with digital imaging tools and video microscopy, but these methodologies cannot provide information about the forces exerted by spermatozoa during the motion and the contribution of every single frequency of flagellar beating to the sperm cell movement. For this purpose, fluidic force microscopy was used to trap single swimming spermatozoa allowing to evaluate these parameters. We observe significant differences between capacitated and non-capacitated spermatozoa in terms of force exerted and beating frequencies. The description of the dynamics of this process is of great interest in the field of reproductive medicine. Such information could be useful to clarify unknown causes of male infertility or for the development of novel methods to assess the quality of semen samples.

Bacillus spizizenii DN and microbial consortia biostimulation followed by gamma irradiation for efficient textile wastewater treatment.

Textile wastewater still poses a huge environmental problem due to its high water consumption and high effluent release that is full of toxic chemicals. In the present study, different approaches were studied to layout an operating procedure for textile wastewater treatment in order to obtain treated wastewater that is safe for non-potable uses. Our approach depended on (1) co-substrate to biostimulate indigenous microbial textile wastewater community by adding Tryptone Soy Broth (TSB) and TSB supplemented with 1% glucose, (2) co-culture (bioaugmentation) with Bacillus spizizenii DN cells (previously isolated, identified and characterized as efficient decolorizing bacteria), and (3) co-metabolites using Bacillus spizizenii DN metabolites. The obtained results show that using Bacillus spizizenii DN cells resulted in 97.78% decolorization while adding Bacillus spizizenii DN metabolites resulted in 82.92% decolorization, both after 48 h incubation under microaerophilic conditions. The phyla identified for all treatments were Bacteroidota, Firmicutes, and Proteobacteria. The dynamic changes in the bacteria showed that both Clostridium and Acinetobacter disappeared for co-substrate, co-culture, and co-metabolite cultures. While Alkalibacterium and Stenotrophomonas appeared after adding Bacillus spizizenii DN cells, Flavobacterium increased for co-substrate and co-metabolic cultures while iron reducing bacteria appeared only for co-metabolic cultures. The use of 25 kGy gamma irradiation as a sterilization dose post bioremediation ensured safe use of treated wastewater. This was confirmed by cytotoxicity assay; the obtained IC50 tested on BJ fibroblasts obtained from skin showed that gamma irradiated treated wastewater are about 80.1% less toxic than non-irradiated treated wastewater. We conclude that (1) we can use combined bioaugmentation and biostimulation as initial steps for in situ bioremediation in collection tanks and that (2) the proposed protocol for bioremediation of industrial wastewater should be tailored based on the required application and level of safety needed for re-use.