Selective pressure leads to an improved synthetic consortium fit for dye degradation.

Microorganisms have great potential for bioremediation as they have powerful enzymes and machineries that can transform xenobiotics. The use of a microbial consortium provides more advantages in application point of view than pure cultures due to cross-feeding, adaptations, functional redundancies, and positive interactions among the organisms. In this study, we screened about 107 isolates for their ability to degrade dyes in aerobic conditions and without additional carbon source. From our screening results, we finally limited our synthetic consortium to Gordonia and Rhodococcus isolates. The synthetic consortium was trained and optimized for azo dye degradation using sequential treatment of small aromatic compounds such as phenols that act as selective pressure agents. After four rounds of optimization with different aims for each round, the consortium was able to decolorize and degrade various dyes after 48 h (80%-100% for brilliant black bn, methyl orange, and chromotrop 2b; 50-70% for orange II and reactive orange 16; 15-30% for chlorazol black e, reactive red 120, and allura red ac). Through rational approaches, we can show that treatment with phenolic compounds at micromolar dosages can significantly improve the degradation of bulky dyes and increase its substrate scope. Moreover, our selective pressure approach led to the production of various dye-degrading enzymes as azoreductase, laccase-like, and peroxidase-like activities were detected from the phenol-treated consortium. Evidence of degradation was also shown as metabolites arising from the degradation of methyl red and brilliant black bn were detected using HPLC and LC-MS analysis. Therefore, this study establishes the importance of rational and systematic screening and optimization of a consortium. Not only can this approach be applied to dye degradation, but this study also offers insights into how we can fully maximize microbial consortium activity for other applications, especially in biodegradation and biotransformation.

Microbial Degradation of Azo Dyes: Approaches and Prospects for a Hazard-Free Conversion by Microorganisms.

Azo dyes have become a staple in various industries, as colors play an important role in consumer choices. However, these dyes pose various health and environmental risks. Although different wastewater treatments are available, the search for more eco-friendly options persists. Bioremediation utilizing microorganisms has been of great interest to researchers and industries, as the transition toward greener solutions has become more in demand through the years. This review tackles the health and environmental repercussions of azo dyes and its metabolites, available biological approaches to eliminate such dyes from the environment with a focus on the use of different microorganisms, enzymes that are involved in the degradation of azo dyes, and recent trends that could be applied for the treatment of azo dyes.

In vitro and in silico analysis of Brilliant Black degradation by Actinobacteria and a Paraburkholderia sp.

The soil bacteria isolated in this study, including three strains of actinobacteria and one Paraburkholderia sp., showed decolorization activity of azo dyes in the resting cell assay and were shown to use methyl red as the sole carbon source to proliferate. Therefore, their ability to degrade, bioabsorb, or a combination of both mechanism was investigated using the substrate brilliant black. The strains DP-A9 and DP-L11, within 24 h of incubation, showed complete biodegradation of 173.54 mg/L brilliant black and the strains DP-D10 and DP-P12 showed partial decolorization of 83.3 mg/L and 36.4 mg/L, respectively, by both biosorption and biodegradation. In addition, the shotgun assembled genome of these strains showed a highly diverse set of genes encoding for candidate dye degrading enzymes, providing avenues to study azo dye metabolism in more detail.

Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase.

Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone - giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).

Improving Biocatalytic Properties of an Azoreductase via the N-Terminal Fusion of Formate Dehydrogenase.

Azoreductases require NAD(P)H to reduce azo dyes but the high cost of NAD(P)H limits its application. Formate dehydrogenase (FDH) allows NAD(P)+ recycling and therefore, the fusion of these two biocatalysts seems promising. This study investigated the changes to the fusion protein involving azoreductase (AzoRo) of Rhodococcus opacus 1CP and FDH (FDHC23S and FDHC23SD195QY196H ) of Candida boidinii in different positions with His-tag as the linker. The position affected enzyme activities as AzoRo activity decreased by 20-fold when it is in the N-terminus of the fusion protein. FDHC23S +AzoRo was the most active construct and was further characterized. Enzymatic activities of FDHC23S +AzoRo decreased compared to parental enzymes but showed improved substrate scope - accepting bulkier dyes. Moreover, pH has an influence on the stability and activity of the fusion protein because at pH 6 (pH that is suboptimal for FDH), the dye reduction decreased to more than 50 % and this could be attributed to the impaired NADH supply for the AzoRo part.

Correction to: Draft genome sequence of Kocuria indica DP-K7, a methyl red degrading actinobacterium.

[This corrects the article DOI: 10.1007/s13205-020-2136-3.].

Draft genome sequence of Kocuria indica DP-K7, a methyl red degrading actinobacterium.

In the present study, we report the draft genome of soil isolate DP-K7 that has the potential to degrade methyl red. The 16S rRNA gene sequencing and whole-genome analysis exposed that the bacterial strain DP-K7 belongs to the species Kocuria indica. The genome annotation of the strain DP-K7 through the bioinformatics tool "Prokka" showed that the genome contains 3,010,594 bp with 69.01% GC content. The genome comprises 57 contigs including 2 rRNA genes, 47 tRNA genes, and 2754 CDS. The plate and broth assay showed that the strain DP-K7 has the potential to utilize methyl red as the sole carbon source for growth. Indeed, the RP-HPLC analysis proved that the strain DP-K7 is capable of degrading methyl red. The genome BLAST against a characterized azoreductase (AzoB-Xenophilus azovorans KF46F) revealed the presence of two azoreductase-like genes (azoKi-1 and azoKi-2). The phylogenetic analysis of the primary amino acid sequence of characterized azoreductases suggested that AzoKi-1 and AzoKi-2 belong to members of the clade IV azoreductase, which are flavin-independent. The multiple sequence alignment of AzoKi-1 and AzoKi-2 with flavin-independent azoreductases showed the presence of NAD(P)H binding like motif (GxxGxxG). In addition, other genes coding for dye degrading enzymes (SodC, SodA, KatA, KatE, and DyP2) were also found in the genome supporting that the strain K. indica DP-K7 is a potential azo dye degrader.

Degradation of Polycyclic Aromatic Hydrocarbons by Moderately Halophilic Bacteria from Luzon Salt Beds.

BACKGROUND: Polycyclic aromatic hydrocarbons (PAHs) are common environmental contaminants which are highly toxic due to their carcinogenic and mutagenic effects. They are released into the environment by incomplete combustion of solid and liquid fuels, accidental spillage of oils and seepage from industrial activities. One of the promising processes mitigating PAHs is through biodegradation. However, conventional microbiological treatment processes do not function well at high salt concentrations. Hence, utilization of halophilic bacteria should be considered. OBJECTIVES: This study aimed to assess the ability of halophilic bacteria isolated from local salt beds in Pangasinan and Cavite, the Philippines, to degrade PAHs pyrene, fluorene and fluoranthene. METHODS: Polycyclic aromatic hydrocarbon-tolerant halophilic bacteria collected from two sampling sites were phenotypically characterized, molecularly identified and tested to determine their potential to degrade the PAHs pyrene, fluorene and fluoranthene at a hypersaline condition. Best PAH degraders were then assayed to identify the optimal degradation using such parameters as pH, temperature and PAH concentration. Testing for enzyme degradation was also done to determine their baseline information. Extraction and analysis of degraded PAHs were performed using centrifugation and UV-vis spectrophotometry. RESULTS: Twelve isolates from both collection sites tolerated and grew in culture with selected PAHs. These were identified into four genera (Halobacillus, Halomonas, Chromohalobacter, and Pontibacillus). Selected best isolates in a series of biodegradation assays with the above-mentioned parameters were Halobacillus B (Collection of Microbial Strains (CMS) 1802) (=trueperi) (Gram-positive) for pyrene and fluoranthene, and Halomonas A (CMS 1901) (Gram-negative) for fluorene. Degrader biomass and PAH degradation were invariably negatively correlated. Qualitative tests with and without peptone as a nitrogen source implied enzymatic degradation. DISCUSSION: Polycyclic aromatic hydrocarbons utilized by these halophilic bacteria served as a sole source of carbon and energy. Implications of biodegradation of the two best isolates show that high molecular weight (HMW) (4-ring) pyrene tends to be degraded better by Gram-positive bacteria and low molecular weight (3-ring) fluorene by Gram-negative degraders. CONCLUSIONS: Halophilic bacteria constitute an untapped natural resource for biotechnology in the Philippines. The present study demonstrated their potential use in bioremediation of recalcitrant hydrocarbons in the environment. COMPETING INTERESTS: The authors declare no competing financial interests.

Decolorization of Selected Synthetic Textile Dyes by Yeasts from Leaves and Fruit Peels.

BACKGROUND: Discharge of textile dyes into the environment poses a significant threat. They are poorly biodegradable and toxic due to their complex composition and aromatic structures. In the search for alternatives to physical and chemical treatments, biodegradation of synthetic dyes by various microbes is emerging as an effective and promising approach. OBJECTIVES: The decolorization of synthetic dyes by yeast co-cultures and consortia from leaves and fruit peels was assessed at a 50 µg/mL dye concentration. METHODS: Yeasts isolates from leaves and fruit peels were screened for potential decolorization of synthetic dyes at 25-50 µg/mL. Decolorization parameters were optimized for synergistic properties and development of yeast co-cultures and consortium. Possible decolorization reactions were initially assessed by cell immobilization, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and Fourier transform infrared spectroscopy (FTIR) analysis. RESULTS: A total of 16 organisms were isolated from rose, mango, and pineapple leaves and pineapple fruit peels. Only 4 organisms showed high decolorization of four synthetic dyes: Direct Pink B, Disperse Yellow 5G, Direct Fast Orange S, and Reactive Turquoise Blue G. The optimum condition for best decolorizers of selected dyes at 50 µg/mL were Candida guilliermondii (Y011) for Direct Pink B at pH 9, 37°C; C. dubliniensis (Y014) for Disperse Yellow 5G at pH 4, 25°C; C. guilliermondii (Y004) for Direct Fast Orange S at pH 7, 25°C, and C. famata (Y003) for Reactive Turquoise Blue G at pH 4, 35°C. None of the 4 yeast isolates showed any antagonistic activity when subjected to the lawn-spotting method for the formation of co-cultures and consortium. The best co-cultures obtained 61% decolorization of Direct Pink B, 65% decolorization of Disperse Yellow 5G, 41% decolorization of Direct Fast Orange S, and 50-51% decolorization of Reactive Turquoise Blue G. Immobilized yeast cells were active in decolorizing the dyes and SDS-PAGE analysis confirmed the presence of an extracellular protein. The results of FTIR also showed changes in the functional group of Direct Pink B, but minimal changes in the functional groups of Reactive Turquoise Blue G, indicating a different decolorization pathway. CONCLUSIONS: Yeasts in co-cultures and consortia can decolorize toxic synthetic dyes through different decolorization pathways such as enzyme degradation and bioaccumulation. This technique may have a use in the treatment of wastewater systems.