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Cellobiose dehydrogenases (CDHs) are a group of enzymes belonging to the hemoflavoenzyme group, which are mostly found in fungi. They play an important role in the production of acid sugar. In this research, CDH annotated from the actinobacterium Cellulomonas palmilytica EW123 ( Cp CDH) was cloned and characterized. The Cp CDH exhibited a domain architecture resembling class-I CDH found in Basidiomycota. The cytochrome c and flavin-containing dehydrogenase domains in Cp CDH showed an extra-long evolutionary distance compared to fungal CDH. The amino acid sequence of Cp CDH revealed conservative catalytic amino acids and a distinct flavin adenine dinucleotide region specific to CDH, setting it apart from closely related sequences. The physicochemical properties of Cp CDH displayed optimal pH conditions similar to those of CDHs but differed in terms of optimal temperature. The Cp CDH displayed excellent enzymatic activity at low temperatures (below 30°C), unlike other CDHs. Moreover, Cp CDH showed the highest substrate specificity for disaccharides such as cellobiose and lactose, which contain a glucose molecule at the non-reducing end. The catalytic efficiency of Cp CDH for cellobiose and lactose were 2.05 x 10 5 and 9.06 x 10 4 (M -1 s -1 ), respectively. The result from the Fourier-transform infrared spectroscopy (FT-IR) spectra confirmed the presence of cellobionic and lactobionic acids as the oxidative products of Cp CDH. This study establishes Cp CDH as a novel and attractive bacterial CDH, representing the first report of its kind in the Cellulomonas genus.

The development of multifunctional particles using polymeric scaffolds is an emerging technology for many nanobiotechnological applications. Here we present a system for the production of multifunctional complexes, based on the high affinity non-covalent interaction of cohesin and dockerin modules complementary fused to decameric Brucella abortus lumazine synthase (BLS) subunits, and selected target proteins, respectively. The cohesin-BLS scaffold was solubly expressed in high yield in Escherichia coli, and revealed a high thermostability. The production of multienzymatic particles using this system was evaluated using the catalytic domain of Cellulomonas fimi endoglucanase CenA recombinantly fused to a dockerin module. Coupling of the enzyme to the scaffold was highly efficient and occurred with the expected stoichiometry. The decavalent enzymatic complexes obtained showed higher cellulolytic activity and association to the substrate compared to equivalent amounts of the free enzyme. This phenomenon was dependent on the multiplicity and proximity of the enzymes coupled to the scaffold, and was attributed to an avidity effect in the polyvalent enzyme interaction with the substrate. Our results highlight the usefulness of the scaffold presented in this work for the development of multifunctional particles, and the improvement of lignocellulose degradation among other applications. KEY POINTS: • New system for multifunctional particle production using the BLS scaffold • Higher cellulolytic activity of polyvalent endoglucanase compared to the free enzyme • Amount of enzyme associated to cellulose is higher for the polyvalent endoglucanase., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cellulomonas flavigena is a saprotrophic bacterium that encodes, within its genome, four predicted lytic polysaccharide monooxygenases (LPMOs) from Auxiliary Activity family 10 (AA10). We showed previously that three of these cleave the plant polysaccharide cellulose by oxidation at carbon-1 (J. Li, L. Solhi, E.D. Goddard-Borger, Y. Mattieu et al., Biotechnol Biofuels 14:29, 2021, https://doi.org/10.1186/s13068-020-01860-3). Here, we present the biochemical characterization of the fourth C. flavigena AA10 member ( Cfla LPMO10D) as a chitin-active LPMO. Both the full-length Cfla LPMO10D-Carbohydrate-Binding Module family 2 (CBM2) and catalytic module-only proteins were produced in Escherichia coli using the native general secretory (Sec) signal peptide. To quantify chitinolytic activity, we developed a high-performance anion-exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method as an alternative to the established hydrophilic interaction liquid ion chromatography coupled with UV detection (HILIC-UV) method for separation and detection of released oxidized chito-oligosaccharides. Using this method, we demonstrated that Cfla LPMO10D is strictly active on the β-allomorph of chitin, with optimal activity at pH 5 to 6 and a preference for ascorbic acid as the reducing agent. We also demonstrated the importance of the CBM2 member for both mediating enzyme localization to substrates and prolonging LPMO activity. Together with previous work, the present study defines the distinct substrate specificities of the suite of C. flavigena AA10 members. Notably, a cross-genome survey of AA10 members indicated that chitinolytic LPMOs are, in fact, rare among Cellulomonas bacteria. IMPORTANCE Species from the genus Cellulomonas have a long history of study due to their roles in biomass recycling in nature and corresponding potential as sources of enzymes for biotechnological applications. Although Cellulomonas species are more commonly associated with the cleavage and utilization of plant cell wall polysaccharides, here, we show that C. flavigena produces a unique lytic polysaccharide monooxygenase with activity on β-chitin, which is found, for example, in arthropods. The limited distribution of orthologous chitinolytic LPMOs suggests adaptation of individual cellulomonads to specific nutrient niches present in soil ecosystems. This research provides new insight into the biochemical specificity of LPMOs in Cellulomonas species and related bacteria, and it raises new questions about the physiological function of these enzymes.

Valorization of the hemicellulose fraction of plant biomass is crucial for the sustainability of lignocellulosic biorefineries. The Cellulomonas genus comprises Gram-positive Actinobacteria that degrade cellulose and other polysaccharides by secreting a complex array of enzymes. In this work, we studied the specificity and synergy of two enzymes, CsXyn10A and CsAbf62A, which were identified as highly abundant in the extracellular proteome of Cellulomonas sp. B6 when grown on wheat bran. To explore their potential for bioprocessing, the recombinant enzymes were expressed and their activities were thoroughly characterized. rCsXyn10A is a GH10 endo-xylanase (EC 3.2.1.8), active across a broad pH range (5 to 9), at temperatures up to 55 °C. rCsAbf62A is an α-L-arabinofuranosidase (ABF) (EC 3.2.1.55) that specifically removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides (AXOS), xylan, and arabinan backbones, but it cannot act on double-substituted residues. It also has activity on pNPA. No differences were observed regarding activity when CsAbf62A was expressed with its appended CBM13 module or only the catalytic domain. The amount of xylobiose released from either wheat arabinoxylan or arabino-xylo-oligosaccharides increased significantly when rCsXyn10A was supplemented with rCsAbf62A, indicating that the removal of arabinosyl residues by rCsAbf62A improved rCsXyn10A accessibility to β-1,4-xylose linkages, but no synergism was observed in the deconstruction of wheat bran. These results contribute to designing tailor-made, substrate-specific, enzymatic cocktails for xylan valorization. KEY POINTS: • rCsAbf62A removes α-1,2 and α-1,3-L-arabinosyl substituents from arabino-xylo-oligosaccharides, xylan, and arabinan backbones. • The appended CBM13 of rCsAbf62A did not affect the specific activity of the enzyme. • Supplementation of rCsXyn10A with rCsAbf62A improves the degradation of AXOS and xylan., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Hexavalent chromium (Cr(VI)) is recognized to be carcinogenic and toxic and registered as a contaminant in many drinking water regulations. It occurs naturally and is also produced by industrial processes. The reduction of Cr(VI) to Cr(III) has been a central topic for chromium remediation since Cr(III) is less toxic and less mobile. In this study, fermentative Fe(III)-reducing bacterial strains (Cellu-2a, Cellu-5a, and Cellu-5b) were isolated from a groundwater sample and were phylogenetically related to species of Cellulomonas by 16S rRNA gene analysis. One selected strain, Cellu-2a showed its capacity of reduction of both soluble iron (ferric citrate) and solid iron (hydrous ferric oxide, HFO), as well as aqueous Cr(VI). The strain Cellu-2a was able to reduce 15 μM Cr(VI) directly with glucose or sucrose as a sole carbon source under the anaerobic condition and indirectly with one of the substrates and HFO in the same incubations. The heterogeneous reduction of Cr(VI) by the surface-associated reduced iron from HFO by Cellu-2a likely assisted the Cr(VI) reduction. Fermentative features such as large-scale cell growth may impose advantages on the application of bacterial Cr(VI) reduction over anaerobic respiratory reduction.

Background: A bacterial consortium SCP comprising three bacterial members, viz. Stenotrophomonas acidaminiphila APG1, Pseudomonas stutzeri APG2 and Cellulomonas sp. APG4 was developed for degradation of the mono-azo dye, Reactive Blue 28. The genomic analysis of each member of the SCP consortium was done to elucidate the catabolic potential and role of the individual organism in dye degradation., Results: The genes for glycerol utilization were detected in the genomes of APG2 and APG4, which corroborated with their ability to grow on a minimal medium containing glycerol as the sole co-substrate. The genes for azoreductase were identified in the genomes of APG2 and APG4, while no such trait could be determined in APG1. In addition to co-substrate oxidation and dye reduction, several other cellular functions like chemotaxis, signal transduction, stress-tolerance, repair mechanisms, aromatic degradation, and copper tolerance associated with dye degradation were also annotated. A model for azo dye degradation is postulated, representing the predominant role of APG4 and APG2 in dye metabolism while suggesting an accessory role of APG1., Conclusions: This exploratory study is the first-ever attempt to divulge the genetic basis of azo-dye co-metabolism by cross-genome comparisons and can be harnessed as an example for demonstrating microbial syntrophy.

Cellobiohydrolases directly convert crystalline cellulose into cellobiose and are of biotechnological interest to achieve efficient biomass utilization. As a result, much research in the field has focused on identifying cellobiohydrolases that are very fast. Cellobiohydrolase A from the bacterium Cellulomonas fimi (CfCel6B) and cellobiohydrolase II from the fungus Trichoderma reesei (TrCel6A) have similar catalytic domains (CDs) and show similar hydrolytic activity. However, TrCel6A and CfCel6B have different cellulose-binding domains (CBDs) and linkers: TrCel6A has a glycosylated peptide linker, whereas CfCel6B's linker consists of three fibronectin type 3 domains. We previously found that TrCel6A's linker plays an important role in increasing the binding rate constant to crystalline cellulose. However, it was not clear whether CfCel6B's linker has similar function. Here we analyze kinetic parameters of CfCel6B using single-molecule fluorescence imaging to compare CfCel6B and TrCel6A. We find that CBD is important for initial binding of CfCel6B, but the contribution of the linker to the binding rate constant or to the dissociation rate constant is minor. The crystal structure of the CfCel6B CD showed longer loops at the entrance and exit of the substrate-binding tunnel compared with TrCel6A CD, which results in higher processivity. Furthermore, CfCel6B CD showed not only fast surface diffusion but also slow processive movement, which is not observed in TrCel6A CD. Combined with the results of a phylogenetic tree analysis, we propose that bacterial cellobiohydrolases are designed to degrade crystalline cellulose using high-affinity CBD and high-processivity CD., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Nakamura et al.)

Aims: This study was done to obtain denitrifiers that could be used for bioaugmentation in woodchip bioreactors to remove nitrate from agricultural subsurface drainage water., Methods and Results: We isolated denitrifiers from four different bioreactors in Minnesota, and characterized the strains by measuring their denitrification rates and analysing their whole genomes. A total of 206 bacteria were isolated from woodchips and thick biofilms (bioslimes) that formed in the bioreactors, 76 of which were able to reduce nitrate at 15°C. Among those, nine potential denitrifying strains were identified, all of which were isolated from the woodchip samples. Although many nitrate-reducing strains were isolated from the bioslime samples, none were categorized as denitrifiers but instead as carrying out dissimilatory nitrate reduction to ammonium., Conclusions: Among the denitrifiers confirmed by 15 N stable isotope analysis and genome analysis, Cellulomonas cellasea strain WB94 and Microvirgula aerodenitrificans strain BE2.4 appear to be promising for bioreactor bioaugmentation due to their potential for both aerobic and anaerobic denitrification, and the ability of strain WB94 to degrade cellulose., Significance and Impact of the Study: Denitrifiers isolated in this study could be useful for bioaugmentation application to enhance nitrate removal in woodchip bioreactors., (© 2020 The Society for Applied Microbiology.)

Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them ( Cf Cel6B and Cf Cel48A) slide on crystalline cellulose. The direction of processive movement of Cf Cel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei ( Tr Cel7A), whose movement was first observed by this technique, while Cf Cel48A moves in the same direction as Tr Cel7A. When Cf Cel6B and Tr Cel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of Cf Cel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded β-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

A facultative exoelectrogen strain Lsc-8 belonging to the Cellulomonas genus with the ability to degrade carboxymethyl cellulose (CMC) coupled with the reduction of Cr(VI), was successfully isolated from rumen content. The maximum output power density of the microbial fuel cells (MFCs) inoculated strain Lsc-8 was 9.56 ± 0.37 mW·m -2 with CMC as the sole carbon source. From the biomass analysis it can be seen that the electricity generation of the MFCs was primarily attributed to the planktonic cells of strain Lsc-8 rather than the biofilm attached on the electrode, which was different from Geobacter sulfurreducens. Especially, during electricity generation of the MFCs using CMC as carbon source in the anode chamber, the Cr(VI) reduction were simultaneously realized. And it is also found that the Cr(VI) reduction ratio by strain Lsc-8 is directly related to the initial Cr(VI) concentration, and it increased with the increase of initial Cr(VI) concentration at first, then started to decrease when the Cr(VI) concentration was above 21 mg ·L -1 . Meanwhile, the highest output power density of 3.47 ± 0.28 mW·m -2 was observed coupling with 95.22 ± 2.72 % of Cr(VI) reduction. These data suggested that the strain Lsc-8 could reduce high toxicity Cr(VI) to low toxicity Cr(III) coupled with electricity generation in MFCs with CMC as the carbon source. Our results also suggested that this study will provide a possibility to simultaneously degrade Cr(VI) and generate electricity by using cellulose as the carbon source via MFCs., Competing Interests: Declaration of Competing Interest All the authors have no competing interests to declare., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The strain of Cellulomonas fimi NBRC 15513 can generate electricity with cellulose as fuel without mediator using a single chamber type microbial fuel cell (MFC) which had 100 mL of chamber and 50 cm 2 of the air cathode. The MFCs were operated over five days and showed the maximum current density of 10.0 ± 1.8 mA/m 2 , the maximum power density of 0.74 ± 0.07 mW/m 2 and the ohmic resistance of 6.9 kΩ. According to the results of cyclic voltammetry, the appearance of the oxidation or reduction peak was not observed from the cell removed solution. The fact is that C. fimi does not secrete mediator-like compounds, while the oxidation peak was observed at +0.68 V from the phosphate buffer containing the washed cell. The peak appearance was caused by the electron transfer activity of which corresponds to cytochrome c, and disappeared after adding antimycin A which inhibits the electron transfer activity. The cell was alive throughout the experiment as the result of a colony forming unit on Luria-Bertani agar plates. This was thought that cytochrome c was on the membrane surface of the living cell and played a role in the direct electron transfer between the cells and anode., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Aims: Lignocellulosic biomass deconstruction is a bottleneck for obtaining biofuels and value-added products. Our main goal was to characterize the secretome of a novel isolate, Cellulomonas sp. B6, when grown on residual biomass for the formulation of cost-efficient enzymatic cocktails., Methods and Results: We identified 205 potential CAZymes in the genome of Cellulomonas sp. B6, 91 of which were glycoside hydrolases (GH). By secretome analysis of supernatants from cultures in either extruded wheat straw (EWS), grinded sugar cane straw (SCR) or carboxymethylcellulose (CMC), we identified which proteins played a role in lignocellulose deconstruction. Growth on CMC resulted in the secretion of two exoglucanases (GH6 and GH48) and two GH10 xylanases, while growth on SCR or EWS resulted in the identification of a diversity of CAZymes. From the 32 GHs predicted to be secreted, 22 were identified in supernatants from EWS and/or SCR cultures, including endo- and exoglucanases, xylanases, a xyloglucanase, an arabinofuranosidase/β-xylosidase, a β-glucosidase and an AA10. Surprisingly, among the xylanases, seven were GH10., Conclusions: Growth of Cellulomonas sp. B6 on lignocellulosic biomass induced the secretion of a diverse repertoire of CAZymes., Significance and Impact of the Study: Cellulomonas sp. B6 could serve as a source of lignocellulose-degrading enzymes applicable to bioprocessing and biotechnological industries., (© 2018 The Society for Applied Microbiology.)

Lignocellulose degradation is a challenging step for value added products and biofuels production. Cellulomonas fimi secretes complex mixtures of carbohydrate active enzymes (CAZymes) which synergistically degrade cellulose and hemicelluloses. Their characterization may provide new insights for enzymatic cocktails implementation. Bioinformatic analysis highlighted 1127 secreted proteins, constituting the in silico secretome, graphically represented in a 2DE map. According to Blast2GO functional annotation, many of these are involved in carbohydrates metabolism. In vivo secretomes were obtained, growing C. fimi on glucose, CMC or wheat straw for 24 h. Zymography revealed degradative activity on carbohydrates and proteomic analysis identified some CAZymes, only in secretomes obtained with CMC and wheat straw. An interaction between cellobiohydrolases is proposed as a strategy adopted by soluble multimodular cellulases. Such approach can be crucial for a better characterization and industrial exploitation of the synergism among C. fimi enzymes., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Amylosucrase, catalyzing the synthesis of α-(1,4)-glucan from sucrose, has been widely studied and used in carbohydrate biotransformation because of its versatile activities. In this study, a novel amylosucrase was characterized from Cellulomonas carboniz T26. The recombinant enzyme was overexpressed in Escherchia coli and purified by nickel affinity chromatography. It was determined to be a monomeric protein with a molecular mass of 72 kDa. The optimum pH and temperature for transglucosylation were measured to be pH 7.0 and 40 °C. The transglucosylation activity was significantly higher than the hydrolytic activity. The main product generated from sucrose was structurally determined to be α-(1,4)-glucan. A small amount of glucose was produced by hydrolysis, and sucrose isomers including turanose and trehalulose were generated as minor products. The ratio of hydrolytic, polymerization, and isomerization reactions was calculated to be 5.8:84.0:10.2. The enzyme favored production of long-chain insoluble α-glucan at lower temperature.

Cellulomonas fimi, Cellulomonas biazotea, and Cellulomonas flavigena are cellulose-degrading microorganisms chosen to compare the degradation of cellulose. C. fimi degraded 2.5 g/L of cellulose within 4 days, which was the highest quantity among the three microorganisms. The electric current generation by the microbial fuel cell (MFC) using the cellulose-containing medium with C. fimi was measured over 7 days. The medium in the MFC was sampled every 24 h to quantify the degradation of cellulose, and the results showed that the electric current increased with the degradation of cellulose. The maximum electric power generated by the MFC was 38.7 mW/m 2 , and this numeric value was 63% of the electric power generated by an MFC with Shewanella oneidensis MR-1, a well-known current-generating microorganism. Our results showed that C. fimi was an excellent candidate to produce the electric current from cellulose via MFCs., (Copyright © 2016 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Cellulomonas uda (DSM 20108/ATCC 21399) is one of the few described cellulolytic facultative anaerobes. Based on these characteristics, we initiated a physiological study of C. uda with the aim to exploit it for cellulase production in simple bioreactors with no or sporadic aeration. Growth, cellulase activity and fermentation product formation were evaluated in different media under both aerobic and anaerobic conditions and in experiments where C. uda was exposed to alternating aerobic/anaerobic growth conditions. Here we show that C. uda behaves as a true facultative anaerobe when cultivated on soluble substrates such as glucose and cellobiose, but for reasons unknown cellulase activity is only induced under aerobic conditions on insoluble cellulosic substrates and not under anaerobic conditions. These findings enhance knowledge on the limited number of described facultative cellulolytic anaerobes, and in addition it greatly limits the utility of C. uda as an 'easy to handle' cellulase producer with low aeration demands., Competing Interests: The authors declare that they have no conflict of interest.

The bacteria in the genus Cellulomonas are known for their ability to degrade plant cell wall biomass. Cellulomonas fimi ATCC 484 and C. flavigena ATCC 482 have been the subject of much research into secreted cellulases and hemicellulases. Recently the genome sequences of both C. fimi ATCC 484 and C. flavigena ATCC 482 were published, and a genome comparison has revealed their full spectrum of possible carbohydrate-active enzymes (CAZymes). Using mass spectrometry, we have compared the proteins secreted by C. fimi and C. flavigena during growth on the soluble cellulose substrate, carboxymethylcellulose (CMC), as well as a soluble xylan fraction. Many known C. fimi CAZymes were detected, which validated our analysis, as were a number of new CAZymes and other proteins that, though identified in the genome, have not previously been observed in the secretome of either organism. Our data also shows that many of these are co-expressed on growth of either CMC or xylan. This analysis provides a new perspective on Cellulomonas enzymes and provides many new CAZyme targets for characterization.

A plasmid transfer-mediated bioaugmentation method for the enhancement of dichlorodiphenyltrichloroethane (DDT) degradation in soil was developed using the catabolic plasmid pDOD from Sphingobacterium sp. D-6. The pDOD plasmid could be transferred to soil bacteria, such as members of Cellulomonas, to form DDT degraders and thus accelerate DDT degradation. The transfer efficiency of pDOD was affected by the donor, temperature, moisture, and soil type. Approximately 50.7% of the DDT in the contaminated field was removed 210 days after the application of Escherichia coli TG I (pDOD-gfp). The results suggested that seeding pDOD into soil is an effective bioaugmentation method for enhancing the degradation of DDT., (Copyright © 2014. Published by Elsevier B.V.)