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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.)

Species of actinobacteria previously isolated from Tyume River in the Eastern Cape Province of South Africa and identified by 16S rDNA sequence as Cellulomonas and Streptomyces species were evaluated as a consortium for the production of bioflocculant. Sucrose, peptone and magnesium chloride were the nutritional sources which supported optimal production of bioflocculant resulting in flocculation activities of 91%, 82% and 78% respectively. Response surface design revealed sucrose, peptone and magnesium chloride as critical media components following Plackett-Burman design, while the central composite design showed optimum concentration of the critical nutritional source as 16.0 g/L (sucrose), 1.5 g/L (peptone) and 1.6g/L (magnesium chloride) yielding optimal flocculation activity of 98.9% and bioflocculant yield of 4.45 g/L. FTIR spectrometry of the bioflocculant indicated the presence of carboxyl, hydroxyl and amino groups, typical for heteropolysaccharide, while SEM imaging revealed an interwoven clump-like structure. The molecular weight distribution of the constituents of the bioflocculants ranged 494.81-18,300.26 Da thus, an indication of heterogeneity in composition. Additionally, the chemical analyses of the purified bioflocculant revealed the presence of polysaccharides and proteins with neutral sugar, amino sugar and uronic acids in the following concentration: 5.7 mg, 9.3mg and 17.8 mg per 100mg. The high flocculation activity of the bioflocculant suggests commercial potential., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Hydrogen production from sugarcane bagasse (SCB) by integrating dark-fermentation by Enterobacter aerogenes MTCC 2822 and photo-fermentation by Rhodopseudomonas BHU 01 was investigated. The SCB was hydrolysed by sulphuric acid and the hydrolysate detoxified by passing through adsorbent resin column (Amberlite XAD-4) to remove the inhibitory furfural, and subjected to dark-fermentation. The cellulosic residue from acid hydrolysis was hydrolysed by the new isolate Cellulomonas fimi to release sugars for H2 production by E. aerogenes, through simultaneous saccharification, filtration and fermentation (SSFF). Cumulative H2 production during dark-fermentation and SSFF was 1000 and 613 ml/L, respectively. The spent media of dark-fermentation and SSFF were utilized for photo-fermentation by Rhodopseudomonas BHU 01. The cumulative H2 production was 755 ml/L for dark-fermentation and 351 ml/L for SSFF spent medium., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The reduction of hexavalent chromium, Cr(VI), to trivalent chromium, Cr(III), can be an important aspect of remediation processes at contaminated sites. Cellulomonas species are found at several Cr(VI) contaminated and uncontaminated locations at the Department of Energy site in Hanford, Washington. Members of this genus have demonstrated the ability to effectively reduce Cr(VI) to Cr(III) fermentatively and therefore play a potential role in Cr(VI) remediation at this site. Batch studies were conducted with Cellulomonas sp. strain ES6 to assess the influence of various carbon sources, iron minerals, and electron shuttling compounds on Cr(VI) reduction rates as these chemical species are likely to be present in, or added to, the environment during in situ bioremediation. Results indicated that the type of carbon source as well as the type of electron shuttle present influenced Cr(VI) reduction rates. Molasses stimulated Cr(VI) reduction more effectively than pure sucrose, presumably due to presence of more easily utilizable sugars, electron shuttling compounds or compounds with direct Cr(VI) reduction capabilities. Cr(VI) reduction rates increased with increasing concentration of anthraquinone-2,6-disulfonate (AQDS) regardless of the carbon source. The presence of iron minerals and their concentrations did not significantly influence Cr(VI) reduction rates. However, strain ES6 or AQDS could directly reduce surface-associated Fe(III) to Fe(II), which was capable of reducing Cr(VI) at a near instantaneous rate. These results suggest the rate limiting step in these systems was the transfer of electrons from strain ES6 to the intermediate or terminal electron acceptor whether that was Cr(VI), Fe(III), or AQDS.

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484(T). For comparative purposes, we also sequenced the genome of the aerobic cellulolytic "Cellvibrio gilvus" ATCC 13127(T). An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that "Cellvibrio gilvus" belongs to the genus Cellulomonas. We thus propose to assign "Cellvibrio gilvus" to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482(T) showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.

The consolidated bioprocessing (CBP) of corn stover pretreated via ammonia fiber expansion (AFEX-CS) into ethanol was investigated in a microbial electrolysis cell (MEC) driven by the exoelectrogen Geobacter sulfurreducens and the CBP bacterium Cellulomonas uda. C. uda was identified in a screening for its ethanologenic potential from AFEX-CS and for producing electron donors for G. sulfurreducens fermentatively. C. uda produced ethanol from AFEX-CS in MECs inoculated simultaneously or sequentially, with the concomitant conversion of the fermentation byproducts into electricity by G. sulfurreducens. The fermentation and electrical conversion efficiencies were high, but much of the AFEX-CS remained unhydrolyzed as nitrogen availability limited the growth of the CBP partner. Nitrogen supplementation stimulated the growth of C. uda, AFEX-CS hydrolysis and ethanologenesis. As a result, the synergistic activities of the CBP and exoelectrogen catalysts resulted in substantial energy recoveries from ethanologenesis alone (ca. 56%). The cogeneration of cathodic H(2) in the MEC further increased the energy recoveries to ca. 73%. This and the potential to optimize the activities of the microbial catalysts via culturing approaches and genetic engineering or adaptive evolution, make this platform attractive for the processing of agricultural wastes.

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

An endo-1,3-β-glucanase was purified from Tunicase®, a crude enzyme preparation from Cellulosimicrobium cellulans DK-1, and determined to be a 383-residue protein (Ala1-Leu383), comprising a catalytic domain of the glycoside hydrolase family 16 and a C-terminal carbohydrate-binding module family 13. The Escherichia coli expression system of the catalytic domain (Ala1-Thr256) was constructed, and the protein with N-terminal polyhistidine tag was purified using a Ni-nitrilotriacetic acid column. We analyzed enzymatic properties of the recombinant catalytic domain, its variants, and the Tunicase®-derived full-length endo-1,3-β-glucanase. Substitution of Glu119 with Ala and deletion of Met123, both of the residues are located in the catalytic motif, resulted in the loss of hydrolytic activity. In comparison between the full-length enzyme and isolated catalytic domain, their hydrolytic activities for soluble substrates such as laminarin and laminarioligosaccharides were similar. In contrast, the hydrolytic activity of the full-length enzyme for insoluble substrates such as curdlan and yeast-glucan was significantly higher than that of the catalytic domain. It should be noted that the acid stabilities for the hydrolysis of laminarin were clearly different. Secondary structure analysis using circular dichroism showed that the full-length enzyme was more acid stable than was the catalytic domain, possibly because of domain interactions between the catalytic domain and the carbohydrate-binding module., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Microbially reduced iron minerals can reductively transform a variety of contaminants including heavy metals, radionuclides, chlorinated aliphatics, and nitroaromatics. A number of Cellulomonas spp. strains, including strain ES6, isolated from aquifer samples obtained at the U.S. Department of Energy's Hanford site in Washington, have been shown to be capable of reducing Cr(VI), TNT, natural organic matter, and soluble ferric iron [Fe(III)]. This research investigated the ability of Cellulomonas sp. strain ES6 to reduce solid phase and dissolved Fe(III) utilizing different carbon sources and various electron shuttling compounds. Results suggest that Fe(III) reduction by and growth of strain ES6 was dependent upon the type of electron donor, the form of iron present, and the presence of synthetic or natural organic matter, such as anthraquinone-2,6-disulfonate (AQDS) or humic substances. This research suggests that Cellulomonas sp. strain ES6 could play a significant role in metal reduction in the Hanford subsurface and that the choice of carbon source and organic matter addition can allow for independent control of growth and iron reduction activity.

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.

Removal of hexavalent uranium (U(VI)) from aqueous solution was studied using a Gram-positive facultative anaerobe, Cellulomonas sp. strain ES6, under anaerobic, non-growth conditions in bicarbonate and PIPES buffers. Inorganic phosphate was released by cells during the experiments providing ligands for formation of insoluble U(VI) phosphates. Phosphate release was most probably the result of anaerobic hydrolysis of intracellular polyphosphates accumulated by ES6 during aerobic growth. Microbial reduction of U(VI) to U(IV) was also observed. However, the relative magnitudes of U(VI) removal by abiotic (phosphate-based) precipitation and microbial reduction depended on the buffer chemistry. In bicarbonate buffer, X-ray absorption fine structure (XAFS) spectroscopy showed that U in the solid phase was present primarily as a non-uraninite U(IV) phase, whereas in PIPES buffer, U precipitates consisted primarily of U(VI)-phosphate. In both bicarbonate and PIPES buffer, net release of cellular phosphate was measured to be lower than that observed in U-free controls suggesting simultaneous precipitation of U and PO₄³⁻. In PIPES, U(VI) phosphates formed a significant portion of U precipitates and mass balance estimates of U and P along with XAFS data corroborate this hypothesis. High-resolution transmission electron microscopy (HR-TEM) and energy dispersive X-ray spectroscopy (EDS) of samples from PIPES treatments indeed showed both extracellular and intracellular accumulation of U solids with nanometer sized lath structures that contained U and P. In bicarbonate, however, more phosphate was removed than required to stoichiometrically balance the U(VI)/U(IV) fraction determined by XAFS, suggesting that U(IV) precipitated together with phosphate in this system. When anthraquinone-2,6-disulfonate (AQDS), a known electron shuttle, was added to the experimental reactors, the dominant removal mechanism in both buffers was reduction to a non-uraninite U(IV) phase. Uranium immobilization by abiotic precipitation or microbial reduction has been extensively reported; however, the present work suggests that strain ES6 can remove U(VI) from solution simultaneously through precipitation with phosphate ligands and microbial reduction, depending on the environmental conditions. Cellulomonadaceae are environmentally relevant subsurface bacteria and here, for the first time, the presence of multiple U immobilization mechanisms within one organism is reported using Cellulomonas sp. strain ES6., (© 2010 Wiley Periodicals, Inc.)

The Cfl xyn11A gene, encoding the endo-1,4-beta-xylanase Cfl Xyn11A from Cellulomonas flavigena, was isolated from a genomic DNA library. The open reading frame of the Cfl xyn11A gene was 999 base pairs long and encoded a polypeptide (Cfl Xyn11A) of 332 amino acids with a calculated molecular mass of 35,110Da. The Cfl xyn11A gene was expressed in Escherichia coli and the recombinant enzyme, with an estimated molecular weight of 31kDa was purified and xylanase activity was measured. Cfl Xyn11A showed optimal activity at pH 6.5 and 55 degrees C. The enzyme demonstrated moderate thermal stability as Cfl Xyn11A maintained 50% of its activity when incubated at 55 degrees C for 1h or at 45 degrees C for 6h. This is the first report describing the cloning, expression and functional characterization of an endo-1,4-beta-xylanase-encoding gene from C. flavigena. Cfl Xyn11A may be suitable for industrial applications in the food and feed industries, or in the pre-treatment of lignocellulosic biomass required to improve the yields of fermentable sugars for bioethanol production., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

To date, rational redesign of glycosidase active-site clefts has been mainly limited to the removal of essential functionalities rather than their introduction. The glycoside hydrolase family 26 endo-beta-1,4-mannanase from the soil bacterium Cellulomonas fimi depolymerizes various abundant plant mannans. On the basis of differences in the structures and hydrolytic action patterns of this wild-type (but recombinantly expressed) enzyme and a homologous mannanase from Cellvibrio japonicus, two nonconserved amino acid residues at two distal glycone-binding subsites of the C. fimi enzyme were substituted, Ala323Arg at subsite -2 and Phe325Ala at subsite -3, to achieve inverted mannosyl affinities in the respective subsites, mimicking the Ce. japonicus enzyme that has an Arg providing mannosyl interactions at subsite -2. The X-ray crystal structure of the C. fimi doubly substituted mannanase was determined to 2.35 A resolution and shows that the introduced Arg323 is in a position suitable for hydrogen bonding to mannosyl at subsite -2. We report steady-state enzyme kinetics and hydrolysis-product analyses using anion-exchange chromatography and a novel rapid mass spectrometric profiling method of (18)O-labeled products obtained using H(2)(18)O as a solvent. The results obtained with oligosaccharide substrates show that although the catalytic efficiency (k(cat)/K(m)) is wild-type-like for the engineered enzyme, it has an altered hydrolytic action pattern that stems from promotion of substrate binding at subsite -2 (due to the introduced Arg323) and demotion of it at subsite -3 (to which removal of Phe325 contributed). However, k(cat)/K(m) decreased approximately 1 order of magnitude with polymeric substrates, possibly caused by spatial repositioning of the substrate at subsite -3 and beyond for the engineered enzyme.

Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

Among four cellulolytic microorganisms examined, Cellulomonas biazotea NCIM-2550 can grow on various cellulosic substrates and produce reducing sugar. The activity of cellulases (endoglucanase, exoglucanase, and cellobiase), xylanase, amylase, and lignin class of enzymes produced by C. biazotea was mainly present extracellularly and the enzyme production was dependent on cellulosic substrates (carboxymethyl cellulose [CMC], sugarcane bagasse [SCB], and xylan) used for growth. Effects of physicochemical conditions on cellulolytic enzyme production were systematically investigated. Using MnCl(2) as a metal additive significantly induces the cellulase enzyme system, resulting in more reducing sugar production. The efficiency of fermentative conversion of the hydrolyzed SCB and xylan into clean H(2) energy was examined with seven H(2)-producing pure bacterial isolates. Only Clostridiumbutyricum CGS5 exhibited efficient H(2) production performance with the hydrolysate of SCB and xylan. The cumulative H(2) production and H(2) yield from using bagasse hydrolysate (initial reducing sugar concentration = 1.545 g/L) were approximately 72.61 mL/L and 2.13 mmol H(2)/g reducing sugar (or 1.91 mmol H(2)/g cellulose), respectively. Using xylan hydrolysate (initial reducing sugar concentration = 0.345 g/L) as substrate could also attain a cumulative H(2) production and H(2) yield of 87.02 mL/L and 5.03 mmol H(2)/g reducing sugar (or 4.01 mmol H(2)/g cellulose), respectively.

A "temperature-shift" strategy was developed to improve reducing sugar production from bacterial hydrolysis of cellulosic materials. In this strategy, production of cellulolytic enzymes with Cellulomonas uda E3-01 was promoted at a preferable temperature (35 degrees C), while more efficient enzymatic cellulose hydrolysis was achieved under an elevated culture temperature (45 degrees C), at which cell growth was inhibited to avoid consumption of reducing sugar. This temperature-shift strategy was shown to markedly increase the reducing sugar (especially, monosaccharide and disaccharide) concentration in the hydrolysate while hydrolyzing pure (carboxymethyl-cellulose, xylan, avicel and cellobiose) and natural (rice husk, rice straw, bagasse and Napier-grass) cellulosic materials. The cellulosic hydrolysates from CMC and xylan were successfully converted to H(2) via dark fermentation with Clostridium butyricum CGS5, attaining a maximum hydrogen yield of 4.79 mmol H(2)/g reducing sugar.

Cellulose is the most abundant polymer on Earth and in recent years, renewed interest has developed in its use for the production of biofuels and other value added products. Cellulose is degraded to glucose by the concerted action of cellulolytic enzymes that include cellulases, cellobiohydrolases, and beta-glucosidases. In many cases, these enzymes are multi-modular, being comprised of distinct catalytic and carbohydrate-binding modules. The latter appear to aid in both the adsorption of the enzymes to the insoluble cellulose substrate and the destabilization of the hydrogen-bonding network within the crystalline substrate. To better understand these dynamic processes, we have engineered a carbohydrate-binding module that can be attached to the probe of an atomic force microscope. Thus, the coding sequence for the leader peptide and carbohydrate-binding module from the Cellulomonas fimi cellulase A (cenA) was cloned and over-expressed in Escherichia coli. Site-directed mutagenesis was used to replace Thr87 of this module with Cys to facilitate covalent binding of the module to gold-plated AFM probes. The recombinant proteins with cleavable N-terminal His-tags were purified to apparent homogeneity by a combination of affinity and anion-exchange chromatographies using Ni(2+)-NTA-agarose and Source Q, respectively. Their ability to bind insoluble cellulose was demonstrated using a cellulose-binding assay involving the micro-crystalline cellulose, Avicel.

Chromate (Cr(VI)) reduction studies were performed in bench scale flow columns using the fermentative subsurface isolate Cellulomonas sp. strain ES6. In these tests, columns packed with either quartz sand or hydrous ferric oxide (HFO)-coated quartz sand, were inoculated with strain ES6 and fed nutrients to stimulate growth before nutrient-free Cr(VI) solutions were injected. Results show that in columns containing quartz sand, a continuous inflow of 2 mg/L Cr(VI) was reduced to below detection limits in the effluent for durations of up to 5.7 residence times after nutrient injection was discontinued proving the ability of strain ES6 to reduce chromate in the absence of an external electron donor. In the HFO-containing columns, Cr(VI) reduction was significantly prolonged and effluent Cr(VI) concentrations remained below detectable levels for periods of up to 66 residence times after nutrient injection was discontinued. Fe was detected in the effluent of the HFO-containing columns throughout the period of Cr(VI) removal indicating that the insoluble Fe(III) bearing solids were being continuously reduced to form soluble Fe(II) resulting in prolonged abiotic Cr(VI) reduction. Thus, growth of Cellulomonas within the soil columns resulted in formation of permeable reactive barriers that could reduce Cr(VI) and Fe(III) for extended periods even in the absence of external electron donors. Other bioremediation systems employing Fe(II)-mediated reactions require a continuous presence of external nutrients to regenerate Fe(II). After depletion of nutrients, contaminant removal within these systems occurs by reaction with surface-associated Fe(II) that can rapidly become inaccessible due to formation of crystalline Fe-minerals or other precipitates. The ability of fermentative organisms like Cellulomonas to reduce metals without continuous nutrient supply in the subsurface offers a viable and economical alternative technology for in situ remediation of Cr(VI)-contaminated groundwater through formation of permeable reactive biobarriers (PRBB).

The diversity of cellulases and xylanases secreted by Cellulomonas flavigena cultured on sugar cane bagasse, Solka-floc, xylan, or glucose was explored by two-dimensional gel electrophoresis. C. flavigena produced the largest variety of cellulases and xylanases on sugar cane bagasse. Multiple extracellular proteins were expressed with these growth substrates, and a limited set of them coincided in all substrates. Thirteen proteins with carboxymethyl cellulase or xylanase activity were liquid chromatography/mass spectrometry sequenced. Proteins SP4 and SP18 were identified as products of celA and celB genes, respectively, while SP20 and SP33 were isoforms of the bifunctional cellulase/xylanase Cxo recently sequenced and characterized in C. flavigena. The rest of the detected proteins were unknown enzymes with either carboxymethyl cellulase or xylanase activities. All proteins aligned with glycosyl hydrolases listed in National Center for Biotechnology Information database, mainly with cellulase and xylanase enzymes. One of these unknown enzymes, protein SP6, was cross-induced by sugar cane bagasse, Solka-floc, and xylan. The differences in the expression maps of the presently induced cultures revealed that C. flavigena produces and secretes multiple enzymes to use a wide range of lignocellulosic substrates as carbon sources. The expression of these proteins depends on the nature of the cellulosic substrate.

We report the purification and characterization of two thermophilic xylanases from the mesophilic bacteria Cellulomonas flavigena grown on sugarcane bagasse (SCB) as the only carbon source. Extracellular xylanase activity produced by C. flavigena was found both free in the culture supernatant and associated with residual SCB. To identify some of the molecules responsible for the xylanase activity in the substrate-bound fraction, residual SCB was treated with 3 M guanidine hydrochloride and then with 6 M urea. Further analysis of the eluted material led to the identification of two xylanases Xyl36 (36 kDa) and Xyl53 (53 kDa). The pI for Xyl36 was 5.0, while the pI for Xyl53 was 4.5. Xyl36 had a Km value of 1.95 mg/ml, while Xyl53 had a Km value of 0.78 mg/ml. In addition to SCB, Xyl36 and Xyl53 were also able to bind to insoluble oat spelt xylan and Avicel, as shown by substrate-binding assays. Xyl36 and Xyl53 showed optimal activity at pH 6.5, and at optimal temperature 65 and 55 degrees C, respectively. Xyl36 and Xyl53 retained 24 and 35%, respectively, of their original activity after 8 h of incubation at their optimal temperature. As far as we know, this is the first study on the thermostability properties of purified xylanases from microorganisms belonging to the genus Cellulomonas.

Microbial enrichments from Cr(VI) contaminated and uncontaminated US Department of Energy Hanford Site sediments produced Cr(VI) reducing consortia when grown in the presence of Cr(VI) with acetate, D-xylose or glycerol as a carbon and energy source. Eight of the nine isolates from the consortia were Gram positive and four of these were identified by 16S rRNA sequence homology and membrane fatty acid composition as belonging to the genus Cellulomonas. Two strains, ES6 and WS01, were further examined for their ability to reduce Cr(VI) under growth and non-growth conditions. During fermentative growth on D-xylose, ES6 and WS01 decreased aqueous Cr(VI) concentrations from 0.04 mM Cr(VI) to below the detection limit (0.002 mM Cr(VI)) in less than three days and retained their ability to reduce Cr(VI) even after four months of incubation. Washed ES6 and WS01 cells also reduced Cr(VI) under non-growth conditions for over four months, both with and without the presence of an exogenous electron donor. K-edge XANES spectroscopy confirmed the reduction of Cr(VI) to Cr(III). The ability to reduce Cr(VI) after growth had stopped and in the absence of an external electron donor, suggests that stimulation of these types of organisms may lead to effective long-term, in situ passive reactive barriers for Cr(VI) removal. Our results indicate that Cr(VI) reduction by indigenous Cellulomonas spp. may be a potential method of in situ bioremediation of Cr(VI) contaminated sediment and groundwater.

The apical domain of the chaperonin, GroEL, fused to the carbohydrate binding module type II, CBD(Cex), of Cellulomonas fimi, was expressed in Escherichia coli. The recombinant protein, soluble or from inclusion bodies, was directly purified and immobilized in microcrystalline cellulose particles or cellulose fabric membranes. Assisted refolding of rhodanese by the immobilized mini-chaperone showed a two-fold improvement as compared to a control. Using chromatographic refolding, 35% of rhodanese activity was recovered in only 5 min (mean residence time) as compared to 17% for spontaneous refolding. This mini-chaperone immobilized in cellulose could be a cost-efficient method to refold recombinant proteins expressed as inclusion bodies.

Cr(VI) is considerably toxic and the detoxification of Cr(VI) is of great importance. This study investigated the effect of iron on Cr(VI) reduction by Cellulomonas flavigena. The results demonstrated that addition of FeCl3 or lepidocrocite promoted Cr(VI) reduction, with the reduction ratio of above 90 and 80% achieved, respectively, but addition of hematite did not lead to the increase of reduction ratio, which suggests that the effect of iron on chromate reduction appears different with the diversity of iron-oxides. In this study, the effect of initial Cr(VI) and Fe(III) concentration on Cr(VI) reduction and the change of pH value were also investigated. The reduction ratio increased with the increase of the initial concentration ratio of Fe(III)/Cr(VI). The value of pH gradually increased from 7.0 to around 9.0.

An alkaliphilic, slightly halotolerant, chemo-organotrophic, Gram-positive, rod-shaped bacterium, strain 69B4(T), was isolated from the sediment of the littoral zone of Lake Bogoria, Kenya. Phylogenetically, it is a member of the genus Cellulomonas, showing less than 97.5 % sequence similarity to the type strains of other Cellulomonas species. The highest level of similarity, albeit moderate, was found with respect to Cellulomonas cellasea DSM 20118(T). Chemotaxonomic properties confirm the 16S rRNA gene-based generic affiliation, i.e. a DNA G+C content of 71.5 mol%, anteiso-C(15:0) and C(16:0) as the major fatty acids, MK-9(H(4)) as the major isoprenoid quinone, a peptidoglycan containing L-ornithine as the diamino acid and D-aspartic acid in the interpeptide bridge and phosphatidylglycerol as the only identified main polar lipid. The strain is aerobic to facultatively anaerobic, being capable of growth under strictly anaerobic conditions. Optimal growth occurs between pH values 9.0 and 10.0. On the basis of its distinct phylogenetic position and metabolic properties, strain 69B4(T) represents a novel species of the genus Cellulomonas, for which the name Cellulomonas bogoriensis sp. nov. is proposed. The type strain is 69B4(T) (=DSM 16987(T)=CIP 108683(T)).

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (beta-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.

Aims: To develop a plate assay for simultaneous screening of polysaccharide-degrading and protein-degrading micro-organisms., Methods and Results: A plate assay, based on the visible solubilization of small substrate particles and the formation of haloes on Petri dishes, containing a mixture of diversely coloured insoluble polysaccharides and dye-labelled collagen as chromogenic substrates, was developed. This method was successfully applied for isolating the diverse polysaccharide- and/or protein-degrading bacteria from soil and sludge samples. Selected strains were identified using 16S rDNA partial sequencing; most of them belong to the genera Bacillus, Cellulomonas and Cellulosimicrobium., Conclusions: This novel approach provides unique and valuable information for direct primary screening when the target of selection is micro-organisms exhibiting protein-degrading activity, polysaccharide-degrading activity or a specific combination of them., Significance and Impact of the Study: This plate assay is convenient and easy to perform, rapid, and more adaptable for screening of a large number of samples, compared with other existing methods in the literature.

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.

A bacterium isolated from a petal of Casa Blanca Lily (ST26 strain) produced a marked amount of extracellular trehalose (alpha- d-glucopyranosyl-[1,1]-alpha- d-glucopyranose) in culture medium containing glucose. 16S rDNA-based phylogeny showed that ST26 belongs to, or is related to, Cellulosimicrobium cellulans, a close relative of Cellulomonas spp. Various Cellulomonas strains obtained from culture collections also showed extracellular trehalose productivity, suggesting that trehalose production is a common property of this bacterial genus. ST26 accumulated trehalose in medium supplied with glucose but not with sucrose, glycerol or maltose. Effective extracellular trehalose production by ST26 was achieved by supplying 0.5-1% ammonium sulfate and 0.5-1% CaCO(3). The addition of CaCO(3) adjusted the pH of the culture to around 5.0. The optimized culture conditions yielded trehalose from glucose at a conversion rate of 61%. The addition of ammonium sulfate greatly reduced the dry cell weight of ST26 and intracellular content of trehalose, which suggests that the addition of ammonium sulfate makes ST26 cells leak trehalose into the medium. ST26 effectively propagated in minimal medium containing trehalose as a sole carbon source, which suggests that trehalose serves as a carbohydrate reserve of this organism.

Microbial communities from three Argentinean saline soils were extracted and tested for their ability to degrade diesel fuel in liquid culture at salinities between 0% and 25%. In each case, the degradation process was continuously monitored by measuring oxygen consumption. Two communities (CR1 and CR2) showed nearly equal degrees of degradation across a salinity range of 0%-10% (the former degrading about 63% of the diesel fuel and the latter about 70% after 53 and 80 d, respectively). Furthermore, the degree of degradation was not significantly lower in the presence of 17.5% salt (58% and 65% degraded, respectively). A third community (El Zorro) showed a maximum turnover at 5% salt (79% diesel fuel degraded) and significant degradation (66%) at a salinity of 10%. However, the degree of degradation by this community clearly dropped at 0% and 15% salt. None of the communities were able to degrade diesel fuel in the presence of 25% salt, but the living cell counts showed that components of the microbial population survived the long-term exposure. The surviving portion is obviously sufficient to allow substantial restoration of the original community, as verified by the BIOLOG method. Isolates of the CR1 community were identified as members of the genera Cellulomonas, Bacillus, Dietzia, and Halomonas. In light of our investigations, the bioremediation of contaminated saline soils should be quite possible if the salinity of the soil water is lower than 15% or if it is reduced below this limit by the addition of water.

A two-membered coculture of strains KYM-7 and KYM-8, identified as Cellulomonas cellulans and Agrobacterium tumefaciens, respectively, produced a large amount of an extracellular polysaccharide, designated APK-78, from starch. Each strain in pure culture produced only very little amount of polysaccharide from starch; the coexistence of the two strains from the early stage of cultivation was indispensable for a large amount of polysaccharide to be produced. The polysaccharide APK-78 was acidic and composed of glucose, galactose, succinic acid, and pyruvic acid with a molar ratio of 8.1:1.0:1.7:1.0, indicating that it is a succinoglycan type of polysaccharide.

Cellulomonas flavigena KU produces large quantities of an insoluble exopolysaccharide (EPS) under certain growth conditions. The EPS has previously been shown to be a glucose polymer and to have solubility properties similar to curdlan, a beta-1,3-D-glucan produced by Alcaligenes faecalis var. myxogenes 10C3K. Furthermore, EPS purified by alkaline extraction stains with aniline blue, a dye specific for curdlan-type polysaccharides. However, EPS-producing colonies of C. flavigena KU do not stain on aniline blue agar as do those of curdlan-producing bacteria. These facts prompted a more thorough structural analysis of the EPS. Here we report that purified EPS is indeed identical to curdlan in primary structure, but that the native form of the EPS may differ from curdlan in physical conformation.

The reduction of Cr(VI), Fe(III), and U(VI) was studied using three recently isolated environmental Cellulomonas sp. (WS01, WS18, and ES5) and a known Cellulomonas strain ( Cellulomonas flavigena ATCC 482) under anaerobic, non-growth conditions. In all cases, these cultures were observed to reduce Cr(VI), Fe(III), and U(VI). In 100 h, with lactate as electron donor, the Cellulomonas isolates (500 mg/l total cell protein) reduced nitrilotriacetic acid chelated Fe(III) [Fe(III)-NTA] from 5 mM to less than 2.2 mM, Cr(VI) from 0.2 mM to less than 0.001 mM, and U(VI) from 0.2 mM to less than 0.12 mM. All Cellulomonas isolates also reduced Cr(VI), Fe(III), and U(VI) in the absence of lactate, while no metal reduction was observed in either the cell-free or heat-killed cell controls. This is the first report of Cellulomonas sp. reducing Fe(III) and U(VI). Further, this is the first report of Cellulomonas spp. coupling the oxidation of lactate, or other unknown electron donors in the absence of lactate, to the reduction of Cr(VI), Fe(III), and U(VI).