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      Diversity of microbial carbohydrate-active enzymes in Danish anaerobic digesters fed with wastewater treatment sludge

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          Abstract

          Background

          Improved carbohydrate-active enzymes (CAZymes) are needed to fulfill the goal of producing food, feed, fuel, chemicals, and materials from biomass. Little is known about how the diverse microbial communities in anaerobic digesters (ADs) metabolize carbohydrates or which CAZymes that are present, making the ADs a unique niche to look for CAZymes that can potentiate the enzyme blends currently used in industry.

          Results

          Enzymatic assays showed that functional CAZymes were secreted into the AD environments in four full-scale mesophilic Danish ADs fed with primary and surplus sludge from municipal wastewater treatment plants. Metagenomes from the ADs were mined for CAZymes with Homology to Peptide Patterns (HotPep). 19,335 CAZymes were identified of which 30% showed 50% or lower identity to known proteins demonstrating that ADs make up a promising pool for discovery of novel CAZymes. A function was assigned to 54% of all CAZymes identified by HotPep. Many different α-glucan-acting CAZymes were identified in the four metagenomes, and the most abundant family was glycoside hydrolase family 13, which contains α-glucan-acting CAZymes. Cellulytic and xylanolytic CAZymes were also abundant in the four metagenomes. The cellulytic enzymes were limited almost to endoglucanases and β-glucosidases, which reflect the large amount of partly degraded cellulose in the sludge. No dockerin domains were identified suggesting that the cellulytic enzymes in the ADs studied operate independently. Of xylanolytic CAZymes, especially xylanases and β-xylosidase, but also a battery of accessory enzymes, were present in the four ADs.

          Conclusions

          Our findings suggest that the ADs are a good place to look for novel plant biomass degrading and modifying enzymes that can potentiate biological processes and provide basis for production of a range of added-value products from biorefineries.

          Electronic supplementary material

          The online version of this article (doi:10.1186/s13068-017-0840-y) contains supplementary material, which is available to authorized users.

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          Most cited references61

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          Hemicelluloses.

          Hemicelluloses are polysaccharides in plant cell walls that have beta-(1-->4)-linked backbones with an equatorial configuration. Hemicelluloses include xyloglucans, xylans, mannans and glucomannans, and beta-(1-->3,1-->4)-glucans. These types of hemicelluloses are present in the cell walls of all terrestrial plants, except for beta-(1-->3,1-->4)-glucans, which are restricted to Poales and a few other groups. The detailed structure of the hemicelluloses and their abundance vary widely between different species and cell types. The most important biological role of hemicelluloses is their contribution to strengthening the cell wall by interaction with cellulose and, in some walls, with lignin. These features are discussed in relation to widely accepted models of the primary wall. Hemicelluloses are synthesized by glycosyltransferases located in the Golgi membranes. Many glycosyltransferases needed for biosynthesis of xyloglucans and mannans are known. In contrast, the biosynthesis of xylans and beta-(1-->3,1-->4)-glucans remains very elusive, and recent studies have led to more questions than answers.
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            Lignocellulose degradation mechanisms across the Tree of Life

            Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.
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              Back to Basics – The Influence of DNA Extraction and Primer Choice on Phylogenetic Analysis of Activated Sludge Communities

              DNA extraction and primer choice have a large effect on the observed community structure in all microbial amplicon sequencing analyses. Although the biases are well known, no comprehensive analysis has been conducted in activated sludge communities. In this study we systematically explored the impact of a number of parameters on the observed microbial community: bead beating intensity, primer choice, extracellular DNA removal, and various PCR settings. In total, 176 samples were subjected to 16S rRNA amplicon sequencing, and selected samples were investigated through metagenomics and metatranscriptomics. Quantitative fluorescence in situ hybridization was used as a DNA extraction-independent method for qualitative comparison. In general, an effect on the observed community was found on all parameters tested, although bead beating and primer choice had the largest effect. The effect of bead beating intensity correlated with cell-wall strength as seen by a large increase in DNA from Gram-positive bacteria (up to 400%). However, significant differences were present at lower phylogenetic levels within the same phylum, suggesting that additional factors are at play. The best primer set based on in silico analysis was found to underestimate a number of important bacterial groups. For 16S rRNA gene analysis in activated sludge we recommend using the FastDNA SPIN Kit for Soil with four times the normal bead beating and V1-3 primers.
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                Author and article information

                Contributors
                cwil@kt.dtu.dk
                pbus@kt.dtu.dk
                bpil@kt.dtu.dk
                wenjing.zhang@wisc.edu
                kln@bio.aau.dk
                phn@bio.aau.dk
                lenl@kt.dtu.dk
                Journal
                Biotechnol Biofuels
                Biotechnol Biofuels
                Biotechnology for Biofuels
                BioMed Central (London )
                1754-6834
                21 June 2017
                21 June 2017
                2017
                : 10
                : 158
                Affiliations
                [1 ]ISNI 0000 0001 2181 8870, GRID grid.5170.3, Center for Bioprocess Engineering, Department of Chemical and Biochemical Engineering, , Technical University of Denmark, ; Søltofts Plads, Building 229, 2800 Kongens Lyngby, Denmark
                [2 ]ISNI 0000 0001 0742 471X, GRID grid.5117.2, Section for Sustainable Biotechnology, Department of Chemistry and Bioscience, , Aalborg University, ; A. C. Meyers Vænge 15, 2450 Copenhagen, Denmark
                [3 ]ISNI 0000 0001 0742 471X, GRID grid.5117.2, Center for Microbial Communities, Section for Biotechnology, Department of Chemistry and Bioscience, , Aalborg University, ; Frederiks Bajer Vej 7, 9220 Aalborg, Denmark
                [4 ]ISNI 0000 0001 2167 3675, GRID grid.14003.36, Department of Animal Sciences, , University of Wisconsin-Madison, ; Madison, WI 53706 USA
                Article
                840
                10.1186/s13068-017-0840-y
                5480151
                28053662
                b1e83206-bf9b-42ba-a367-c8542e4fe3c8
                © The Author(s) 2017

                Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License ( http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

                History
                : 30 January 2017
                : 6 June 2017
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100008398, Villum Fonden;
                Award ID: VKR022796
                Award ID: VKR022796
                Award Recipient :
                Categories
                Research
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                © The Author(s) 2017

                Biotechnology
                carbohydrate-active enzymes,anaerobic digester,wastewater treatment,bacteria,glycoside hydrolase,glycosyltransferase,carbohydrate esterase,polysaccharide lyase,carbohydrate-binding module,metagenomics,enzyme discovery,peptide pattern recognition,carbohydrate hydrolysis

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