Evaluation of the production of exopolysaccharides by two strains of the thermophilic bacterium Rhodothermus marinus

AbstractThe thermophile Rhodothermus marinus produces extracellular polysaccharides (EPSs) that forms a distinct cellular capsule. Here, the first data on EPS production in strains DSM4252T and MAT493 are reported and compared. Cultures of both strains, supplemented with either glucose, sucrose, lactose or maltose showed that the EPS were produced both in the exponential and stationary growth phase and that production in the exponential phase was boosted by maltose supplementation, while stationary phase production was boosted by lactose. The latter was higher, resulting in 8.8 (DSM4252T) and 13.7mg EPS/g cell dry weight (MAT493) in cultures in marine broth supplemented with 10g/L lactose. The EPSs were heteropolymeric with an average molecular weight of 8×104Da and different monosaccharides, including arabinose and xylose. FT-IR spectroscopy revealed presence of hydroxyl, carboxyl, N-acetyl, amine, and sulfate ester groups, showing that R. marinus produces unusual sulfated EPS with high arabinose and xylose content

Life-cycle assessment of yeast-based single-cell protein production with oat processing side-stream

Production of fish meal and plant-based feed proteins continues to increase to meet the growing demand for seafood, leading to impacts on marine and terrestrial ecosystems. Microbial proteins such as single-cell proteins (SCPs) have been introduced as feed alternatives since they can replace current fish feed ingredients, e.g., soybean, which are associated with negative environmental impacts. Microbial protein production also enables utilization of grain processing side-streams as feedstock sources. This study assesses the environmental impacts of yeast-based SCP using oat side-stream as feedstock (OS-SCP). Life-cycle assessment with a cradle-to-gate approach was used to quantify global warming, freshwater eutrophication, marine eutrophication, terrestrial acidification, land use, and water consumption of OS-SCP production in Finland. Dried and wet side-streams of oat were compared with each other to identify differences in energy consumption and transportation effects. Sensitivity analysis was performed to examine the difference in impacts at various locations and fermentation times. Benchmarking was used to evaluate the environmental impacts of OS-SCP and other feed products, including both conventional and novel protein products. Results highlight the importance of energy sources in quantifying the environmental performance of OS-SCP production. OS-SCP produced with dried side-streams resulted in higher global warming (16.3 %) and water consumption (7.5 %) than OS-SCP produced from wet side-streams, reflecting the energy and water requirements for the drying process. Compared with conventional products, such as soy protein concentrates, OS-SCP resulted in 61 % less land use, while exacerbating the environmental impacts in all the other categories. OS-SCP had more impact on global warming (205–754 %), water consumption (166–1401 %), freshwater eutrophication (118–333 %), and terrestrial acidification (85–340 %) than other novel products, including yeast protein concentrate, methanotrophic bacterial SCP, and insect meal, while lowering global warming (11 %) and freshwater eutrophication (20 %) compared with dry microalgae biomass.Peer reviewe

Composition analysis and minimal treatments to solubilize polysaccharides from the brown seaweed Laminaria digitata for microbial growth of thermophiles

Publisher's version (útgefin grein)Brown macroalgae (Phaeophyta) hold high potential as feedstock for biorefineries due to high biomass productivity and carbohydrate content. They are, however, a challenging, unconventional feedstock for microbial refining and several processing problems need to be solved to make them a viable option. Pre-treatment is necessary to enhance accessibility and solubility of the biomass components but should be minimal and mild to assure sustainable and cost-effective processing. Here, two routes to pre-treatLaminaria digitata to release polysaccharides were investigated: hot water pre-treatment by autoclaving (121 °C, 20 min or 60 min) and a two-step extraction with mild acid (0.1 M HCl) followed by alkaline treatment. Hot water pre-treatment resulted in partial extraction of a mixture of polysaccharides consisting of alginate, fucoidan and laminarin. After mild acid pre-treatment, alginate was found in the remaining insoluble residues and was extracted in a second step via alkaline treatment using Na2CO3 (0.15 M) at 80 °C and CaCl2 (10%) for the precipitation. In addition to carbohydrates, a fraction of other components such as proteins, phenolic compounds, minerals and trace elements was detected in the extracts. Cultivation of the thermophilic bacterial strains Rhodothermus marinus DSM 16675 and Bacillus methanolicus MGA3 (ATCC 53907) in media supplemented with the respective extracts resulted in growth of both strains, indicating that they were able to utilize the available carbon source for growth. R. marinus displayed the highest cell density in the medium containing the extract from acid pre-treatment, whereas B. methanolicus growth was highest with the extract from hot water pre-treatment.Open access funding provided by Lund University. The authors received financial support for the projects Thermofactories, (Era-net MBT1, grant agreement No 604814), MacroVal (the Swedish research council Formas, grant 2015-769), Marine food resources for new markets (Formas, grant 2018-01863), Macro cascade (Bio-Based Industries Joint Undertaking under EU Horizon 2020, grant agreement No 720755), the Novo Nordisk foundation (grant agreement No NNF18OC00349792) and ProSeaFood, (Era-net SusFood2, grant agreement No 727473).Peer Reviewe

Exploring a novel β-1,3-glucanosyltransglycosylase, MlGH17B, from a marine Muricauda lutaonensis strain for modification of laminari-oligosaccharides

The marine environment, contains plentiful renewable resources, e.g. macroalgae with unique polysaccharides, motivating search for enzymes from marine microorganisms to explore conversion possibilities of the polysaccharides. In this study, the first GH17 glucanosyltransglycosylase, MlGH17B, from a marine bacterium (Muricauda lutaonensis), was characterized. The enzyme was moderately thermostable with Tm at 64.4 ◦C and 73.2 ◦C, but an activity optimum at 20 ◦C, indicating temperature sensitive active site interactions. MlGH17B uses β-1,3 laminari-oligosaccharides with a degree of polymerization (DP) of 4 or higher as donors. Two glucose moieties (bound in the aglycone +1 and +2 subsites) are cleaved off from the reducing end of the donor while the remaining part (bound in the glycone subsites) is transferred to an incoming β-1,3 glucan acceptor, making a β-1,6-linkage, thereby synthesizing branched or kinked oligosaccharides. Synthesized oligosaccharides up to DP26 were detected by mass spectrometry analysis, showing that repeated transfer reactions occurred, resulting in several β-1,6-linked branches. The modeled structure revealed an active site comprising five subsites: three glycone (−3, −2 and −1) and two aglycone (+1 and +2) subsites, with significant conservation of substrate interactions compared to the only crystallized 1,3-β-glucanosyltransferase from GH17 (RmBgt17A from the compost thriving fungus Rhizomucor miehei), suggesting a common catalytic mechanism, despite different phylogenetic origin, growth environment, and natural substrate. Both enzymes lacked the subdomain extending the aglycone subsites, found in GH17 endo-β-glucanases from plants, but this extension was also missing in bacterial endoglucanases (modeled here), showing that this feature does not distinguish transglycosylation from hydrolysis, but may rather relate to phylogeny

Characterization of Sigma Factor Genes in Streptomyces lividans TK24 Using a Genomic Library-Based Approach for Multiple Gene Deletions

Alternative sigma factors control numerous aspects of bacterial life, including adaptation to physiological stresses, morphological development, persistence states and virulence. This is especially true for the physiologically complex actinobacteria. Here we report the development of a robust gene deletions system for Streptomyces lividans TK24 based on a BAC library combined with the λ-Red recombination technique. The developed system was validated by systematically deleting the most highly expressed genes encoding alternative sigma factors and several other regulatory genes within the chromosome of S. lividans TK24. To demonstrate the possibility of large scale genomic manipulations, the major part of the undecylprodigiosin gene cluster was deleted as well. The resulting mutant strains were characterized in terms of morphology, growth parameters, secondary metabolites production and response to thiol-oxidation and cell-wall stresses. Deletion of SLIV_12645 gene encoding S. coelicolor SigR1 ortholog has the most prominent phenotypic effect, resulted in overproduction of actinorhodin and coelichelin P1 and increased sensitivity to diamide. The secreted proteome analysis of SLIV_12645 mutant revealed SigR1 influence on trafficking of proteins involved in cell wall biogenesis and refactoring. The reported here gene deletion system will further facilitate work on S. lividans strain improvement as a host for either secondary metabolites or protein production and will contribute to basic research in streptomycetes physiology, morphological development, secondary metabolism. On the other hand, the systematic deletion of sigma factors encoding genes demonstrates the complexity and conservation of regulatory processes conducted by sigma factors in streptomycetes

Bioactive effect of sulphated polysaccharides derived from orange-footed sea cucumber (Cucumaria frondosa) toward THP-1 macrophages

To access publisher's full text version of this article click on the hyperlink belowAtlantic sea cucumber (Cucumaria frondosa) is a marine invertebrate that occurs naturally in the North Atlantic. Its collagen rich body walls make the sea cucumber an appreciated food source in many countries. The heteroglycans derived from the cartilage of Atlantic sea cucumbers have shown potential as a source of bioactive compounds, but further investigation is needed to confirm this hypothesis. The objectives of this work were to test the immunomodulating properties of heteroglycan fractions (FCFs) derived from Atlantic sea cucumbers toward phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophages. Oxygen Radical Absorbance Capacity (ORAC) was determined and cell culture media was collected and analyzed for presence of pro-inflammatory and anti-inflammatory cytokines (TNF-α, IL-6, and IL-10). FCFs toxicity toward macrophages was evaluated by light microscopy and confirmed by XTT proliferation assay. Heteroglycans derived from cartilage of Atlantic sea cucumber showed immunomodulatory activity in THP-1 macrophages. FCF-1, −2, and − 3) at concentrations 0.1–100 µg/mL induced levels of TNF-α, IL-6, and IL-10 proofing their bioactivity towards THP-1 macrophages. However, fractions of FCF-1 and FCF-2 may have induced oxidative stress in the macrophages, which possibly could lead to decreased cellular viability

Characterization and diversity of the complete set of GH family 3 enzymes from Rhodothermus marinus DSM 4253

The genome of Rhodothermus marinus DSM 4253 encodes six glycoside hydrolases (GH) classified under GH family 3 (GH3): RmBgl3A, RmBgl3B, RmBgl3C, RmXyl3A, RmXyl3B and RmNag3. The biochemical function, modelled 3D-structure, gene cluster and evolutionary relationships of each of these enzymes were studied. The six enzymes were clustered into three major evolutionary lineages of GH3: β-N-acetyl-glucosaminidases, β-1,4-glucosidases/β-xylosidases and macrolide β-glucosidases. The RmNag3 with additional β-lactamase domain clustered with the deepest rooted GH3-lineage of β-N-acetyl-glucosaminidases and was active on acetyl-chitooligosaccharides. RmBgl3B displayed β-1,4-glucosidase activity and was the only representative of the lineage clustered with macrolide β-glucosidases from Actinomycetes. The β-xylosidases, RmXyl3A and RmXyl3B, and the β-glucosidases RmBgl3A and RmBgl3C clustered within the major β-glucosidases/β-xylosidases evolutionary lineage. RmXyl3A and RmXyl3B showed β-xylosidase activity with different specificities for para-nitrophenyl (pNP)-linked substrates and xylooligosaccharides. RmBgl3A displayed β-1,4-glucosidase/β-xylosidase activity while RmBgl3C was active on pNP-β-Glc and β-1,3-1,4-linked glucosyl disaccharides. Putative polysaccharide utilization gene clusters were also investigated for both R. marinus DSM 4253 and DSM 4252T (homolog strain). The analysis showed that in the homolog strain DSM 4252T Rmar_1080 (RmXyl3A) and Rmar_1081 (RmXyl3B) are parts of a putative polysaccharide utilization locus (PUL) for xylan utilization

Cloning and Characterization of a Novel <i>N</i>-Acetyl-<span style="font-variant: small-caps">D</span>-galactosamine-4-<i>O</i>-sulfate Sulfatase, SulA1, from a Marine <i>Arthrobacter</i> Strain

Sulfation is gaining increased interest due to the role of sulfate in the bioactivity of many polysaccharides of marine origin. Hence, sulfatases, enzymes that control the degree of sulfation, are being more extensively researched. In this work, a novel sulfatase (SulA1) encoded by the gene sulA1 was characterized. The sulA1-gene is located upstream of a chondroitin lyase encoding gene in the genome of the marine Arthrobacter strain (MAT3885). The sulfatase was produced in Escherichia coli. Based on the primary sequence, the enzyme is classified under sulfatase family 1 and the two catalytic residues typical of the sulfatase 1 family—Cys57 (post-translationally modified to formyl glycine for function) and His190—were conserved. The enzyme showed increased activity, but not improved stability, in the presence of Ca2+, and conserved residues for Ca2+ binding were identified (Asp17, Asp18, Asp277, and Asn278) in a structural model of the enzyme. The temperature and pH activity profiles (screened using p-nitrocatechol sulfate) were narrow, with an activity optimum at 40–50 °C and a pH optimum at pH 5.5. The Tm was significantly higher (67 °C) than the activity optimum. Desulfation activity was not detected on polymeric substrates, but was found on GalNAc4S, which is a sulfated monomer in the repeated disaccharide unit (GlcA–GalNAc4S) of, e.g., chondroitin sulfate A. The position of the sulA1 gene upstream of a chondroitin lyase gene and combined with the activity on GalNAc4S suggests that there is an involvement of the enzyme in the chondroitin-degrading cascade reaction, which specifically removes sulfate from monomeric GalNAc4S from chondroitin sulfate degradation products

Evaluation of Laminaria Digitata Hydrolysate for the Production of Bioethanol and Butanol by Fermentation

Seaweeds (macroalgae) are gaining attention as potential sustainable feedstock for the production of fuels and chemicals. This comparative study focuses on the characterization of the microbial production of alcohols from fermentable carbohydrates in the hydrolysate of the macroalgae Laminaria digitata as raw material. The potential of a hydrolysate as a carbon source for the production of selected alcohols was tested, using three physiologically different fermentative microbes, in two main types of processes. For the production of ethanol, Saccharomyces cerevisiae was used as a benchmark microorganism and compared with the strictly anaerobic thermophile Thermoanaerobacterium strain AK17. For mixed production of acetone/isopropanol, butanol, and ethanol (A/IBE), three strictly anaerobic Clostridium strains were compared. All strains grew well on the hydrolysate, and toxicity constraints were not observed, but fermentation performance and product profiles were shown to be both condition- and strain-specific. S. cerevisiae utilized only glucose for ethanol formation, while strain AK17 utilized glucose, mannitol, and parts of the glucan oligosaccharides. The clostridia strains tested showed different nutrient requirements, and were able to utilize glucan, mannitol, and organic acids in the hydrolysate. The novelty of this study embodies the application of different inoculates for fermenting a common brown seaweed found in the northern Atlantic Ocean. It provides important information on the fermentation properties of different microorganisms and pinpoints the value of carbon source utilization when selecting microbes for efficient bioconversion into biofuel and chemical products of interest

Evaluation of Laminaria Digitata Hydrolysate for the Production of Bioethanol and Butanol by Fermentation

Seaweeds (macroalgae) are gaining attention as potential sustainable feedstock for the production of fuels and chemicals. This comparative study focuses on the characterization of the microbial production of alcohols from fermentable carbohydrates in the hydrolysate of the macroalgae Laminaria digitata as raw material. The potential of a hydrolysate as a carbon source for the production of selected alcohols was tested, using three physiologically different fermentative microbes, in two main types of processes. For the production of ethanol, Saccharomyces cerevisiae was used as a benchmark microorganism and compared with the strictly anaerobic thermophile Thermoanaerobacterium strain AK17. For mixed production of acetone/isopropanol, butanol, and ethanol (A/IBE), three strictly anaerobic Clostridium strains were compared. All strains grew well on the hydrolysate, and toxicity constraints were not observed, but fermentation performance and product profiles were shown to be both condition- and strain-specific. S. cerevisiae utilized only glucose for ethanol formation, while strain AK17 utilized glucose, mannitol, and parts of the glucan oligosaccharides. The clostridia strains tested showed different nutrient requirements, and were able to utilize glucan, mannitol, and organic acids in the hydrolysate. The novelty of this study embodies the application of different inoculates for fermenting a common brown seaweed found in the northern Atlantic Ocean. It provides important information on the fermentation properties of different microorganisms and pinpoints the value of carbon source utilization when selecting microbes for efficient bioconversion into biofuel and chemical products of interest