Towards bacterial valorization of low molecular weight lignin

Lignin is one of the major constituents in lignocellulosic biomass and is the mostabundant source of renewable aromatics on terrestrial ecosystems. The workcarried out in this thesis concerns bacterial conversion of low molecular weightlignin. This thesis is divided into three major sections, with an initial emphasison screening and characterization of selected bacterial species on lignin modelcompounds, followed by testing their performance on treated lignin substrates,and finally progressing towards strain improvement via metabolic engineering. During screening for bacteria using samples from natural and man-madeenvironments, Pseudomonas species were found dominant. Some of theseisolates, and the well-known aromatic degrader – Pseudomonas putida KT2440– were cultivated on lignin model compounds. P. putida and Pseudomonas sp.isolate 9.1 attained specific growth rates of about 0.21-0.27 h-1 and 0.12-0.30 h-1 respectively, on several compounds from the coniferyl, p-coumaryl and benzoyl branches of the funnelling pathways. Meanwhile, a contaminant was found growing on syringate plates, and was later identified to be a bacterium belonging to the Microbacterium genus. This Gram-positive bacterium, named RG1, was able to consume syringate and syringaldehyde besides other aromatic compounds from the coniferyl and p-coumaryl branches. Due to its interesting abilities to assimilate syringyl compounds, the genome of this strain was sequenced to identify genes involved in a putative syringyl pathway.To assess the performance of selected bacteria on lignin substrates, cultivationswere performed using alkaline- and oxidatively-treated Kraft lignin. P. putida and P. fluorescens consumed 4-HBA, vanillin, and vanillate in the complex ligninmixture that likely contained various toxic products. In addition, Rhodococcusopacus and Sphingobium sp. SYK-6 assimilated guaiacol and acetovanillonerespectively, from the lignin mixture. Interestingly, P. fluorescens was able tobreak down the higher molecular weight lignin and produce several smallermolecules.P. putida was selected as a host organism for genetic engineering aimed atexpanding the range of substrates utilized. Heterologous expression of thecytochrome P450 and oxidoreductase genes from R. rhodochrous enabled P.putida to assimilate guaiacol – one of the major depolymerization products fromsoftwood lignin – as the sole carbon source. Furthermore, the identification anddeletion of an aldehyde reductase in a P. putida strain that converts ferulate tovanillin, increased the yield of vanillin by eliminating the formation of vanillylalcohol as by-product

A case study for cloud based high throughput analysis of NGS data using the globus genomics system

AbstractNext generation sequencing (NGS) technologies produce massive amounts of data requiring a powerful computational infrastructure, high quality bioinformatics software, and skilled personnel to operate the tools. We present a case study of a practical solution to this data management and analysis challenge that simplifies terabyte scale data handling and provides advanced tools for NGS data analysis. These capabilities are implemented using the “Globus Genomics” system, which is an enhanced Galaxy workflow system made available as a service that offers users the capability to process and transfer data easily, reliably and quickly to address end-to-endNGS analysis requirements. The Globus Genomics system is built on Amazon's cloud computing infrastructure. The system takes advantage of elastic scaling of compute resources to run multiple workflows in parallel and it also helps meet the scale-out analysis needs of modern translational genomics research

Oxidative Depolymerization of Kraft Lignin for Microbial Conversion

The valorization of lignin is being increasingly recognized as crucial to improve the economic viability of integrated biorefineries. Because of its inherent heterogeneity and recalcitrance, lignin has been treated as a waste product in the pulp and paper industry, but new technologies are now being explored to transform lignin into a sustainable resource and enhance its value chain. In the present study, alkaline oxidative depolymerization was investigated as a potential form of pretreatment to enable further biological conversion of LignoBoost kraft lignin (LB). LB lignin oxidation reactions were studied at various temperatures (120-200 °C) and O2 partial pressures (3-15 bar) to identify the optimal conditions for obtaining a biocompatible, oxidatively depolymerized lignin (ODLB) stream. The low molecular weight compounds resulting from this treatment consisted mainly of aromatic monomers and carboxylic acids. The highest yield of aromatic monomers, 3 wt %, was obtained at 160 °C and 3 bar O2. The yield of carboxylic acids increased with both increasing temperature and O2 pressure, exceeding 13% under the harshest conditions investigated. The growth of four aromatic-catabolizing bacterial strains was examined on reaction product mixtures, all of which showed growth on agar plates utilizing ODLB as the sole source of carbon and energy. Rhodococcus opacus and Sphingobium sp. SYK-6 were found to consume most of the aromatic monomers present in the ODLB (e.g., vanillin, vanillate, acetovanillone, and guaiacol). The findings of this study indicate that pretreatment by oxidative depolymerization has potential in the biological valorization of technical lignin streams, for the production of valuable chemicals and materials

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Improving Adsorption Deinking by Identifying the Optimum Balance between Polymer Beads and Deinking Chemistry

Ink removal from recovered paper is a very important process in paper and board recycling. The current deinking processes have made obvious contributions to the use of raw materials for the paper and board industries. In contrast to the flotation deinking process, in which small air bubbles are used to remove ink from the pulp, the novel and more energy-efficient method of adsorption deinking technique depends on the attachment and adsorption of ink particles on small polymer beads. The energy savings of adsorption deinking results from the fact that the process is efficient at greater stock consistencies, thus providing water conservation and savings. The present study was carried out to improve the adsorption deinking method by identifying the optimum balance between the deinking chemistry and the polymer beads. Different types of deinking solutions and polymer beads were used for this study with newsprints and mixture of newsprints and magazines. It was found that EGA 3000 solution and polyethylene terephthalate beads worked well with newspaper pulp

Conversion of lignin model compounds by Pseudomonas putida KT2440 and isolates from compost

Starting from mature vegetable compost, four bacterial strains were selected using a lignin-rich medium. 16S ribosomal RNA identification of the isolates showed high score similarity with Pseudomonas spp. for three out of four isolates. Further characterization of growth on mixtures of six selected lignin model compounds (vanillin, vanillate, 4-hydroxybenzoate, p-coumarate, benzoate, and ferulate) was carried out with three of the Pseudomonas isolates and in addition with the strain Pseudomonas putida KT2440 from a culture collection. The specific growth rates on benzoate, p-coumarate, and 4-hydroxybenzoate were considerably higher (0.26–0.27 h−1) than those on ferulate and vanillate (0.21 and 0.22 h−1), as were the uptake rates.There was no direct growth of P. putida KT2440 on vanillin, but instead, vanillin was rapidly converted in to vanillate at a rate of 4.87 mmol (gCDW h)−1 after which the accumulated vanillate was taken up. The growth curve reflected a diauxic growthwhen mixtures of the model compounds were used as carbon source. Vanillin, 4-hydroxybenzoate, and benzoate were preferentially consumed first, whereas ferulate was always the last substrate to be taken in. These results contribute to a better understanding of the aromatic metabolism of P. putida in terms of growth and uptake rates, which will be helpful for the utilization of these bacteria as cell factories for upgrading lignin-derived mixtures of aromatic molecules

Mapping the diversity of microbial lignin catabolism : experiences from the eLignin database

Lignin is a heterogeneous aromatic biopolymer and a major constituent of lignocellulosic biomass, such as wood and agricultural residues. Despite the high amount of aromatic carbon present, the severe recalcitrance of the lignin macromolecule makes it difficult to convert into value-added products. In nature, lignin and lignin-derived aromatic compounds are catabolized by a consortia of microbes specialized at breaking down the natural lignin and its constituents. In an attempt to bridge the gap between the fundamental knowledge on microbial lignin catabolism, and the recently emerging field of applied biotechnology for lignin biovalorization, we have developed the eLignin Microbial Database (www.elignindatabase.com), an openly available database that indexes data from the lignin bibliome, such as microorganisms, aromatic substrates, and metabolic pathways. In the present contribution, we introduce the eLignin database, use its dataset to map the reported ecological and biochemical diversity of the lignin microbial niches, and discuss the findings

Biological conversion of aromatic monolignol compounds by a Pseudomonas isolate from sediments of the Baltic Sea

Bacterial strains were isolated from the sediments of the Baltic Sea using ferulic acid, guaiacol or a lignin-rich softwood waste stream as substrate. In total nine isolates were obtained, five on ferulic acid, two on guaiacol and two on a lignin-rich softwood stream as a carbon source. Three of the isolates were found to be Pseudomonas sp. based on 16S rRNA sequencing. Among them, isolate 9.1, which showed the fastest growth in defined M9 medium, was tentatively identified as a Pseudomonas deceptionensis strain based on the gyrB sequencing. The growth of isolate 9.1 was further examined on six selected lignin model compounds (ferulate, p-coumarate, benzoate, syringate, vanillin and guaiacol) from different upper funneling aromatic pathways and was found able to grow on four out of these six compounds. No growth was detected on syringate and guaiacol. The highest specific growth and uptake rates were observed for benzoate (0.3 h−1 and 4.2 mmol gCDW −1 h−1) whereas the lowest were for the compounds from the coniferyl branch. Interestingly, several pathway intermediates were excreted during batch growth. Vanillyl alcohol was found to be excreted during growth on vanillin. Several other intermediates like cis,cis-muconate, catechol, vanillate and 4-hydroxybenzoate from the known bacterial catabolic pathways were excreted during growth on the model compounds

Identification of the two-component guaiacol demethylase system from Rhodococcus rhodochrous and expression in Pseudomonas putida EM42 for guaiacol assimilation

A diversity of softwood lignin depolymerization processes yield guaiacol as the main low molecular weight product. This key aromatic compound can be utilized as a carbon source by several microbial species, most of which are Gram positive bacteria. Microbial degradation of guaiacol is known to proceed initially via demethylation to catechol, and this reaction is catalyzed by cytochrome P450 monooxygenases. These enzymes typically require a set of redox partner proteins, whose number and identities were not described until very recently in the case of guaiacol. In this work we identified two proteins involved in guaiacol demethylation by the actinomycete Rhodococcus rhodochrous. Additionally, we constructed four different polycistronic operons carrying combinations of putative redox partners of this guaiacol demethylation system in an inducible expression plasmid that was introduced into the Gram negative host Pseudomonas putida EM42, and the guaiacol consumption dynamics of each resulting strain were analyzed. All the polycistronic operons, expressing a cytochrome P450 together with a putative ferredoxin reductase from R. rhodochrous and putative ferredoxins from R. rhodochrous or Amycolatopsis ATCC 39116 enabled P. putida EM42 to metabolize and grow on guaiacol as the sole carbon source