Engineering of ecological niches to create stable artificial consortia for complex biotransformations

The design of controllable artificial microbial consortia has attracted considerable interest in recent years to capitalize on the inherent advantages in comparison to monocultures such as the distribution of the metabolic burden by division of labor, the modularity and the ability to convert complex substrates. One promising approach to control the consortia composition, function and stability is the provision of defined ecological niches fitted to the specific needs of the consortium members. In this review, we discuss recent examples for the creation of metabolic niches by biological engineering of resource partitioning and syntrophic interactions. Moreover, we introduce a complementing process engineering approach to provide defined spatial niches with differing abiotic conditions (e.g. O2, T, light) in stirred tank reactors harboring biofilms. This enables the co-cultivation of microorganisms with non-overlapping abiotic requirements and the control of the strain ratio in consortia characterized by substrate competition

Slowing the Kinetics of Alumina Sol-Gel Chemistry for Controlled Catalyst Overcoating and Improved Catalyst Stability and Selectivity

Catalyst overcoating is an emerging approach to engineer surface functionalities on supported metal catalyst and improve catalyst selectivity and durability. Alumina deposition on high surface area material by sol–gel chemistry is traditionally difficult to control due to the fast hydrolysis kinetics of aluminum‐alkoxide precursors. Here, sol–gel chemistry methods are adapted to slow down these kinetics and deposit nanometer‐scale alumina overcoats. The alumina overcoats are comparable in conformality and thickness control to overcoats prepared by atomic layer deposition even on high surface area substrates. The strategy relies on regulating the hydrolysis/condensation kinetics of Al(sBuO)3 by either adding a chelating agent or using nonhydrolytic sol–gel chemistry. These two approaches produce overcoats with similar chemical properties but distinct physical textures. With chelation chemistry, a mild method compatible with supported base metal catalysts, a conformal yet porous overcoat leads to a highly sintering‐resistant Cu catalyst for liquid‐phase furfural hydrogenation. With the nonhydrolytic sol–gel route, a denser Al2O3 overcoat can be deposited to create a high density of Lewis acid–metal interface sites over Pt on mesoporous silica. The resulting material has a substantially increased hydrodeoxygenation activity for the conversion of lignin‐derived 4‐propylguaiacol into propylcyclohexane with up to 87% selectivity

A mild biomass pretreatment using gamma-valerolactone for concentrated sugar production

Here we report that gamma-valerolactone (GVL), a biomass-derived solvent, can be used to facilitate the mild pretreatment of lignocellulosic biomass. An 80% GVL, 20% water solvent system was used to pretreat hardwood at the mild temperature of 120 degrees C with an acid loading of 75 mM H2SO4. Up to 80% of original lignin was removed with 96-99% of original cellulose retained in the pretreated substrates. The use of a mild temperature and low acid concentrations caused negligible degradation of sugars. Up to 99% of the original glucan and 96% of the original xylan could be recovered after pretreatment. The pretreated substrate was quantitatively converted to sugars (99% and 100% total glucose and xylose yield) with an enzyme loading of 15 FPU g(-1) glucan. These digestibilities were three times higher than those obtained when using other organic solvents such as tetrahydrofuran or ethanol, and 20 times higher than when pure water was used during pretreatment. Over 99.5% of GVL could be recovered by liquid-CO2 extraction of the pretreated slurries while removing less than 1% of the sugars. This approach produced pretreatment slurries that could easily undergo high-solids (30% w/v) enzymatic hydrolysis without any substrate washing or drying. We obtained glucose and xylose yields of up to 90% and 97%, respectively, and generated sugar streams with sugar concentrations up to 182 g L-1

Highly Selective Oxidation and Depolymerization of α,γ-Diol Protected Lignin

Lignin oxidation offers a potential sustainable pathway to oxygenated aromatic molecules. However, current methods that use real lignin tend to have low selectivity and a yield that is limited by lignin degradation during its extraction. We developed stoichiometric and catalytic oxidation methods using 2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone (DDQ) as oxidant/catalyst to selectively deprotect the acetal and oxidize the α‐OH into a ketone. The oxidized lignin was then depolymerized using a formic acid/sodium formate system to produce aromatic monomers with a 36 mol % (in the case of stoichiometric oxidation) and 31 mol % (in the case of catalytic oxidation) yield (based on the original Klason lignin). The selectivity to a single product reached 80 % (syringyl propane dione, and 10–13 % to guaiacyl propane dione). These high yields of monomers and unprecedented selectivity are attributed to the preservation of the lignin structure by the acetal

Dual Valorization of Lignin as a Versatile and Renewable Matrix for Enzyme Immobilization and (Flow) Bioprocess Engineering

Lignin has emerged as an attractive alternative in the search for more eco-friendly and less costly materials for enzyme immobi- lization. In this work, the terephthalic aldehyde-stabilization of lignin is carried out during its extraction to develop a series of functionalized lignins with a range of reactive groups (epoxy, amine, aldehyde, metal chelates). This expands the immobiliza- tion to a pool of enzymes (carboxylase, dehydrogenase, trans- aminase) by different binding chemistries, affording immobiliza- tion yields of 64–100%. As a proof of concept, a ω- transaminase reversibly immobilized on polyethyleneimine- lignin is integrated in a packed-bed reactor. The stability of the immobilized biocatalyst is tested in continuous-flow deamina- tion reactions and maintains the same conversion for 100 cycles. These results outperform previous stability tests carried out with the enzyme covalently immobilized on methacrylic resins, with the advantage that the reversibility of the immobilized enzyme allows recycling and reuse of lignin beyond the enzyme inactivation. Additionally, an in-line system also based on lignin is added into the downstream process to separate the reaction products by catch-and-release. These results demonstrate a fully closed-loop sustainable flow- biocatalytic system based exclusively on lignin

Controlled deposition of titanium oxide overcoats by non-hydrolytic sol gel for improved catalyst selectivity and stability

Advances in the synthetic control of surface nanostructures could improve the activity, selectivity and stability of heterogeneous catalysts. Here, we present a technique for the controlled deposition of TiO2 overcoats based on non-hydrolytic sol-gel chemistry. Continuous injection of Ti(iPrO)4 and TiCl4 mixtures led to the formation of conformal TiO2 overcoats with a growth rate of 0.4 nm/injected monolayer on several materials including high surface area SBA-15. Deposition of TiO2 on SBA-15 generated medium-strength Lewis acid sites, which catalyzed 1-phenylethanol dehydration at high selectivities and decreased deactivation rates compared to typically used HZSM-5. When supported metal nanoparticles were similarly overcoated, the intimate contact between the metal and acid sites at the support-overcoat interface significantly increased propylcyclohexane selectivity during the deoxygenation of lignin-derived propyl guaiacol (89% at 90% conversion compared to 30% for the uncoated catalyst). For both materials, the surface reactivity could be tuned with the overcoat thickness

Protection Group Effects During α,γ-Diol Lignin Stabilization Promote High-Selectivity Monomer Production

Protection groups were introduced during biomass pretreatment to stabilize lignin's α,γ-diol group during its extraction and prevent its condensation. Acetaldehyde and propionaldehyde stabilized the α,γ-diol without any aromatic ring alkylation, which significantly increased final product selectivity. The subsequent hydrogenolysis catalyzed by Pd/C generated lignin monomers at near-theoretical yields based on Klason lignin (48 % from birch, 20 % from spruce, 70 % from high-syringyl transgenic poplar), and with high selectivity to a single 4-n-propanolsyringol product (80 %) in the case of the poplar. Unlike direct hydrogenation of native wood, hydrogenolysis of protected lignin with Ni/C also led to high selectivity to this single product (78 %), paving the way to high-selectivity lignin upgrading with base metal catalysts. The use of extracted lignin facilitated valorization of polysaccharides, leading to high yields of all three major biomass polymers to a single major product

Consolidated bioprocessing of lignocellulosic biomass to lactic acid by a synthetic fungal-bacterial consortium

Consolidated bioprocessing (CBP) of lignocellulosic feedstocks to platform chemicals requires complex metabolic processes, which are commonly executed by single genetically engineered microorganisms. Alternatively, synthetic consortia can be employed to compartmentalize the required metabolic functions among different specialized microorganisms as demonstrated in this work for the direct production of lactic acid from lignocellulosic biomass. We composed an artificial cross‐kingdom consortium and co‐cultivated the aerobic fungus Trichoderma reesei for the secretion of cellulolytic enzymes with facultative anaerobic lactic acid bacteria. We engineered ecological niches to enable the formation of a spatially structured biofilm. Up to 34.7 gL−1 lactic acid could be produced from 5% (w/w) microcrystalline cellulose. Challenges in converting pretreated lignocellulosic biomass include the presence of inhibitors, the formation of acetic acid and carbon catabolite repression. In the CBP consortium hexoses and pentoses were simultaneously consumed and metabolic cross‐feeding enabled the in situ degradation of acetic acid. As a result, superior product purities were achieved and 19.8 gL−1 (85.2% of the theoretical maximum) of lactic acid could be produced from non‐detoxified steam‐pretreated beech wood. These results demonstrate the potential of consortium‐based CBP technologies for the production of high value chemicals from pretreated lignocellulosic biomass in a single step

A heterogeneous microbial consortium producing short-chain fatty acids from lignocellulose.

Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes. Clostridium tyrobutyricum, Veillonella criceti, or Megasphaera elsdenii were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals

Prominent role of mesopore surface area and external acid sites for the synthesis of polyoxymethylene dimethyl ethers (OME) on a hierarchical H-ZSM-5 zeolite

H-ZSM-5 zeolite has been shown to be an active catalyst for the synthesis of polyoxymethylene dimethyl ethers (OME). However, we demonstrated – by passivation of the zeolite's external surface – that the reaction rate is limited due to severe internal diffusion limitations of the reactants and products. External acid sites thus played a more prominent role in the observed overall reaction rate compared to the acid sites in the zeolite's micropores. Through controlled introduction of an intercrystalline network of mesopores the zeolite's activity was significantly enhanced by allowing a more significant part of the reaction to take place within the zeolite's micropores. By optimising alkaline treatment and consequent acid wash of H-ZSM-5, we achieved a two-fold increase in the initial reaction rate and a 10% increase in selectivity towards OME with 3 to 5 oxymethylene units (OME3–5), which are the more desirable products