Bioproduction of Eriodictyol By Escherichia Coli Engineered Co-Culture

Eriodictyol is a flavonoid in the flavanones subclass. It is abundantly present in a wide range of medicinal plants, citrus fruits, and vegetables. In addition, eriodictyol owns numerous importantly medicinal bioactivities such as inhibition of proliferation, metastasis and induction of apoptosis in glioma cells or inhibition of glioblastoma migration, and invasion. This study described the heterologous production of eriodictyol by E. coli based co-culture engineering system from the initial substrate as D-glucose. Notably, the upstream module was composed of genes for synthesis of p-coumaric acid (pCA) from D-glucose. The downstream module consisted of genes for the synthesis of eriodictyol from p-coumaric acid. The maximal result of eryodictyol was achieved 51.5 mg/L using optimal culture conditions, while mono-culture was only achieved 21.3 mg/L. In conclusion, co-culture was the efficiently alternative approach for the synthesis of eriodictyol and other natural products.

Improvement of the Transglycosylation Efficiency of a Lacto-N-Biosidase from Bifidobacterium bifidum by Protein Engineering

The lacto-N-biosidase LnbB from Bifidobacterium bifidum JCM 1254 was engineered to improve its negligible transglycosylation efficiency with the purpose of enzymatically synthesizing lacto-N-tetraose (LNT; Gal-β1,3-GlcNAc-β1,3-Gal-β1,4-Glc) in one enzymatic step. LNT is a prebiotic human milk oligosaccharide in itself and constitutes the structural core of a range of more complex human milk oligosaccharides as well. Thirteen different LnbB variants were expressed and screened for transglycosylation activity by monitoring transglycosylation product formation using lacto-N-biose 1,2-oxazoline as donor substrate and lactose as acceptor substrate. LNT was the major reaction product, yet careful reaction analysis revealed the formation of three additional LNT isomers, which we identified to have a β1,2-linkage, a β1,6-linkage, and a 1,1-linkage, respectively, between lacto-N-biose (Gal-β1,3-GlcNAc) and lactose. Considering both maximal transglycosylation yield and regioselectivity as well as minimal product hydrolysis, the best variant was LnbB W394H, closely followed by W465H and Y419N. A high transglycosylation yield was also obtained with W394F, yet the substitution of W394 and W465 of the subsite −1 hydrophobic platform in the enzyme with His dramatically impaired the undesirable product hydrolysis as compared to substitution with Phe; the effect was most pronounced for W465. Using p-nitrophenyl-β-lacto-N-bioside as donor substrate manifested W394 as an important target position. The optimization of the substrate concentrations confirmed that high initial substrate concentration and high acceptor-to-donor ratio both favor transglycosylation.

Catalytic Transfer Hydrogenation and Acid Reactions of Furfural and 5-(Hydroxymethyl)Furfural over Hf-TUD-1 Type Catalysts

Heterogeneous catalysis, which has served well the petrochemical industry, may valuably contribute towards a bio-based economy by sustainably enabling selective reactions to renewable chemicals. Carbohydrate-containing matter may be obtained from various widespread sources and selectively converted to furanic platform chemicals: furfural (Fur) and 5-(hydroxymethyl)furfural (Hmf). Valuable bioproducts may be obtained from these aldehydes via catalytic transfer hydrogenation (CTH) using alcohols as H-donors under relatively moderate reaction conditions. Hafnium-containing TUD-1 type catalysts were the first of ordered mesoporous silicates explored for the conversion of Fur and Hmf via CTH/alcohol strategies. The materials promoted CTH and acid reactions leading to the furanic ethers. The bioproducts spectrum was broader for the reaction of Fur than of Hmf. A Fur reaction mechanism based on literature data was discussed and supported by kinetic modelling. The influence of the Hf loading and reaction conditions (catalyst load, type of alcohol H-donor, temperature, initial substrate concentration) on the reaction kinetics was studied. The reaction conditions were optimized to maximize the yields of 2-(alkoxymethyl)furan ethers formed from Fur; up to 63% yield was reached at 88% Fur conversion, 4 h/150 °C, using Hf-TUD-1(75), which was a stable catalyst. The Hf-TUD-1(x) catalysts promoted the selective conversion of Hmf to bis(2-alkoxymethyl)furan; e.g., 96% selectivity at 98% Hmf conversion, 3 h/170 °C for Hf-TUD-1(50).

Mathematical Modelling of Canola Oil Biodegradation and Optimisation of Biosurfactant Production by an Antarctic Bacterial Consortium Using Response Surface Methodology

An Antarctic soil bacterial consortium (reference BS14) was confirmed to biodegrade canola oil, and kinetic studies on this biodegradation were carried out. The purpose of this study was to examine the ability of BS14 to produce biosurfactants during the biodegradation of canola oil. Secondary mathematical equations were chosen for kinetic analyses (Monod, Haldane, Teissier–Edwards, Aiba and Yano models). At the same time, biosurfactant production was confirmed through a preliminary screening test and further optimised using response surface methodology (RSM). Mathematical modelling demonstrated that the best-fitting model was the Haldane model for both waste (WCO) and pure canola oil (PCO) degradation. Kinetic parameters including the maximum degradation rate (μmax) and maximum concentration of substrate tolerated (Sm) were obtained. For WCO degradation these were 0.365 min−1 and 0.308%, respectively, while for PCO they were 0.307 min−1 and 0.591%, respectively. The results of all preliminary screenings for biosurfactants were positive. BS14 was able to produce biosurfactant concentrations of up to 13.44 and 14.06 mg/mL in the presence of WCO and PCO, respectively, after optimisation. The optimum values for each factor were determined using a three-dimensional contour plot generated in a central composite design, where a combination of 0.06% salinity, pH 7.30 and 1.55% initial substrate concentration led to the highest biosurfactant production when using WCO. Using PCO, the highest biosurfactant yield was obtained at 0.13% salinity, pH 7.30 and 1.25% initial substrate concentration. This study could help inform the development of large-scale bioremediation applications, not only for the degradation of canola oil but also of other hydrocarbons in the Antarctic by utilising the biosurfactants produced by BS14.

Prediction of Epitaxial Grain Growth in Single-Track Laser Melting of IN718 Using Integrated Finite Element and Cellular Automaton Approach

The mechanical properties of selective laser melting (SLM) components are fundamentally dependent on their microstructure. Accordingly, the present study proposes an integrated simulation framework consisting of a three-dimensional (3D) finite element model and a cellular automaton model for predicting the epitaxial grain growth mode in the single-track SLM processing of IN718. The laser beam scattering effect, melt surface evolution, powder volume shrinkage, bulk heterogeneous nucleation, epitaxial growth, and initial microstructure of the substrate are considered. The simulation results show that during single-track SLM processing, coarse epitaxial grains are formed at the melt–substrate interface, while fine grains grow at the melt–powder interface with a density determined by the intensity of the heat input. During the solidification stage, the epitaxial grains and bulk nucleated grains grow toward the top surface of the melt pool along the temperature gradient vectors. The rate of the epitaxial grain growth varies as a function of the orientation and size of the partially melted grains at the melt–substrate boundary, the melt pool size, and the temperature gradient. This is observed that by increasing heat input from 250 J/m to 500 J/m, the average grain size increases by ~20%. In addition, the average grain size reduces by 17% when the initial substrate grain size decreases by 50%. In general, the results show that the microstructure of the processed IN718 alloy can be controlled by adjusting the heat input, preheating conditions, and initial substrate grain size.

The Dynamics of Mass Loss and Nutrient Release of Decomposing Fine Roots, Needle Litter and Standard Substrates in Hemiboreal Coniferous Forests

Litter decomposition is a key process that drives carbon and nutrient cycles in forest soils. The decomposition of five different substrate types was analyzed in hemiboreal coniferous forests, focusing on the mass loss and nutrient (N, P, and K) release of fine roots (FR) and needle litter in relation to the initial substrate and soil chemistry. A litterbag incubation experiment with site-specific FR and needle litter and three standard substrates (green and rooibos tea, α-cellulose) was carried out in four Norway spruce and four Scots pine-dominated stands in Estonia. Substrate type was the primary driver of mass loss and the decay rate of different substrates did not depend on the dominant tree species of the studied stands. Alpha-cellulose lost 98 ± 1% of the mass in 2-years, while the FR mass loss was on average 23 ± 2% after 3-years of decomposition. The FR decomposition rate could be predicted using a corresponding model of green tea, although the rate of FR decomposition is approximately five times lower than the rate of green tea in the first 3-years. The annual decomposition rate of the needle litter is rather constant in hemiboreal coniferous forests in the first 3 years. The initial substrate of fine roots or needle litter and soil chemistry jointly had a significant effect on mass loss in the later stage of decomposition. The critical N concentration for N release was lower for pine FR and needle litter (0.9–1.3% and 0.7–1.1%) compared to spruce (1.2–1.6% and 1.5–1.9%, respectively). The release rate of K depended on the initial K of substrate, while the release of N and P was significantly related to the initial C:N and N:P ratios, respectively. The results show the central role of soil and substrate initial chemistry in the decomposition of fine roots and needle litter across hemiboreal forests, especially at later stage (after 2 years) of decomposition. The slower decomposition and higher retention of N in the fine roots relative to needle litter suggests that fine roots have a substantial role in the carbon and nitrogen accumulation in boreal and hemiboreal forest ecosystems.

Biodegradation of Indanthrene Blue RS dye in immobilized continuous upflow packed bed bioreactor using corncob biochar

The current study describes the aerobic biodegradation of Indanthrene Blue RS dye by a microbial consortium immobilized on corn-cob biochar in a continuous up-flow packed bed bioreactor. The adsorption experiments were performed without microbes to monitor the adsorption effects on initial dye decolorization efficiency. The batch experiments were carried out to estimate the process parameters, and the optimal values of pH, temperature, and inoculum volume were identified as 10.0, 30 °C, and 3.0 × 106 CFU mL−1, respectively. During the continuous operation, the effect of flow rate, initial substrate concentration, inlet loading rate of Indanthrene Blue RS on the elimination capacity, and its removal efficiency in the bioreactor was studied. The continuous up-flow packed bed bioreactor was performed at different flow rates (0.25 to 1.25 L h−1) under the optimal parameters. The maximum removal efficiency of 90% was observed, with the loading rate varying between 100 and 300 mg L−1 day−1. The up-flow packed bed bioreactor used for this study was extremely useful in eliminating Indanthrene Blue RS dye using both the biosorption and biodegradation process. Therefore, it is a potential treatment strategy for detoxifying textile wastewater containing anthraquinone-based dyes.

Recombinant Enzymatic Redox Systems for Preparation of Aroma Compounds by Biotransformation

The aim of this study was to develop immobilized enzyme systems that reduce carbonyl compounds to their corresponding alcohols. The demand for natural aromas and food additives has been constantly growing in recent years. However, it can no longer be met by extraction and isolation from natural materials. One way to increase the availability of natural aromas is to prepare them by the enzymatic transformation of suitable precursors. Recombinant enzymes are currently being used for this purpose. We investigated trans-2-hexenal bioreduction by recombinant Saccharomyces cerevisiae alcohol dehydrogenase (ScADH1) with simultaneous NADH regeneration by recombinant Candida boidinii formate dehydrogenase (FDH). In a laboratory bioreactor with two immobilized enzymes, 88% of the trans-2-hexenal was transformed to trans-2-hexenol. The initial substrate concentration was 3.7 mM. The aldehyde destabilized ScADH1 by eluting Zn2+ ions from the enzyme. A fed-batch operation was used and the trans-2-hexenal concentration was maintained at a low level to limit the negative effect of Zn2+ ion elution from the immobilized ScADH1. Another immobilized two-enzyme system was used to reduce acetophenone to (S)-1-phenylethanol. To this end, the recombinant alcohol dehydrogenase (RrADH) from Rhodococcus ruber was used. This biocatalytic system converted 61% of the acetophenone to (S)-1-phenylethanol. The initial substrate concentration was 8.3 mM. All enzymes were immobilized by poly-His tag to Ni2+, which formed strong but reversible bonds that enabled carrier reuse after the loss of enzyme activity.

The Use of Response Surface Methodology as a Statistical Tool for the Optimisation of Waste and Pure Canola Oil Biodegradation by Antarctic Soil Bacteria

Hydrocarbons can cause pollution to Antarctic terrestrial and aquatic ecosystems, both through accidental release and the discharge of waste cooking oil in grey water. Such pollutants can persist for long periods in cold environments. The native microbial community may play a role in their biodegradation. In this study, using mixed native Antarctic bacterial communities, several environmental factors influencing biodegradation of waste canola oil (WCO) and pure canola oil (PCO) were optimised using established one-factor-at-a-time (OFAT) and response surface methodology (RSM) approaches. The factors include salinity, pH, type of nitrogen and concentration, temperature, yeast extract and initial substrate concentration in OFAT and only the significant factors proceeded for the statistical optimisation through RSM. High concentration of substrate targeted for degradation activity through RSM compared to OFAT method. As for the result, all factors were significant in PBD, while only 4 factors were significant in biodegradation of PCO (pH, nitrogen concentration, yeast extract and initial substrate concentration). Using OFAT, the most effective microbial community examined was able to degrade 94.42% and 86.83% (from an initial concentration of 0.5% (v/v)) of WCO and PCO, respectively, within 7 days. Using RSM, 94.99% and 79.77% degradation of WCO and PCO was achieved in 6 days. The significant interaction for the RSM in biodegradation activity between temperature and WCO concentration in WCO media were exhibited. Meanwhile, in biodegradation of PCO the significant factors were between (1) pH and PCO concentration, (2) nitrogen concentration and yeast extract, (3) nitrogen concentration and PCO concentration. The models for the RSM were validated for both WCO and PCO media and it showed no significant difference between experimental and predicted values. The efficiency of canola oil biodegradation achieved in this study provides support for the development of practical strategies for efficient bioremediation in the Antarctic environment.

Resisting the Heat: Bacterial Disaggregases Rescue Cells From Devastating Protein Aggregation

Bacteria as unicellular organisms are most directly exposed to changes in environmental growth conditions like temperature increase. Severe heat stress causes massive protein misfolding and aggregation resulting in loss of essential proteins. To ensure survival and rapid growth resume during recovery periods bacteria are equipped with cellular disaggregases, which solubilize and reactivate aggregated proteins. These disaggregases are members of the Hsp100/AAA+ protein family, utilizing the energy derived from ATP hydrolysis to extract misfolded proteins from aggregates via a threading activity. Here, we describe the two best characterized bacterial Hsp100/AAA+ disaggregases, ClpB and ClpG, and compare their mechanisms and regulatory modes. The widespread ClpB disaggregase requires cooperation with an Hsp70 partner chaperone, which targets ClpB to protein aggregates. Furthermore, Hsp70 activates ClpB by shifting positions of regulatory ClpB M-domains from a repressed to a derepressed state. ClpB activity remains tightly controlled during the disaggregation process and high ClpB activity states are likely restricted to initial substrate engagement. The recently identified ClpG (ClpK) disaggregase functions autonomously and its activity is primarily controlled by substrate interaction. ClpG provides enhanced heat resistance to selected bacteria including pathogens by acting as a more powerful disaggregase. This disaggregase expansion reflects an adaption of bacteria to extreme temperatures experienced during thermal based sterilization procedures applied in food industry and medicine. Genes encoding for ClpG are transmissible by horizontal transfer, allowing for rapid spreading of extreme bacterial heat resistance and posing a threat to modern food production.