Formation and modulation of multi-pulse femtosecond laser-induced periodic surface structures by electromagnetic and partial differential equation simulations.

In order to demonstrate the formation of laser-induced periodic surface structures (LIPSS), simulations were performed to investigate the effect of multiple femtosecond laser pulses with different laser energy densities on a Ti6Al4V surface. In this work, a set of partial differential equations calculating the electron and lattice temperature variations, followed by coupling with an electric field, is used to analyze the evolution of the periodic surface structure induced by the interaction of the femtosecond laser with the material. As the number of pulses increases, the surface structure of the material changes from none to produce LIPSS structure and from low spatial frequency LIPSS (LSFL) structure to high spatial frequency LIPSS (HSFL) structure. In order to compare the results, single-point laser scanning ablation experiments were carried out at femtosecond laser energy. The experimental results are consistent with the simulation results.

Genome sequencing of 15 acid-tolerant yeasts.

We report draft genome sequences for 15 non-conventional Saccharomycotina yeast strains obtained from public culture repositories. Included in our collection are eight strains of Pichia with broad tolerance to dicarboxylic acids. The genome sequences of these strains will enable comparative genomics of acid-tolerant phenotypes and strain engineering of non-conventional hosts.

Peculiarity of the Mechanism of Early Stages of Photo-Oxidative Degradation of Linear Low-Density Polyethylene Films in the Presence of Ferric Stearate.

Ferric stearate (FeSt3) is very efficient in accelerating polyethylene (PE) degradation, but there is a lack of exploration of its role in accelerating the early stages of polyethylene photo-oxidative degradation. This study aimed to investigate the effect of FeSt3 on the photo-oxidative degradation of PE films, especially in the early stages of photo-oxidative degradation. The results show that FeSt3 not only promotes the oxidative degradation of PE but also contributes significantly to the early behavior of photo-oxidative degradation. Moreover, the results of the density functional theory (DFT) calculations proved that the C-H in the FeSt3 ligand was more easily dissociated compared with the PE matrix. The generated H radicals participate in the coupling reaction of the primary alkyl macro radicals leading to the molecular weight reduction, thus significantly increasing the initial rate of molecular weight reduction of PE. Meanwhile, the transfer reaction of the dissociation-generated C-centered radicals induced the PE matrix to produce more secondary alkyl macroradicals, which shortened the time to enter the oxidative degradation stage. This finding reveals the mechanism by which FeSt3 promotes the degradation of PE at the early stage of photo-oxidative degradation. It provides guiding significance for the in-depth study of the early degradation behavior in photo-oxidative degradation on polyolefin/FeSt3 films.

Screening non-conventional yeasts for acid tolerance and engineering Pichia occidentalis for production of muconic acid.

Saccharomyces cerevisiae is a workhorse of industrial biotechnology owing to the organism's prominence in alcohol fermentation and the suite of sophisticated genetic tools available to manipulate its metabolism. However, S. cerevisiae is not suited to overproduce many bulk bioproducts, as toxicity constrains production at high titers. Here, we employ a high-throughput assay to screen 108 publicly accessible yeast strains for tolerance to 20 g L-1 adipic acid (AA), a nylon precursor. We identify 15 tolerant yeasts and select Pichia occidentalis for production of cis,cis-muconic acid (CCM), the precursor to AA. By developing a genome editing toolkit for P. occidentalis, we demonstrate fed-batch production of CCM with a maximum titer (38.8 g L-1), yield (0.134 g g-1 glucose) and productivity (0.511 g L-1 h-1) that surpasses all metrics achieved using S. cerevisiae. This work brings us closer to the industrial bioproduction of AA and underscores the importance of host selection in bioprocessing.

Study on the Mechanism of Molecular Weight Reduction of Polyethylene Based on Fe-Montmorillonite and Its Potential Application.

The reactions occurring in the oxidative degradation phase during the photo-oxidative degradation of polyethylene (PE) are the factors leading to molecular weight reduction. However, the mechanism of molecular weight reduction before oxidative degradation has not been clarified. The present study aims to investigate the photodegradation of PE/Fe-montmorillonite (Fe-MMT) films, especially molecular weight change. The results show the rate of photo-oxidative degradation of each PE/Fe-MMT film is much faster than that of the pure linear low-density polyethylene (LLDPE) film. A decrease in the molecular weight of polyethylene was also found in the photodegradation phase. Based on this, it was found that the transfer and coupling of primary alkyl radicals originating from photoinitiation lead to a decrease in the molecular weight of polyethylene, and the kinetic results validate this new mechanism well. This new mechanism is an improvement on the existing mechanism of molecular weight reduction during the photo-oxidative degradation of PE. In addition, Fe-MMT can greatly accelerate the reduction of PE molecular weight into small oxygen-containing molecules as well as induce cracks on the surface of polyethylene films, all of which can accelerate the biodegradation process of polyethylene microplastics. The excellent photodegradation properties of PE/Fe-MMT films will be useful in the design of more environmentally friendly degradable polymers.

Impact of incineration slag co-disposed with municipal solid waste on methane production and methanogens ecology in landfills.

Co-landfill of incineration slag and municipal solid waste (MSW) is a main method for disposal of slag, and it has the potential of promoting methane (CH4) production and accelerating landfill stabilization. Four simulated MSW landfill columns loaded with different amount of slag (A, 0%; B, 5%; C, 10%; D, 20%) were established, and the CH4 production characteristics and methanogenic mechanisms were investigated. The maximum CH4 concentration in columns A, B, C and D was 10.8%, 23.3%, 36.3% and 34.3%, respectively. Leachate pH and refuse pH were positively correlated with CH4 concentration. Methanosarcina was the dominant genus with abundance of 35.1%∼75.2% and it was positively correlated with CH4 concentration. CO2-reducing and acetoclastic methanogenesis were the main types of methanogenesis pathway, and the methanogenesis functional abundance increased with slag proportion during stable methanogenesis process. This research can help understanding the impact of slag on CH4 production characteristics and microbiological mechanisms in landfills.

Insights into the landfill leachate properties and bacterial structure succession resulting from the colandfilling of municipal solid waste and incineration bottom ash.

Four simulated bioreactors were loaded with only MSW, 5 % BA + MSW, 10 % BA + MSW and 20 % BA + MSW to investigate the leachate property and bacterial community change trends during the colandfilling process. The results showed that with increasing BA addition proportion (5 %∼20 %), the leachate oxidation-reduction potential (ORP) was lower, the leachate pH quickly entered the neutral stage, and the chemical oxygen demand (COD), volatile fatty acids (VFA), NH4+-N, Ca2+ and SO42- presented faster downward trends. The leachate SUVA254 and E300/400 confirmed that BA can accelerate the leachate humification process. BA can quickly increase bacterial diversity, and the higher the addition proportion of BA, the more significant the change in microbial community structure during the landfilling process. The leachate pH and COD greatly influenced the bacterial community structure. A low BA proportion can increase metabolism pathway abundance during the initial stage, but a high BA proportion had an inhibitory effect on the metabolism pathway.

Kinetic characterization and structure analysis of an altered polyol dehydrogenase with d-lactate dehydrogenase activity.

During adaptive metabolic evolution a native glycerol dehydrogenase (GDH) acquired a d-lactate dehydrogenase (LDH) activity. Two active-site amino acid changes were detected in the altered protein. Biochemical studies along with comparative structure analysis using an X-ray crystallographic structure model of the protein with the two different amino acids allowed prediction of pyruvate binding into the active site. We propose that the F245S alteration increased the capacity of the glycerol binding site and facilitated hydrogen bonding between the S245 γ-O and the C1 carboxylate of pyruvate. To our knowledge, this is the first GDH to gain LDH activity due to an active site amino acid change, a desired result of in vivo enzyme evolution.

Corrosion resistance and antibacterial activity of zinc-loaded montmorillonite coatings on biodegradable magnesium alloy AZ31.

A Zinc-loaded montmorillonite (Zn-MMT) coating was hydrothermally prepared using Zn2+ ion intercalated sodium montmorillonite (Na-MMT) upon magnesium (Mg) alloy AZ31 as bone repairing materials. Biodegradation rate of the Mg-based materials was studied via potentiodynamic polarization curves, electrochemical impedance spectroscopy (EIS) and hydrogen evolution tests. Results revealed that both Na-MMT and Zn-MMT coatings exhibited better corrosion resistance in Dulbecco's modified eagle medium (DMEM) + 10% calf serum (CS) than bare Mg alloy AZ31 counterparts. Hemolysis results demonstrated that hemocompatibility of the Na-MMT and Zn-MMT coatings were 5%, and lower than that of uncoated Mg alloy AZ31 pieces. In vitro MTT tests and live-dead stain of osteoblast cells (MC3T3-E1) indicated a significant improvement in cytocompatibility of both Na-MMT and Zn-MMT coatings. Antibacterial properties of two representative bacterial strains associated with device-related infection, i.e. Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), were employed to explore the antibacterial behavior of the coatings. The measured inhibitory zone and bacterial growth rate confirmed that Zn-MMT coatings exhibited higher suppression toward both E. coli and S. aureus than that of Na-MMT coatings. The investigation on antibacterial mechanism through scanning electron microscopy (SEM) and lactate dehydrogenase (LDH) release assay manifested that Zn-MMT coating led to severe breakage of bacterial membrane of E. coli and S. aureus, which resulted in a release of cytoplasmic materials from the bacterial cells. In addition, the good inhibition of Zn-MMT coatings against E. coli and S. aureus might be attributed to the slow but sustainable release of Zn2+ ions (up to 144 h) from the coatings into the culture media. This study provides a novel coating strategy for manufacturing biodegradable Mg alloys with good corrosion resistance, biocompatibility and antibacterial activity for future orthopedic applications. STATEMENT OF SIGNIFICANCE: The significance of the current work is to develop a corrosion-resistant and antibacterial Zn-MMT coating on magnesium alloy AZ31 through a hydrothermal method. The Zn-MMT coating on magnesium alloy AZ31 shows better corrosion resistance, biocompatibility and excellent antibacterial ability than magnesium alloy AZ31. This study provides a novel coating on Mg alloys for future orthopedic applications.

Metabolic engineering of Bacillus subtilis for production of D-lactic acid.

Poly lactic acid (PLA) based plastics is renewable, bio-based, and biodegradable. Although present day PLA is composed of mainly L-LA, an L- and D- LA copolymer is expected to improve the quality of PLA and expand its use. To increase the number of thermotolerant microbial biocatalysts that produce D-LA, a derivative of Bacillus subtilis strain 168 that grows at 50°C was metabolically engineered. Since B. subtilis lacks a gene encoding D-lactate dehydrogenase (ldhA), five heterologous ldhA genes (B. coagulans ldhA and gldA101, and ldhA from three Lactobacillus delbrueckii) were evaluated. Corresponding D-LDHs were purified and biochemically characterized. Among these, D-LDH from L. delbrueckii subspecies bulgaricus supported the highest D-LA titer (about 1M) and productivity (2 g h-1 g cells-1 ) at 37°C (B. subtilis strain DA12). The D-LA titer at 48°C was about 0.6 M at a yield of 0.99 (g D-LA g-1 glucose consumed). Strain DA12 also fermented glucose at 48°C in mineral salts medium to lactate at a yield of 0.89 g g-1 glucose and the D-lactate titer was 180 ± 4.5 mM. These results demonstrate the potential of B. subtilis as a platform organism for metabolic engineering for production of chemicals at 48°C that could minimize process cost.

An ancient Chinese wisdom for metabolic engineering: Yin-Yang.

In ancient Chinese philosophy, Yin-Yang describes two contrary forces that are interconnected and interdependent. This concept also holds true in microbial cell factories, where Yin represents energy metabolism in the form of ATP, and Yang represents carbon metabolism. Current biotechnology can effectively edit the microbial genome or introduce novel enzymes to redirect carbon fluxes. On the other hand, microbial metabolism loses significant free energy as heat when converting sugar into ATP; while maintenance energy expenditures further aggravate ATP shortage. The limitation of cell "powerhouse" prevents hosts from achieving high carbon yields and rates. Via an Escherichia coli flux balance analysis model, we further demonstrate the penalty of ATP cost on biofuel synthesis. To ensure cell powerhouse being sufficient in microbial cell factories, we propose five principles: 1. Take advantage of native pathways for product synthesis. 2. Pursue biosynthesis relying only on pathways or genetic parts without significant ATP burden. 3. Combine microbial production with chemical conversions (semi-biosynthesis) to reduce biosynthesis steps. 4. Create "minimal cells" or use non-model microbial hosts with higher energy fitness. 5. Develop a photosynthesis chassis that can utilize light energy and cheap carbon feedstocks. Meanwhile, metabolic flux analysis can be used to quantify both carbon and energy metabolisms. The fluxomics results are essential to evaluate the industrial potential of laboratory strains, avoiding false starts and dead ends during metabolic engineering.

Amino acid substitutions at glutamate-354 in dihydrolipoamide dehydrogenase of Escherichia coli lower the sensitivity of pyruvate dehydrogenase to NADH.

Pyruvate dehydrogenase (PDH) of Escherichia coli is inhibited by NADH. This inhibition is partially reversed by mutational alteration of the dihydrolipoamide dehydrogenase (LPD) component of the PDH complex (E354K or H322Y). Such a mutation in lpd led to a PDH complex that was functional in an anaerobic culture as seen by restoration of anaerobic growth of a pflB, ldhA double mutant of E. coli utilizing a PDH- and alcohol dehydrogenase-dependent homoethanol fermentation pathway. The glutamate at position 354 in LPD was systematically changed to all of the other natural amino acids to evaluate the physiological consequences. These amino acid replacements did not affect the PDH-dependent aerobic growth. With the exception of E354M, all changes also restored PDH-dependent anaerobic growth of and fermentation by an ldhA, pflB double mutant. The PDH complex with an LPD alteration E354G, E354P or E354W had an approximately 20-fold increase in the apparent K(i) for NADH compared with the native complex. The apparent K(m) for pyruvate or NAD(+) for the mutated forms of PDH was not significantly different from that of the native enzyme. A structural model of LPD suggests that the amino acid at position 354 could influence movement of NADH from its binding site to the surface. These results indicate that glutamate at position 354 plays a structural role in establishing the NADH sensitivity of LPD and the PDH complex by restricting movement of the product/substrate NADH, although this amino acid is not directly associated with NAD(H) binding.

Metabolic engineering of thermophilic Bacillus licheniformis for chiral pure D-2,3-butanediol production.

2,3-Butanediol is an important compound that can be used in many areas, especially as a platform chemical and liquid fuel. But traditional 2,3-butanediol producing microorganisms, such as Klebsiella pneumonia and K. xoytoca, are pathogens and they can only ferment sugars at 37°C. Here, we reported a newly developed Bacillus licheniformis. A protoplast transformation system was developed and optimized for this organism. With this transformation method, a marker-less gene deletion protocol was successfully used to knock out the ldh gene of B. licheniformis BL1 and BL3. BL1 was isolated earlier from soil for lactate production and it was further evolved to BL3 for xylose utilization. Combined with pH and aeration control, ldh mutant BL5 and BL8 can efficiently ferment glucose and xylose to D-(-) 2,3-butanediol at 50°C, pH 5.0. For glucose and xylose, the specific 2,3-butanediol productivities are 29.4 and 26.1 mM/h, respectively. The yield is 0.73 mol/mol for BL8 in xylose and 0.9 mol/mol for BL5 and BL8 in glucose. The D-(-) 2,3-butanediol optical purity is more than 98%. As far as we know, this is the first reported high temperature butanediol producer to match the simultaneous saccharification and fermentation conditions. Therefore, it has potential to further lower butanediol producing cost with low cost lignocellulosic biomass in the near future.

Evolution of D-lactate dehydrogenase activity from glycerol dehydrogenase and its utility for D-lactate production from lignocellulose.

Lactic acid, an attractive, renewable chemical for production of biobased plastics (polylactic acid, PLA), is currently commercially produced from food-based sources of sugar. Pure optical isomers of lactate needed for PLA are typically produced by microbial fermentation of sugars at temperatures below 40 °C. Bacillus coagulans produces L(+)-lactate as a primary fermentation product and grows optimally at 50 °C and pH 5, conditions that are optimal for activity of commercial fungal cellulases. This strain was engineered to produce D(-)-lactate by deleting the native ldh (L-lactate dehydrogenase) and alsS (acetolactate synthase) genes to impede anaerobic growth, followed by growth-based selection to isolate suppressor mutants that restored growth. One of these, strain QZ19, produced about 90 g L(-1) of optically pure D(-)-lactic acid from glucose in < 48 h. The new source of D-lactate dehydrogenase (D-LDH) activity was identified as a mutated form of glycerol dehydrogenase (GlyDH; D121N and F245S) that was produced at high levels as a result of a third mutation (insertion sequence). Although the native GlyDH had no detectable activity with pyruvate, the mutated GlyDH had a D-LDH specific activity of 0.8 µmoles min(-1) (mg protein)(-1). By using QZ19 for simultaneous saccharification and fermentation of cellulose to D-lactate (50 °C and pH 5.0), the cellulase usage could be reduced to 1/3 that required for equivalent fermentations by mesophilic lactic acid bacteria. Together, the native B. coagulans and the QZ19 derivative can be used to produce either L(+) or D(-) optical isomers of lactic acid (respectively) at high titers and yields from nonfood carbohydrates.

Isolation, characterization and evolution of a new thermophilic Bacillus licheniformis for lactic acid production in mineral salts medium.

The high fermentation cost of lactic acid is a barrier for polylactic acid (PLA) to compete with the petrochemical derived plastics. In order to lower the cost of lactic acid, the industry needs a microorganism that can ferment various sugars at high temperature (50°C) and at the same time using low cost mineral salts (MS) medium. One such bacterium, BL1, was isolated at 50°C and identified as Bacillus licheniformis. BL1 can ferment glucose to optically pure l-lactate with a maximum specific productivity of 7.8 g/hl in LB medium and 0.7 g/hl in MS medium at 50°C. BL1 can also consume 10% and 15% glucose in 20 and 48 h, respectively. After serial transfer of BL1 and BL2 in different concentrations of xylose and MS medium respectively, the final mutant BL3 could efficiently ferment glucose and xylose with specific productivity of 1.9 g/hl and 1.2g/hl in strict MS medium.

Metabolic flux control at the pyruvate node in an anaerobic Escherichia coli strain with an active pyruvate dehydrogenase.

During anaerobic growth of Escherichia coli, pyruvate formate-lyase (PFL) and lactate dehydrogenase (LDH) channel pyruvate toward a mixture of fermentation products. We have introduced a third branch at the pyruvate node in a mutant of E. coli with a mutation in pyruvate dehydrogenase (PDH*) that renders the enzyme less sensitive to inhibition by NADH. The key starting enzymes of the three branches at the pyruvate node in such a mutant, PDH*, PFL, and LDH, have different metabolic potentials and kinetic properties. In such a mutant (strain QZ2), pyruvate flux through LDH was about 30%, with the remainder of the flux occurring through PFL, indicating that LDH is a preferred route of pyruvate conversion over PDH*. In a pfl mutant (strain YK167) with both PDH* and LDH activities, flux through PDH* was about 33% of the total, confirming the ability of LDH to outcompete the PDH pathway for pyruvate in vivo. Only in the absence of LDH (strain QZ3) was pyruvate carbon equally distributed between the PDH* and PFL pathways. A pfl mutant with LDH and PDH* activities, as well as a pfl ldh double mutant with PDH* activity, had a surprisingly low cell yield per mole of ATP (Y(ATP)) (about 7.0 g of cells per mol of ATP) compared to 10.9 g of cells per mol of ATP for the wild type. The lower Y(ATP) suggests the operation of a futile energy cycle in the absence of PFL in this strain. An understanding of the controls at the pyruvate node during anaerobic growth is expected to provide unique insights into rational metabolic engineering of E. coli and related bacteria for the production of various biobased products at high rates and yields.

Modern biotechnology in China.

In recent years, with the booming economy, the Chinese government has increased its financial input to biotechnology research, which has led to remarkable achievements by China in modern biotechnology. As one of the key parts of modern biotechnology, industrial biotechnology will be crucial for China's sustainable development in this century. This review presents an overview of Chinese industrial biotechnology in last 10 years. Modern biotechnology had been classified into metabolic engineering and systems biology framework. Metabolic engineering is a field of broad fundamental and practical concept so we integrated the related technology achievements into the real practices of many metabolic engineering cases, such as biobased products production, environmental control and others. Now metabolic engineering is developing towards the systems level. Chinese researchers have also embraced this concept and have contributed invaluable things in genomics, transcriptomics, proteomics and related bioinformatics. A series of advanced laboratories or centers were established which will represent Chinese modern biotechnology development in the near future. At the end of this review, metabolic network research advances have also been mentioned.

[The research progress of succinic acid fermentation strains].

The potential of succinic acid as an important chemical intermediates had been realized and fermentation is one of the best ways to make it possible in economical aspect. Fermentation organism is the key part of the fermentation method. The updated research developments of fermentation organisms and the fermentation characteristics and problems of them were reviewed and analyzed in this paper. Finally,the development future of fermenation organism was forecasted.

Metabolic network properties help assign weights to elementary modes to understand physiological flux distributions.

MOTIVATION: Elementary modes (EMs) analysis has been well established. The existing methodologies for assigning weights to EMs cannot be directly applied for large-scale metabolic networks, since the tremendous number of modes would make the computation a time-consuming or even an impossible mission. Therefore, developing more efficient methods to deal with large set of EMs is urgent. RESULT: We develop a method to evaluate the performance of employing a subset of the elementary modes to reconstruct a real flux distribution by using the relative error between the real flux vector and the reconstructed one as an indicator. We have found a power function relationship between the decrease of relative error and the increase of the number of the selecting EMs, and a logarithmic relationship between the increases of the number of non-zero weighted EMs and that of the number of the selecting EMs. Our discoveries show that it is possible to reconstruct a given flux distribution by a selected subset of EMs from a large metabolic network and furthermore, they help us identify the 'governing modes' to represent the cellular metabolism for such a condition.

Genome-scale in silico aided metabolic analysis and flux comparisons of Escherichia coli to improve succinate production.

In the post-genome era, it is one challenge to understand the cellular metabolism at the systematic levels. Mathematical modeling of microorganisms and subsequent computer simulation are effective tools for systems biology. In this paper, based on the genome-scale Escherichia coli stoichiometric model iJR904, through the GAMS linear programming package, the in silico maximal succinate yield was estimated to be 1.714 mol/mol glucose. When another two constraints were added, the maximal succinate yield dropped to 1.60 mol/mol glucose. Further analysis substantiated the uniqueness of the flux distribution under such constraints. After comparisons with the metabolic flux analysis (MFA) results computed from the wet experimental data of the three kinds of E. coli, three potential improvement target sites, the glucose phosphotransferase transport system, the pyruvate carboxylase, and the glyoxylate shunt, were identified and selected for the genetic modifications. All the three genetic modified strains showed increased succinate yield. The final strain TUQ19/pQZ6 had a high yield of 1.29 mol succinate/mol glucose and high productivity. The success of the above experiments proved that this in silico optimal succinate production pathway is reasonable and practical. This method may also be used as a general strategy to help enhance the yields of other favorable metabolites in E. coli.