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Background Aromatic α-hydroxy ketones such as S-2-hydroxypropiophenine (S-2-HPP) are highly valuable chiral building blocks useful for the synthesis of various pharmaceuticals and natural products. In the present study, enantioselective synthesis of S-2-HPP was investigated by free and immobilized whole cells of Pseudomonas putida ATCC 12633 starting from readily-available aldehyde substrates. Whole resting cells of P. putida grown in a culture medium containing ammonium mandelate are a source of native benzoylformate decarboxylase (BFD) activity. BFD synthesized by induced P. putida resting cells is a highly active biocatalyst without any further treatment in comparison with partially purified preparation. These cells can convert benzaldehyde and acetaldehyde into the acyloin compound 2-HPP by BFD catalyzed enantioselective cross-coupling reaction. Results The reaction was carried out in the presence of exogenous benzaldehyde (20 mM) and acetaldehyde (600 mM) as substrates in 6 ml of 200 mM phosphate buffer (pH 7) for 3 hours. The optimal biomass concentration was assessed to be 0.006 g DCW/ml. 2-HPP titer, yield and productivity using the free cells were 1.2 g L-1, 0.56 g 2-HPP/g benzaldehyde (0.4 mol 2-HPP/mol benzaldehyde), 0.067 g 2-HPP/g DCW h, respectively, under optimized biotransformation conditions (30 ⁰C, 200 rpm). Calcium alginate (CA)–polyvinyl alcohol (PVA)-boric acid (BA)-beads were used for the cell entrapment. Encapsulated whole-cells were successfully employed in four consecutive cycles for 2-HPP production under aerobic conditions without any noticeable degradation of the beads. Moreover, there was no production of benzyl alcohol as an unwanted by-product. Conclusion Thus, bioconversion by P. putida whole resting cells is an efficient strategy for the production of 2-HPP and other α-hydroxyketones.

In this study, we used electrospun polycaprolactone (PCL) or a mixture of PCL and gelatin (Gel) in a mixed acidic solvent to develop antimicrobial electrospun nanofibers. Carboxyl-modified mesoporous silica nanoparticles (CMSNs) or CMSNs loaded with antibiotic drugs polymyxin B and vancomycin (CMSNs/ABs) were mixed with the electrospinning solution in concentrations of 1%, 2.5% and 5%. The nanofibers diameter measured between 122 and138 nm. Higher concentrations of gelatin or CMSNs increased hydrophilicity and degradability of the nanofibers. CMSNs enhanced nanofibers mechanical strength. PCL/Gel nanofibers incorporated with CMSNs/ABs (2.5% and 5%) showed high antibacterial efficiency against Pseudomonas aeruginosa and Staphylococcus aureus. Also bacterial cell adhesion decreased when 2.5% and 5% of CMSNs/ABs were incorporated in PCL/Gel mats. MTT and hemolysis assays indicated excellent biocompatibility of all types of electrospun nanofibers. This study confirms that a proper mixture of PCL, gelatin and CMSNs loaded with two antibiotics could offer antimicrobial activities with high biocompatibility and biodegradability properties. Graphic abstract: [Figure not available: see fulltext.].

Costly cofactors such as nicotinamide adenine dinucleotide hydrogen (NADH) are essential to have high activity in many redox enzymatic processes. Cofactor regeneration methods have been suggested to improve the economic aspects of the system. Here, we introduce a microbial electrosynthesis process to regenerate NADH in a two-chamber set-up with Enterobacter aerogenes biofilm as the bio-cathode. The effects of several important factors on the regeneration efficiency were studied and the highest NADH regeneration yield was achieved equal to 65 % at the potential of -1.5 V and the initial NAD

The enzymatic production of formate from CO2 with the immobilized NADH-dependent formate dehydrogenase (FDH) on the activated electrospun polystyrene nanofibers (EPSNF) was investigated to develop a sustainable process for CO2 reduction. Direct electrochemical regeneration on a Cu foam electrode was employed to supply the reaction with the reduced cofactor (NADH). The formate production was studied in two modes of batch and semi-continuous operations with the cofactor recycle. Results indicated that the regenerated cofactor concentrations in both systems were nearly identical (0.5 mM) which ensured the desirable activity of the immobilized enzyme. This showed that the electrochemical regeneration system was effective even in the semi-continuous operation. Although the cumulative formate concentration in the batch operation was higher, the total amounts of the produced formate were higher for the semi-continuous mode for more than 42% which was justified by the fact that the lower formate concentration in the semi-continuous mode would be favorable to the progress of the enzymatic CO2 to formate conversion. Finally, it was concluded that the proposed semi-continuous process in this work could be considered as a promising process for the enzymatic CO2 conversion.

Methylation of DNA at carbon 5 of cytosines is the most common epigenetic modification of human genome. Due to its critical role in many normal cell processes such as growth and development, any aberrant methylation pattern in a particular locus may lead to abnormal functions and diseases such as cancer. Development of methods to detect methylation state of DNA which may eliminate labor-intensive chemical or enzymatic treatments has received considerable attention in recent years. Herein, we report a DNA methylation detection procedure based on fluorescence turn-on strategy. Target sequence was selected from Sept9 promoter region that has been reported as one of the most frequently methylated sites in colorectal cancer. Probe DNA was designed to be complementary to this sequence with an additional six cytosines in the middle to form an internal loop to host silver nanoclusters. The fluorescence intensity of the synthesized silver nanoclusters with the duplexes of probe-non-methylated target was significantly different from that of probe-methylated target. The fluorescence enhanced with increasing the methylated DNA concentration with a linear relation in the range of 1.0 × 10−8 M to 5.0 × 10−7 M with the detection limit of 8.2 × 10−9 M, and quenched with non-methylated ones. The method was very specific in the presence of non-complementary sequences with maximum similarity of 40%. Circular dichroism spectra indicated that silver ions significantly affected the structure of methylated and non-methylated DNA into different extents which could further influence the nanocluster fluorescence. Finally, a method was introduced to meet the concerns in the applicability of the proposed method in real situation.

Pretreatment is the key factor for producing fermentable sugars and biofuels from lignocellulosic biomass. However, during pretreatment lignin repolymerization occurs which reduces the efficiency of carbohydrates hydrolysis. In the present study, three additives (2-naphthol-7-sulfonate, 2-naphthol, and mannitol) were evaluated for boosting the effectiveness of combined pretreatment (liquid hot water + green liquor) of pine-wood biomass. The results revealed that supplied additives into combined pretreatment changed the physicochemical structure of lignin, therefore, alleviating the inhibitory effect of lignin repolymerization and notably raised sugars yield (glucose, xylose) ranging from 85% to 93%. XPS and FTIR analyses confirmed that −OH groups of additives led to lower formation of phenolic hydroxyl (PhOH) and higher hydrophilic groups of lignin, accounting for reduced non-specific binding of lignin into cellulase enzymes. Furthermore, additives increased surface lignin coverage, biomass porosity, and fiber swelling by 1.96–2.59, 1.66–1.86, and 1.13–1.29 folds respectively, comparing to corresponding sample without additives caused more liquified and extractable lignin, which notably reduced the surface lignin barrier. Hence, both factors including non-specific binding effect and surface lignin barrier, contributed to reducing lignin repolymerization, thereby increased cellulose accessibility for high production of fermentable sugars and bioethanol in softwood biomass.

Enzymatic conversion of CO2 to formate was carried out in the cathodic cell of a two-chamber electrochemical apparatus where NAD+ was reduced on the surface of a Copper foam electrode. Formate dehydrogenase (FDH) was used as the biocatalyst in both free form and immobilized on the modified electrospun polystyrene nanofibers (EPSNF). The fabricated EPSNF were modified by a multistage procedure including acid treatment, silanization followed by activation with glutaraldehyde. The effects of regenerated NADH concentration and time of enzymatic reaction on the formate production in the both systems were studied. The results indicated that the EPSNF immobilized FDH had a desirable activity, long-term storage stability (41% after 20 days) and reusability after eight cycles of successive reactions (53% of the initial activity). Moreover, it was revealed that the increase of cofactor concentration at the early times of reaction was favorable to the formate production. However, an inhibitory effect was observed at higher concentrations of NADH, and the optimum values of 0.45 mM and 0.51 mM were obtained for the maximum enzyme activity by the free and immobilized enzymes respectively. The produced formate at the optimum cofactor concentration after 300 min was 0.61 mM and 0.31 mM for the free and immobilized enzyme systems. Finally, it can be concluded that the presented process is a promising approach to the enzymatic conversion of CO2.

Sweet sorghum plant, a widely grown energy crop, was utilized through a biorefinery process, by which its grains were hydrolyzed by the crude amylases produced from its bagasse. The hemicellulosic part of the bagasse was hydrolyzed with 0.5–1.0% sulfuric acid at 140–180 °C for 30–60 min and applied for amylase production using halotolerant bacterium Nesterenkonia sp. strain F. In the hydrolysate obtained at 140 °C for 60 min using 1% acid, Nesterenkonia showed 73.3 U/mL amylase activity by the consumption of 16.2 g/L xylose and 8.3 g/L other sugars. Supplementation of the hydrolysates with sorghum grain resulted in 38–67% higher amylase production. Furthermore, addition of biocompatible surfactants of Tween 20 and Tween 80 (0.1 g/L) increased the activity to 93 and 97 U/mL, respectively. The resulting crude enzyme was used in the process of ethanol production from both tannin-containing and tannin-free sorghum grains (6%), leading to 17.7 and 17.0 g/L bioethanol production, respectively. Through the cultivation of Nesterenkonia on the hemicellulosic hydrolysates, 5–10 g/L volatile fatty acids (VFA), 0.36–0.69 g/L acetone-butanol-ethanol (ABE), and 468–721 mg/L single cell protein (SCP) were also produced. The obtained SCP contained most of the essential amino acids and relatively high amounts of phenylalanine (8%), threonine (7%), methionine (6%), and lysine (6%).

Polymyxin B is a polycationic antibiotic used as the last line treatment against antibiotic-resistant Gram negative bacteria. However, application of polymyxin B is limited because of its toxicity effects. Herein, we used bare and surface modified mesoporous silica nanoparticles (MSNs) with an average diameter of 72.29 +/- 8.17 nm as adsorbent for polymyxin B to improve its therapeutic properties. The polymyxin B adsorption onto MSN surfaces was explained as a function of pH, type of buffer and surface charge of nanoparticles, according to the zeta-potential of silica nanoparticles and adsorption kinetics results. The highest value of the adsorption capacity (about 401 +/- 15.38 mg polymyxin B/g silica nanoparticles) was obtained for the bare nanoparticles in Tris buffer, pH 9. Release profiles of polymyxin B showed a sustained release pattern, fitting Power law and Hill models. The antibiotic molecules-loaded nanoparticles showed enhanced antibacterial activity compared to free antibiotic against different Gram negative bacteria. Biocompatibility evaluation results revealed that loading of polymyxin B onto MSNs can decrease the cytotoxicity effects of the drug by reducing ROS generation. Our results suggest that formulation of drugs by adsorption onto MSNs may offer a way forward to overcome the adverse effects of some antibiotics such as polymyxin B without compromising their antimicrobial properties.

Industrial scale production of biobutanol has been hampered by substrate cost and availability. Sweet sorghum grain is an inexpensive substrate for acetone-butanol-ethanol (ABE) production by Clostridium acetobutylicum. Amylolytic activity of C. acetobutylicum eliminates the need for the hydrolysis of starchy grain prior to fermentation. However, untreated grain contains phenolic compounds, i.e. tannins, which exhibit inhibitory effects against amylolytic activity and ABE fermentation. Less than 3 g/L ABE was obtained from untreated sweet sorghum grain at different substrate concentrations. Concentration of 0.2 mM gallic acid equivalent (GAE) of sorghum tannins was detected as the critical concentration which inhibits severely ABE fermentation. Applying a multi-stage hot water treatment resulted in tannins removal and significant enhancement in total ABE production up to 18 g/L. For efficient butanol production from 40, 60, and 80 g/L sorghum grain, hot water treatment with two, five, and six stages were found to be essential for efficient butanol production, respectively. Moreover, the amylolytic activity of C. acetobutylicum was inhibited by sorghum grain tannins, more than twice as high as the effects on the ABE fermentation pathway. Furthermore, unlike most substrates, sweet sorghum grain could provide all nutrients required for ABE fermentation, eliminating the need for supplementing expensive additional nutrients.

Herein, Cu foam and bimetallic Cu foams are introduced as the cost efficient and easy to scale up electrode for efficient electrochemical regeneration of NADH. The porous copper foam was electrochemically deposited at the copper foil. Cu foam was used as a template for preparation of Ag and Pt coated Cu foam by galvanic replacement of Cu with silver and platinum. The nature and structure of the prepared foams were characterized by energy dispersive X-ray spectrometry and scanning electron microscopy. Electrochemical behavior of NAD+ was studied by cyclic voltammetry at the surface of prepared foams. Also regeneration of the enzymatic cofactor NADH in a batch electrochemical reactor using Cu foil, Cu foam, Ag coated and Pt coated Cu foam electrodes has been investigated. The effect of thickness of Cu foam and the reduction potential of NAD+ on the recovery percent of 1,4-NADH was investigated to achieve the most efficient conditions for electrochemical regeneration of NADH. The regeneration yields of enzymatically active NADH were about 80%, 70% and 75% using the Cu foam, Ag and Pt coated Cu foam electrodes, respectively which all of them were more than that observed at Cu foil electrode 54%.

The yeast cells were coated with Fe3O4 magnetic nanoparticles and employed as biocatalyst for the microbial biotransformation of benzaldehyde into l-phenylacetylcarbinol (l-PAC). Saccharomyces cerevisiae CEN.PK113-7D yeast cells were coated with magnetic nanoparticles to facilitate the cells separation process. Transmission electron microscopy, powder XRD diffraction, and vibrating sample magnetometer were used to characterize magnetic nanoparticles and magnetic nanoparticle-coated yeast cells. Then the reusability of magnetically recoverable cells in production of l-PAC was investigated. Results show that coating yeast cells with magnetic nanoparticles does not affect their size and structure. Coated cells were also used in seven consecutive batch cycles and no significant reduction for l-PAC titer was observed in any of the cycles. Coating yeast cells with magnetic nanoparticles enabled rapid separation and reuse of cells in several successive batch cycle without affecting their ability to produce l-PAC.

Clostridium acetobutylicum is widely used for the microbial production of butanol in a process known as acetone-butanol-ethanol (ABE) fermentation. However, this process suffers from several disadvantages including high oxygen sensitivity of the bacterium which makes the process complicated and necessitate oxygen elimination in the culture medium. Nesterenkonia sp. strain F has attracted interests as the only known non-Clostridia microorganism with inherent capability of butanol production even in the presence of oxygen. This bacterium is not delimited by oxygen sensitivity, a challenge in butanol biosynthesis, but the butanol titer was far below Clostridia. In this study, Nesterenkonia sp. strain F was cocultivated with C. acetobutylicum to form a powerful "coculture" for butanol production thereby eliminating the need for oxygen removal before fermentation. The response surface method was used for obtaining optimal inoculation amount/time and media formulation. The highest yield, 0.31 g/g ABE (13.6 g/L butanol), was obtained by a coculture initiated with 1.5 mg/L Nesterenkonia sp. strain F and inoculated with 15 mg/L C. acetobutylicum after 1.5 hr in a medium containing 67 g/L glucose, 2.2 g/L yeast extract, 4 g/L peptone, and 1.4% (vol/vol) P2 solution. After butanol toxicity assessment, where Nesterenkonia sp. strain F showed no butanol toxicity, the coculture was implemented in a 2 L fermenter with continual aeration leading to 20 g/L ABE.

L-Phenylacetylcarbinol (L-PAC) which is used as a precursor for the production of ephedrine and pseudoephedrine is the first reported biologically produced α-hydroxy ketone compound. l-PAC is commercially produced by the yeast Saccharomyces cerevisiae. Yeast cells transform exogenously added benzaldehyde into l-PAC by using the action of pyruvate decarboxylase (PDC) enzyme. In this work, genome-scale model and flux balance analysis were used to identify novel target genes for the enhancement of l-PAC production in yeast. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using OptGene and minimization of metabolic adjustments. Six single gene deletion strains, namely Δrpe1, Δpda1, Δadh3, Δadh1, Δzwf1 and Δpdc1, were predicted in silico and further tested in vivo by using knock-out strains cultivated semi-anaerobically on glucose and benzaldehyde as substrates. Δzwf1 mutant exhibited the highest l-PAC formation (2.48 g/L) by using 2 g/L of benzaldehyde which is equivalent to 88 % of the theoretical yield.

Silver ions can influence DNA structure. The extent of this structural perturbation is different in methylated and non-methylated DNA. Here, we explored the interaction of eight convenient DNA interacting molecules with methylated and non-methylated short GC rich oligonucleotides in the presence and absence of silver ions. Acridine orange, DAPI, Doxorubicin, Ethidium bromide, Hoechst 33342, Methylene blue, PicoGreen, and Propidium iodide are tested for their ability to distinguish methylated and non-methylated DNA. Among them, Ethidium bromide, Methylene blue, and PicoGreen were able to discriminate between methylated and non-methylated DNA, while DAPI and Hoechst 33342 were only able to discriminate with the aid of silver ions. A detection method is proposed using Ethidium bromide in which the silver-treated sample of DNA exposed different fluorescence intensity from the untreated one on the base of its methylation state. This phenomenon was sequence-dependent and could provide a sensing platform with a detection limit of about 40 nM.

Poly(lactic-co-glycolic acid) (PLGA) and PLGA/gelatin random nanofibrous scaffolds embedded with different amounts of mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. To evaluate the effects of nanoparticles on the scaffolds, physical, chemical, and mechanical properties as well as in vitro degradation behavior of scaffolds were investigated. The mean diameters of nanofibers were 974±68nm for the pure PLGA scaffolds vs 832±70, 764±80, and 486±64 for the PLGA/gelatin, PLGA/10wt% MSNPs, and the PLGA/gelatin/10wt% MSNPs scaffolds, respectively. The results suggested that the incorporation of gelatin and MSNPs into PLGA-based scaffolds enhances the hydrophilicity of scaffolds due to an increase of hydrophilic functional groups on the surface of nanofibers. With porosity examination, it was concluded that the incorporation of MSNPs and gelatin decrease the porosity of scaffolds. Nanoparticles also improved the tensile mechanical properties of scaffolds. Using in vitro degradation analysis, it was shown that the addition of nanoparticles to the nanofibers matrix increases the weight loss percentage of PLGA-based samples, whereas it decreases the weight loss percentage in the PLGA/gelatin composites. Cultivation of rat pheochromocytoma cell line (PC12), as precursor cells of dopaminergic neural cells, on the scaffolds demonstrated that the introduction of MSNPs into PLGA and PLGA/gelatin matrix leads to improved cell attachment and proliferation and enhances cellular processes.

The γ-decalactone is one of the lactones with peachy aroma which has been approved as food additive by FDA. The aim of this study was to optimize media composition and conditions for microbial biotransformation of ricinoleic acid and castor oil as substrates to γ-decalactone using the yeast Yarrowia lipolytica. The Y. lipolytica DSM 3286 strain was used as biotransformation agent in different trails designed by Taguchi method. The highest concentration of γ-decalactone was 62.4 and 52.9 mg/L from 1.5% ricinoleic acid and 2.5% castor oil, respectively. Nitrogen sources exhibited significant effect on the biotransformation. The maximum γ-decalactone production occurred at pH 6. The results showed that the composition of biotransformation medium composition is important for γ-decalactone production.

The yeast Saccharomyces cerevisiae is able to biotransform benzaldehyde into L-phenylacetylcarbinol (L-PAC), a key intermediate in the production of ephedrine and pseudoephedrine, by the action of pyruvate decarobxylase (PDC) enzyme. This biotransformation can alternatively be performed by acetohydroxyacid synthase (AHAS) which is a mitochondrial enzyme. In the yeast petite mutants, AHAS accumulates in the cytosol. In the current study, wild-type yeast cells and yeast petite mutants were examined for L-PAC biosynthesis. The results showed higher L-PAC titers in the yeast petite mutants. In addition, the effect of cell immobilization and carbon source (glucose or molasses) on L-PAC production was investigated. It was found that cell immobilization enhances L-PAC formation. The highest L-PAC concentration (2.4 g/l) was obtained at 2 g/l of benzaldehyde using the immobilized petite mutants grown on molasses.

Effluent sludge from an anaerobic digester was used as a source of nitrogen, phosphate, sulfur, and other nutrients in the culture medium of ethanol production by the yeast Saccharomyces cerevisiae. Several pretreatments (mechanical, chemical, thermal, and thermo-chemical) were performed on the anaerobic digested sludge (ADS) to make the nutrients accessible to the yeast cells. Preliminary experiments revealed that S. cerevisiae is not able to assimilate the carbon content of the ADS. However, when glucose was added to the medium, ethanol production was observed. The yield of ethanol using untreated ADS was only 10 % of the theoretical yield, but alkaline pretreatment improved it up to 43 %. By separating the hydrolysate of alkaline-treated ADS from the suspended solids, the ethanol yield from the supernatant was further improved up to 65 % of theoretical yield. Alkaline-treated ADS exhibited competitive performance with the mixture of yeast extract and mineral salts in ethanol fermentation.

Substrate cost and availability are the major bottlenecks that limit the commercialization of next generation biofuels. The possibility of using waste acorn as an abundant and inexpensive feedstock in Iran for acetone–butanol–ethanol (ABE) production with Clostridium acetobutylicum was investigated. No solvent was produced using untreated acorn due to its high contents of growth-inhibitory tannic acid. To circumvent this problem, a simple extraction in close-to-boiling water was employed to remove tannic acid prior to using acorn powder in fermentation. This pretreatment significantly improved the fermentability of acorn powder by C. acetobutylicum. The maximum yields of solvents (g ABE per g substrate) achieved in serum-bottle experiments were 0.43 ± 0.02, 0.40 ± 0.02, and 0.34 ± 0.02 for glucose, pure starch, and treated acorn starch, respectively. Volumetric ABE productivity with tannin-free acorn powder was 0.21 ± 0.01 g L−1 h−1, slightly higher than that achieved with pure starch (0.18 ± 0.05 g L−1 h−1). When scaled up in 5 L fermentors, ABE production using pretreated acorn powder and pure starch resulted in solvent profiles and yields similar to those observed in the serum bottle experiments. This study demonstrates that acorn powder can be a viable feedstock for biobutanol production in Iran. Future work warrants optimization of a two-step process including extraction of tannic acid as a valuable by-product followed by fermentation of tannin-free acorn powder to improve the economic feasibility of acorn to biobutanol conversion process.

The microbial pathway of butanol biosynthesis is a unique route for the production of a biomass-derived advanced biofuel with high potential to be utilized in place of the fossil fuels. In the present study, Nesterenkonia sp. strain F was applied as a beneficial partner for Clostridium acetobutylicum in “starch-to-butanol process”. The capability of Nesterenkonia sp. strain F in secreting organic solvent-tolerant amylase was utilized for upgrading the yield of solvent, i.e., acetone, butanol, and ethanol (ABE), production under aerobic conditions. Monitoring the amylolytic activity and glucose concentration throughout the micro-aerobic co-culture revealed higher amylase activity and glucose concentration in comparison with the monoculture. The co-culture led to 63% higher amylase activity through the microaerobic cultivation on starch. After optimizing the conditions with response surface methodology (RSM), 10.6 g/L butanol was produced from untreated potato starch (UPS) with a high yield of 0.23 g ABE/g starch, leading to 30% improvement in ABE production. To assess its performance at larger scale, the fermentation was conducted in a 5 L fermenter continually aerated with a rate of 0.05 vvm and 150 rpm. This led to production of 9.7 g/L butanol, 5.0 g/L acetone, and 0.3 g/L ethanol with a yield and productivity of 0.20 g/g and 0.21 g/L.h, respectively. Furthermore, ABE production from tannin-containing sorghum grain was improved by about 3-fold.

Hemicellulose is a cheap and abundant substrate for biofuel production. However, industrial scale production of biofuels from hemicellulose is relatively inefficient because of expensive pretreatment processes and poor pentoses utilization by most microorganisms. In this study, a cost-effective autohydrolysis process using water as the only reagent to hydrolyze lignocellulose was exploited. Sweet sorghum stalk was also utilized as an economic source of hemicellulose. The autohydrolyzed lignocellulosic compounds from sweet sorghum were used as substrate in acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum which is able to ferment pentoses and hexoses into ABE. Separation of 79 % of hemicellulose and roughly 20 % of cellulose from sweet sorghum stalk was detected as a desirable result for autohydrolysis at 210 °C; however, over-production of inhibitors made it an inappropriate pretreatment for ABE fermentation. No butanol production was detected in autohydrolysates of 210 °C, even after using detoxification methods for inhibitory compounds removal. On the other hand, only 20 % of sweet sorghum’s bagasse hemicellulose was separated at 150 °C; yet, it was detected as the most desirable hydrolysate for ABE fermentation. Nevertheless, inherent inefficiency of C. acetobutylicum to ferment xylo-oligomers (as the sole carbon source) led to less than 1 g/L of ABE production in autohydrolysates at 150 °C. Co-fermentation of these hydrolysates with sorghum grain starch was investigated as a solution and it significantly increased the ABE production up to 8.3 g/L. Furthermore, the synergistic effect of co-fermentation was investigated where 35 % improvement in ABE production was detected. Accordingly, xylose utilization increased from 45 % to 80 %.

Treatment of polymicrobial infections requires combination therapy with drugs that have different antimicrobial spectra and possibly work in synergy. However, the different pharmacokinetics and adverse side effects challenge the simultaneous delivery of multiple drugs at the appropriate concentrations to the site of infection. Formulation of multiple drugs in nano-carrier systems may improve therapeutic efficacy by increasing the local concentration and lowering the systemic concentration, leading to fewer side effects. In this study, we loaded polymyxin B and vancomycin on bare and carboxyl-modified mesoporous silica nanoparticles (B-MSNs and C-MSNs, respectively) to achieve simulataneous local delivery of antibiotics against Gram-positive and –negative bacteria. Polymyxin B adsorbed preferentially to nanoparticles compared to vancomycin. The total antibiotic loading was 563 μg and 453 μg per mg B-MSNs or C-MSNs, respectively. Both B-MSNs and C-MSNs loaded with antibiotics were effective against Gram-negative and Gram-positive bacteria. The antibiotics had synergistic interactions against Gram-negative bacteria, and the antimicrobial efficacy was higher for antibiotic-loaded C-MSNs compared to free antibiotics at the same concentration even though the cytotoxicity was lower. Our study shows that formulations of existing antibiotics in nanocarrier systems can improve their therapeutic efficiency, indicating that combination therapy with drug-loaded silica nanoparticles may provide a better treatment outcome for infections that require high concentrations of multiple drugs.

Determining methylation state of a particular DNA sequence is an essential task in many epigenetic investigations. Here a facile method based on silver nanocluster (AgNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region that its hypermethylation is demonstrated as a reliable biomarker of colorectal cancer. Probe DNA was complementary to a 25 nucleotide of the target region and possessed twelve additional cytosines in the middle to grant the formation of AgNCs. After probe strands were hybridized with methylated and non-methylated targets separately, AgNCs were synthesized, and their fluorescence intensities were recorded. Fluorescence intensity enhanced when the target strands were methylated and quenched when they were non-methylated. The Linear range of fluorescence enhancement was from 1.0 × 10-7 M to 5.0 × 10-7 M with the detection limit of 7.6 × 10-8 M. Sensor specificity was checked with non-complementary strands with the maximum similarity of 40%. Further experiments explored various characteristics of methylated and non-methylated DNAs carrying AgNC and indicated that structure of methylated and non-methylated DNAs was affected differently by silver ions that could then influence AgNC fluorescence. This effect was strongly sequence-dependent, and either fluorescence enhancement or quenching was observed with two different sequences.

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l(-1) in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l(-1).Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

Aligned poly lactic-co-glycolic acid (PLGA) and PLGA/gelatin nanofibrous scaffolds embedded with mesoporous silica nanoparticles (MSNPs) were fabricated using electrospinning method. The mean diameters of nanofibers were 641±24 nm for the pure PLGA scaffolds vs 418±85 nm and 267±58 nm for the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively. The contact angle measurement results (102°±6.7 for the pure PLGA scaffold vs 81°±6.8 and 18°±8.7 for the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds, respectively) revealed enhanced hydrophilicity of scaffolds upon incorporation of gelatin and MSNPs. Besides, embedding the scaffolds with MSNPs resulted in improved tensile mechanical properties. Cultivation of PC12 cells on the scaffolds demonstrated that introduction of MSNPs into PLGA and PLGA/gelatin matrices leads to the improved cell attachment and proliferation as well as long cellular processes. DAPI staining results indicated that cell proliferations on the PLGA/10 wt% MSNPs and the PLGA/gelatin/10 wt% MSNPs scaffolds were strikingly (nearly 2.5 and 3 folds, respectively) higher than that on the aligned pure PLGA scaffolds. These results suggest superior properties of silica nanoparticles-incorporated PLGA/gelatin eletrospun nanofibrous scaffolds for the stem cell culture and tissue engineering applications.

Cheese whey lactose was used as a carbon source for xanthan gum production with Xanthomonas campestris and Xanthomonas pelargonii. Proteins were precipitated and removed from whey prior to fermentation. Box-Behnken response surface methodology was used for optimization of the carbon, magnesium, and phosphate source concentrations in the culture medium to maximize xanthan gum production. After 48 h of fermentation using X. campestris, the highest xanthan concentration (16.4 g/L) was achieved at 65.2 g/L of cheese whey (39.1 g/L of lactose), 14.8 g/L of phosphate (K H2PO4), and 1.1 g/L of magnesium (MgSO4·7H2O). The corresponding optimum cheese whey, phosphate, and magnesium concentrations in cultures of X. pelargonii were 80.0, 6.7, and 0.8 g/L, respectively, which resulted in a xanthan production of 12.8 g/L. The xanthan gum yield (g of xanthan/g of lactose) was 0.42 for X. campestris and 0.27 for X. pelargonii.

Production of acetone–butanol–ethanol (ABE) from potato waste starch using free and immobilized cells of Clostridium acetobutylicum was investigated. The starch was directly fermented to ABE by C. acetobutylicum without hydrolysis, and the results were compared with those obtained from glucose as a reference. Maximum butanol yields from waste starch and glucose were 0.21 and 0.26 g/g of initial carbon source (20 g/L), respectively. Batch fermentation of 60 g/L of waste starch in a 5 L bioreactor with free cells resulted in production of 9.9 g/L butanol. In a repeated batch fermentation, using immobilized cells of the bacterium in calcium alginate–polyvinyl alcohol (PVA)–boric acid beads and 60 g/L of the waste starch, a final butanol concentration of 15.3 g/L was achieved.

γ-Decalactone is an industrially important flavor compound with a peachy aroma which has been approved by FDA as a food additive. The aim of this study was to compare batch and fed-batch cultivation for production of γ-decalactone using Yarrowia lipolytica and castor oil as substrate. Microbial production of γ-decalactone from castor oil using the obligate aerobic yeast Y. lipolytica was investigated in a 3 l bioreactor. Batch and fed-batch fermentations were compared for the production of γ-decalactone. Also the effect of enhancing oxygen transfer rate by using higher agitation rates or pure oxygen for aeration was investigated. The highest γ-decalactone concentration (220 mg/l) was obtained in the fed-batch fermentation using pure oxygen which was 3-fold more compared to the batch cultivation. Using pure oxygen instead of atmospheric air in the fed-batch fermentation also resulted in 60% increase in γ-decalactone production.

A method for the controllable synthesis of silver nanoparticles based on a complexing agent method was developed. Citric acid was used as a complexing agent. The effect of pH (1.6 to 5.17) on the size and net height (as obtained from XRD analyses) of silver nanoparticles was investigated. The nanoparticles (10 to 40 nm) were characterized using XRD, TEM, SEM, EDX, UV-Vis spectroscopy and TG/DTG instrument. It was found that pH has a significant influence on both size and crystallinity of the nanoparticles. This is due to the effect of pH on the distribution of citrate ion species which in turn affects the size and crystallinity of the nanoparticles. Increasing the pH value enhances the percentage of [citrate3-] ion species which allows lower size and higher crystallinity of the nanoparticles. Thus, we were able to develop a method for the controllable synthesis of nanoparticles based on the pH.

Among epigenetic modifications of DNA, methylation of cytosine at its carbon 5 is the most common mark that is usually associated with gene silencing in human. Determining whether a particular DNA molecule is methylated or not, is an indispensable task in many epigenetic investigations. Presenting detection methods with less labor-intensive and time-consuming procedures has substantial value. Here a facile method based on gold nanocluster (AuNCs) fluorescence enhancement is presented. Target sequences were selected from Sept9 promoter region as its hypermethylation is demonstrated as a reliable biomarker of colorectal cancer. DNA probe was complementary to a 25 nucleotide of the target region and possessed 9 additional cytosines in the middle to allow the formation of AuNCs. Probe-AuNCs strands were first hybridized with methylated and non-methylated targets separately, and then their fluorescence intensities were recorded. Fluorescence intensity was higher with methylated targets than non-methylated one. Applying silver ions reversed the situation and fluorescence intensities of non-methylated DNA got higher than methylated one.

A genome-scale metabolic model was used to identify new target genes for enhanced biosynthesis of sesquiterpenes in the yeast Saccharomyces cerevisiae. The effect of gene deletions on the flux distributions in the metabolic model of S. cerevisiae was assessed using Opt Gene as the modeling framework and minimization of metabolic adjustments (MOMA) as objective function. Deletion of NADPH-dependent glutamate dehydrogenase encoded by GDH1 was identified as the best target gene for the improvement of sesquiterpene biosynthesis in yeast. Deletion of this gene enhances the available NADPH in the cytosol for other NADPH requiring enzymes, including HMG-CoA reductase. However, since disruption of GDH1 impairs the ammonia utilization, simultaneous over-expression of the NADH-dependent glutamate dehydrogenase en coded by GDH2 was also considered in this study. Deletion of GDH1 led to an approximately 85% increase in the final cubebol titer. However, deletion of this gene also caused a significant decrease in the maximum specific growth rate. Over-expression of GDH2 did not show a further effect on the final cubebol titer but this alteration significantly improved the growth rate compared to the GDH1 deleted strain. (C) 2009 Elsevier Inc. All rights reserved.

Production of xanthan gum using immobilized cells of Xanthomonas campestris and Xanthomonas pelargonii grown on glucose or hydrolyzed starch as carbon sources was investigated. Calcium alginate (CA) and calcium alginate-polyvinyl alcohol-boric acid (CA-PVA) beads were used for the immobilization of cells. Xanthan titers of 8.2 and 9.2g/L were obtained for X. campestris cells immobilized in CA-PVA beads using glucose and hydrolyzed starch, respectively, whereas those for X. pelargonii were 8 and 7.9 g/L, respectively. Immobilized cells in CA-PVA beads were successfully employed in three consecutive cycles for xanthan production without any noticeable degradation of the beads whereas the CA beads were broken after the first cycle. The results of this study suggested that immobilized cells are advantageous over the free cells for xanthan production. Also it was shown that the cells immobilized in CA-PVA beads are more efficient than cells immobilized in CA beads for xanthan production.

Metallothioneins (MTs) are a family of Cys-rich, low molecular weight, cytoplasmic metal binding proteins. MTs are present in all eukaryotes as well as some prokaryotes. Plant MTs are divided into four types based on Cys distribution pattern in their amino acid sequences. In the present work, the gene encoding OsMTI-2b, a type 2 MT found in rice, was cloned into pET41a vector. The resulting construct was transformed into Escherichia coli strain Rosetta (DE3). Following the induction with Isopropyl β-d-1-thiogalactopyranoside the OsMTI-2b was expressed as carboxyl-terminal extensions of glutathione-S-transferase (GST-tag), a 6His-tag, and an S-tag. The expressed recombinant fusion protein was named GST-OsMTI-2b. As compared with control, transgenic E. coli cells expressing GST-OsMTI-2b accumulated more Pb(2+), Ni(2+), Cd(2+), Zn(2+) and Cu(2+) from culture medium and showed increased tolerance against these metals. Furthermore the E. coli cells expressing OsMTI-2b accumulated significantly higher Pb(2+) than previously made strains which expressing other rice OsMT isoforms. The recombinant GST-OsMTI-2b was purified using affinity chromatography. According to in vitro assays the protein GST-OsMTI-2b was able to form complexes with Pb(2+), Ni(2+), Cd(2+) and Zn(2+). However, the binding ability for the different metals differed in the order: Pb(2+)>Cd(2+)>Zn(2+)>Ni(2+).

A new model for the excess Gibbs energy named UNIQUAC-NRF is presented to study the phase behavior of aqueous two-phase polymer–polymer systems. The assumptions of the NRTL-NRF model and the local composition expression of the UNIQUAC model have been used in the development of the new model. Using the random state as a reference state, a model for g E as function of the nonrandom factor has been developed for binary and multicomponent mixtures. The new model was applied for the prediction of the phase behavior of aqueous two-phase polymer–polymer systems of polyethylene glycol+dextran. The interaction parameters were obtained for 25 systems. Comparing the results of the UINQUAC and UNIQUAC-NRF models, it was shown that the present model is more accurate.

Isoprenoids represent a wide group of chemically active compounds that can find a wide range of applications as flavors, perfumes, vitamins, nutraceuticals, and pharmaceuticals. Many isoprenoids are naturally produced in very low quantities by plants, which make their use in broader perspectives difficult. Microbial production of plant-originating isoprenoids quickly appeared as an alternative of choice. Metabolic engineering methods were applied and proved successful to improve the supply of precursors to derive toward isoprenoid compounds of interest. Combinations between metabolic engineering and the flourishing field of synthetic biology have also been observed with researchers attempting to reconstruct and optimize complex biosynthetic pathways in well-characterized and tractable microbial hosts. In this chapter, we review recent metabolic engineering studies for isoprenoid production in yeast and try to show, through key examples, that the fields of synthetic biology and metabolic engineering can go hand in hand to establish microbial cell factories for isoprenoid production.

Background The need for rapid and efficient microbial cell factory design and construction are possible through the enabling technology, metabolic engineering, which is now being facilitated by systems biology approaches. Metabolic engineering is often complimented by directed evolution, where selective pressure is applied to a partially genetically engineered strain to confer a desirable phenotype. The exact genetic modification or resulting genotype that leads to the improved phenotype is often not identified or understood to enable further metabolic engineering. Results In this work we performed whole genome high-throughput sequencing and annotation can be used to identify single nucleotide polymorphisms (SNPs) between Saccharomyces cerevisiae strains S288c and CEN.PK113-7D. The yeast strain S288c was the first eukaryote sequenced, serving as the reference genome for the Saccharomyces Genome Database, while CEN.PK113-7D is a preferred laboratory strain for industrial biotechnology research. A total of 13,787 high-quality SNPs were detected between both strains (reference strain: S288c). Considering only metabolic genes (782 of 5,596 annotated genes), a total of 219 metabolism specific SNPs are distributed across 158 metabolic genes, with 85 of the SNPs being nonsynonymous (e.g., encoding amino acid modifications). Amongst metabolic SNPs detected, there was pathway enrichment in the galactose uptake pathway (GAL1, GAL10) and ergosterol biosynthetic pathway (ERG8, ERG9). Physiological characterization confirmed a strong deficiency in galactose uptake and metabolism in S288c compared to CEN.PK113-7D, and similarly, ergosterol content in CEN.PK113-7D was significantly higher in both glucose and galactose supplemented cultivations compared to S288c. Furthermore, DNA microarray profiling of S288c and CEN.PK113-7D in both glucose and galactose batch cultures did not provide a clear hypothesis for major phenotypes observed, suggesting that genotype to phenotype correlations are manifested post-transcriptionally or post-translationally either through protein concentration and/or function. Conclusions With an intensifying need for microbial cell factories that produce a wide array of target compounds, whole genome high-throughput sequencing and annotation for SNP detection can aid in better reducing and defining the metabolic landscape. This work demonstrates direct correlations between genotype and phenotype that provides clear and high-probability of success metabolic engineering targets. The genome sequence, annotation, and a SNP viewer of CEN.PK113-7D are deposited at http://www.sysbio.se/cenpk.

The mevalonate pathway in the yeast Saccharomyces cerevisiae was deregulated in order to enhance the intracellular pool of farnesyl diphosphate (FPP), the direct precursor for the biosynthesis of sesquiterpenes. Over-expression of the catalytic domain of HMG1, both from the genome and plasmid, resulted in higher production of cubebol, a plant originating sesquiterpene, and increased squalene accumulation. Down-regulation of ERG9 by replacing its native promoter with the regulatable MET3 promoter, enhanced cubebol titers but simultaneous over-expression of tHMG1 and repression of ERG9 did not further improve cubebol production. Furtheremore, the concentrations of squalene and ergosterol were measured in the engineered strains. Unexpectedly, significant accumulation of squalene and restoring the ergosterol biosynthesis were observed in the ERG9 repressed strains transformed with the plasmids harboring cubebol synthase gene. This could be explained by a toxicity effect of cubebol, possibly resulting in higher transcription levels for the genes under control of MET3 promoter, which could lead to accumulation of squalene and ergosterol.

The yeast Saccharomyces cerevisiae was chosen as a microbial host for heterologous biosynthesis of three different plant sesquiterpenes, namely valencene, cubebol, and patchoulol. The volatility and low solubility of the sesquiterpenes were major practical problems for quantification of the excreted sesquiterpenes. In situ separation of sesquiterpenes in a two-phase fermentation using dodecane as the secondary phase was therefore performed in order to enable quantitative evaluation of different strains. In order to enhance the availability of the precursor for synthesis of sesquiterpenes, farnesyl diphosphate (FPP), the ERG9 gene which is responsible for conversion of FPP to squalene was downregulated by replacing the native ERG9 promoter with the regulatable MET3 promoter combined with addition of 2 mM methionine to the medium. This strategy led to a reduced ergosterol content of the cells and accumulation of FPP derived compounds like target sesquiterpenes and farnesol. Adjustment of the methionine level during fermentations prevented relieving MET3 promoter repression and resulted in further improved sesquiterpene production. Thus, the final titer of patchoulol and farnesol in the ERG9 downregulated strain reached 16.9 and 20.2 mg/L, respectively. The results obtained in this study revealed the great potential of yeast as a cell factory for production of sesquiterpenes.

Saving energy, cost efficiency, producing less waste, improving the biodegradability of products, potential for producing novel and complex molecules with improved properties, and reducing the dependency on fossil fuels as raw materials are the main advantages of using biotechnological processes to produce chemicals. Such processes are often referred to as green chemistry or white biotechnology. Metabolic engineering, which permits the rational design of cell factories using directed genetic modifications, is an indispensable strategy for expanding green chemistry. In this chapter, the benefits of using metabolic engineering approaches for the development of green chemistry are illustrated by the recent advances in microbial production of isoprenoids, a diverse and important group of natural compounds with numerous existing and potential commercial applications. Accumulated knowledge on the metabolic pathways leading to the synthesis of the principal precursors of isoprenoids is reviewed, and recent investigations into isoprenoid production using engineered cell factories are described.