Glycine is extensively applied in the field of food, medicine, agrochemistry, etc. It is usually commercially produced by the chemosynthesis method, which generates large amounts of by-product ammonium chloride.
In this paper, the separation of glycine and ammonium chloride was performed with chromatographic column, and deionized water as eluent. The adsorption equilibrium constant K of glycine and ammonium chloride was evaluated by frontal analysis. Based on the equilibrium-dispersive model and a linear driving force of chromatography, the overall mass transfer coefficient km, axial dispersion coefficient DL, and bed voidage εt of the column were obtained by moment analysis.
At 50°C, the equilibrium constants measured were found to be 0.72 and 0.19 for glycine and ammonium chloride, respectively. At 60°C, the equilibrium constants increased to 0.80 and 0.21 for glycine and ammonium chloride, respectively. The value of axial dispersion coefficient DL of glycine had the same order of magnitude with ammonium chloride and was about two times larger than that of ammonium chloride. Their km at 50°C and 60°C were 1.30 and 0.77 and 2.41 and 0.84 min−1 for glycine and ammonium chloride, respectively.
The obtained parameters used to simulate the elution curve and the simulation and experimental results matched well, which showed that the parameters obtained were effective. The results make foundation for further study on large-scale separation of glycine from ammonium chloride by SMB chromatography.
1,3-Propanediol is the starting point of a new-generation polymer with superior properties which can be used in many industrial fields. 3-Hydroxypropionaldehyde and lactate have been identified as two important metabolites in the biological route of 1,3-propanediol bioconversion from glycerol. Here, influence of lactate on the inhibition caused by 3-hydroxypropionaldehyde of 1,3-propanediol fermentation by Klebsiella pneumoniae is reported.
The influences of 3-hydroxypropionaldehyde and lactate on 1,3-propanediol production were investigated in normal and lactate pathway deficient strains with different fermentation conditions.
By using the strains KG1 and L-type lactate dehydrogenase-deficient mutant (KG1Δldh), the results indicated that an early accumulation of 3-hydroxypropionaldehyde directly inhibited the 1,3-propanediol production rather than through lactate accumulation during the late stage of fermentation. Then, the influence of extra addition of lactate on the late stage of fermentation was investigated, and the inhibitory effect of lactate did not appear. At last, it was found that by reducing 3-hydroxypropionaldehyde accumulation in the early stage of fermentation, the concentration and yield of 1,3-propanediol increased by 18% and 16%, respectively, over the initial experimental levels.
An early accumulation of 3-hydroxypropionaldehyde directly decreased the final 1,3-propanediol concentration rather than through lactate accumulation during the late stage of fermentation.
In recent years, green synthesis of silver nanoparticles (AgNPs) has gained much interest from chemists and researchers. In this concern, Indian flora has yet to divulge innumerable sources of cost-effective non-hazardous reducing and stabilizing compounds utilized in preparing AgNPs. This study investigates an efficient and sustainable route of AgNP preparation from 1 mM aqueous AgNO3 using leaf extracts of three plants, Musa balbisiana (banana), Azadirachta indica (neem) and Ocimum tenuiflorum (black tulsi), well adorned for their wide availability and medicinal property.
AgNPs were prepared by the reaction of 1 mM silver nitrate and 5% leaf extract of each type of plant separately. the AgNPs were duely characterized and tested for their antibacterial activity and toxicity.
The AgNPs were characterized by UV-visible (vis) spectrophotometer, particle size analyzer (DLS), scanning electron microscopy (SEM), transmission electron microscopy (TEM) and energy-dispersive spectroscopy (EDS). Fourier transform infrared spectrometer (FTIR) analysis was carried out to determine the nature of the capping agents in each of these leaf extracts. AgNPs obtained showed significantly higher antimicrobial activities against Escherichia coli (E. coli) and Bacillus sp. in comparison to both AgNO3 and raw plant extracts. Additionally, a toxicity evaluation of these AgNP containing solutions was carried out on seeds of Moong Bean (Vigna radiata) and Chickpea (Cicer arietinum). Results showed that seeds treated with AgNP solutions exhibited better rates of germination and oxidative stress enzyme activity nearing control levels, though detailed mechanism of uptake and translocation are yet to be analyzed.
In totality, the AgNPs prepared are safe to be discharged in the environment and possibly utilized in processes of pollution remediation. AgNPs may also be efficiently utilized in agricultural research to obtain better health of crop plants as shown by our study.
Hydrolysis of 2-amino phenylpropionitrile by nitrilase is a fundamental biochemical reaction that produces chiral phenylalanine. For practical application of this biochemical reaction, researchers have attempted to improve enzyme enantioselectivity and the reaction rate.
The substrate concentration was increased from 100 to 200 mM without substrate inhibition because of the formation of a substrate-hydroxypropyl-β-cyclodextrin (HP-β-CD) complex. Meanwhile, the activity of recombinant nitrilase increased 2.5 times because the addition of HP-β-CD solubilized hydrophobic substrates in the aqueous system. Furthermore, the formation of the substrate-HP-β-CD inclusion improved the enantioselectivity of the enzymatic reaction toward producing l-phenylalanine (l-Phe). The enantiomeric excess (e.e.) value of l-Phe increased from 65% to 83% when the conversion rate reached 50%.
The recombinant nitrilase enantioselectively hydrolyzed 2-amino phenylpropionitrile to produce l-Phe. The addition of HP-β-CD to the reaction system enhanced the solubility and bioavailability of hydrophobic substrates as well as the enantioselectivity. The results showed that this additive has potential advantages in biochemical reactions of hydrophobic substrates, particularly for enantioselective biosynthesis.
Ursodeoxycholic acid is an important clinical drug in the treatment of liver disease. In our previous work, ursodeoxycholic acid was prepared by electroreduction of 7-ketolithocholic acid. The separation of ursodeoxycholic acid from the electroreduction product (47% (w/w) ursodeoxycholic acid) by silylation crystallization is described herein.
N,N-dimethylformamide was used as the solvent, whereas hexamethyldisilazane was the reaction agent. The optimal material ratio of electroreduction product/N,N-dimethylformamide/hexamethyldisilazane was found to be 1:10:2 (w/v/v). The reaction proceeded for 2 h at 60°C, and the corresponding silylation derivative was separated by crystallization and pure ursodeoxycholic acid was recovered by 5% acid hydrolysis at 50°C for 0.5 h. The maximum recovery and purity of ursodeoxycholic acid were 99.8% and 99.5%, respectively.
Ursodeoxycholic acid with high purity and high recovery can be prepared directly. The developed method offers a potential application for large-scale production of ursodeoxycholic acid.
d-Galactose dehydrogenase (GalDH; EC 1.1.1.48) belongs to the family of oxidoreductases that catalyzes the reaction of β-d-galactopyranose in the presence of NAD+ to d-galacto-1,5-lactone and NADH. The enzyme has been used in diagnostic kits to neonatal screen for galactosemia diseases. This article reports the partitioning optimization of recombinant Pseudomonas fluorescens GalDH in aqueous two-phase systems (ATPS).
Preliminary two-phase experiments exhibited that the polyethylene glycol (PEG) concentration, pH value, and concentration of salt had a significant influence on the partitioning efficiency of recombinant enzyme. According to these data, response surface methodology (RSM) with a central composite rotatable design (CCRD) was performed to condition optimization.
The optimal partition conditions were found using the 14.33% PEG-4000 and 11.79% ammonium sulfate with pH 7.48 at 25°C. Yield, purity, recovery, and specific activity were achieved 92.8%, 58.9, 268.75%, and 373.9 U/mg, respectively. PEG and ammonium sulfate concentration as well as pH indicated to have a significant effect on GalDH partitioning. Enzyme activity assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated the suitability of predicted optimal ATPS as well. The Km and molecular weight values for the purified GalDH were 0.32 mM and 34 kDa, respectively.
Ultimately, our data showed the feasibility of using ATPS for partitioning and recovery of recombinant GalDH enzyme.
Biotransformation of quinic acid to shikimic acid was attempted using whole cells of Bacillus megaterium as a biocatalyst.
Physico-chemical parameters such as temperature (37°C), pH (7.0), agitation (200 rpm), substrate (5 mM) and cell mass concentrations (200 kg/m 3) and reaction time (3 h) were found optimum to enhance the bioconversion. Maximum conversion (89%) of quinic acid to shikimic acid was achieved using the above optimized parameters. Shikimic acid was extracted from the reaction mixture by a pH-dependent method and maximum recovery (76%) was obtained with petroleum ether.
Biotransformation of quinic acid to shikimic acid seems to be a better alternative over its fermentative production.
Proteases from bacteria are among the most important hydrolytic enzymes that have been studied due to their extracellular nature and high yield of production. Of these, alkaline proteases have potential for application in detergent, leather, food, and pharmaceutical industries. However, their usefulness in industry is limited by low activity and stability at high temperatures, extreme pH, presence of organic solvents and detergent ingredients. It is therefore very crucial to search for new alkaline proteases with novel properties from a variety of microbial sources.
In the present study, 21 Bacillus species isolated from organic waste sites were screened for proteolytic activity on casein agar. Bacillus brevis MWB-01 exhibited highest proteolytic activity with a clear zone diameter of 35 mm. Production of protease from B. brevis MWB-01 was investigated in optimized media after 48 h of cultivation with shaking (180 rpm) at 37°C. The protease was partially purified in a two-step procedure using ammonium sulphate precipitation and gel filtration chromatography on Sephadex G-200 column. The enzyme was purified 2.1-fold with yield of 4.6%. The purified protease had optimum temperature of 40°C with relative activity of about 50% at 50°C and was uniquely stable up to 60°C after 30 min of incubation exhibiting 63% residual activity. The enzyme had optimum pH of 8.0 and remarkably showed relative activity above 70% at pH 9.0 to 11.0 and 53% at pH 12.0, respectively and was very stable over a wide pH range (6.0 to 12.0). Ca2+ and Mn2+ increased protease activity with 9.8% and 3.5%, respectively; Hg2+ and Zn2+ strongly inhibited protease activity by 89% and 86%. The almost complete inhibition of the enzyme by phenylmethylsulphonyl fluoride (PMSF) and ethylene diamine tetra acetic acid (EDTA) confirmed the enzyme as a serine metalloprotease. The enzyme had highest compatibility with Sunlight, a commercial laundry detergent.
The characteristics of purified protease from B. brevis MWB-01 reveal the enzyme as a thermotolerant serine alkaline metalloprotease compatible with detergent formulation aids. Results suggest that protease from B. brevis MWB-01 is a good bioresource for industrial applications.
In the present study, we aim to utilize the ecological diversity of soil for the isolation and screening for poly β-hydroxybutyrate (PHB)-accumulating bacteria and production of cost-effective bioplastic using cardboard industry effluent.
A total of 120 isolates were isolated from different soil samples and a total of 62 isolates showed positive results with Nile blue A staining, a specific dye for PHB granules and 27 isolates produced PHB using cardboard industry effluent. The selected isolate NA10 was identified as Bacillus sp. NA10 by studying its morphological, biochemical, and molecular characteristics. The growth pattern for the microorganism was studied by logistic model and exactly fitted in the model. A maximum cell dry weight (CDW) of 7.8 g l−1 with a PHB concentration of 5.202 g l−1 was obtained when batch cultivation was conducted at 37°C for 72 h, and the PHB content was up to 66.6% and productivity was 0.072 g l−1 h−1 in 2.0 L fermentor. Chemical characterization of the extracted PHB was done by H1NMR, Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), Gas chromatography–mass spectrometry (GC-MS) analysis to determine the structure, melting point, and molecular mass of the purified PHB. The polymer sheet of extracted polymer was prepared by blending the polymer with starch for packaging applications.
The isolate NA10 can be a good candidate for industrial production of PHB from cardboard industry waste water cost-effectively and ecofriendly.
Limonene is an important monoterpene used as a chemical commodity and precursor for producing biofuels, flavor and medicinal compounds.
In this paper, we engineered Escherichia coli by embedding two exogenous genes encoding a limonene synthase (LS) and a geranyl diphosphate synthase (GPPS) for production of limonene. Out of 12 E. coli strains transformed with various plasmids, the best one with p15T7-ls-gpps produced limonene with a titer of 4.87 mg/L. In order to enhance the limonene production, two rate-limiting enzymes in the endogenous MEP pathway of E. coli, 1-deoxy-xylulose-5-phosphate synthase (DXS) and isopentenyl diphosphate isomerase (IDI), were overexpressed consecutively on vector pET21a+, resulting in a production of 17.4 mglimonene/L at 48 h.
After the preliminary optimization of the medium in a two-phase culture system composed of n-hexadecane (1/50, Vorg/Vaq), the final production of limonene was raised up to 35.8 mg/L, representing approximately a 7-fold improvement compared to the initial titer.
Novozym 435, a commercial lipase from Candida antarctica, recombinant, expressed in Aspergillus niger, immobilized on macroporous acrylic resin, has been already described in the obtention of biodiesel. It is here evaluated in the production of a new biofuel that integrates the glycerol as monoglyceride (MG) together with two fatty acid ethyl esters (FAEE) molecules by the application of 1,3-selective lipases in the ethanolysis reaction of sunflower oil.
Response surface methodology (RSM) is employed to estimate the effects of main reaction. Optimum conditions for the viscosity, selectivity, and conversion were determined using a multifactorial design of experiments with three factors run by the software Stat Graphics version XV.I. The selected experimental parameters were reaction temperature, oil/ethanol ratio and alkaline environment. On the basis of RSM analysis, the optimum conditions for synthesis were 1/6 oil/EtOH molar ratio, 30°C, and 12.5 μl of NaOH 10 N aqueous solutions, higher stirring than 300 rpm, for 2 h and 0.5 g of biocatalyst.
These obtained results have proven a very good efficiency of the biocatalyst in the studied selective process. Furthermore, it was allowed sixteen times the successive reuse of the biocatalyst with good performance.
levo-Menthol is an important flavoring chemical, which can be prepared by enantioselective enzymatic hydrolysis of dl-menthyl esters. A recombinant esterase (BsE) cloned from Bacillus subtilis 0554 shows excellent enantioselectivity to dl-menthyl acetate and has been immobilized using cross-linked enzyme aggregates. Though BsE has relatively high substrate tolerance, the conversion of dl-menthyl acetate decreased sharply with the increase of substrate loading from 1 to 3 M in mono-aqueous system, which might be due to the severe inhibition of enzyme activity at extremely high load of substrate or product. In this work, enzymatic hydrolysis of dl-menthyl acetate with an extremely high load using the immobilized CLEA-BsE was investigated in an organic-aqueous biphasic system containing surfactant to establish a promising bioprocess for large-scale production of l-menthol.
An efficient biphasic reaction system of pentanol-water containing sodium dodecyl sulfate (SDS) was developed for improving enantioselective hydrolysis of dl-menthyl acetate to produce l-menthol by immobilized BsE. Under the optimized reaction conditions, l-menthol was produced in >97% enantiomeric excess (ee) at a substrate load of up to 3.0 M with >40% conversion.
All the positive features demonstrate the potential applicability of the bioprocess for the large-scale production of l-menthol.
In acetone-butanol-ethanol (ABE) fermentation by Clostridium acetobutylicum ATCC 824 using corn-based substrate, the solvents are generally produced at a ratio of 3:6:1 (A:B:E, w/w).
A higher butanol/acetone ratio of 2.9:1 was found when cassava was used as the substrate of an in-situ extractive fermentation by C. acetobutylicum. This ratio had a 64% increment compared to that on corn-based substrate. The metabolic flux and (key enzymes) genes transcriptional analysis indicated that weakened metabolic fluxes in organic acids (especially butyrate) formation and re-assimilation pathways, which associated with lower buk and ctfAB transcriptional levels, contributed to higher butanol and lower acetone production rate in fermentations on cassava. Moreover, NADH generation was enhanced under the enriched reductive environment of using cassava-based substrate, which converted more carbon flux to butanol synthesis pathway, also leading to a higher ratio of butanol/acetone. To further increase butanol/acetone ratio, tiny amount of electron carrier, neutral red was supplemented into cassava-based substrate at 60 h when butonal production rate reached maximal level. However, neutral red addition enhanced NADH production, followed with strengthening the metabolic fluxes of organic acids formation/re-assimilation pathways, resulted in unchanged in butanol/acetone ratio.
A further increase in butanol/acetone ratio could be realized when NADH regeneration was enhanced and the metabolic fluxes in organic acids formation/reutilization routes were controlled at suitably low levels simultaneously.
Protopectinases which were consisted of various different enzymes can promote the solubilization of protopectin from the plant cell and can be applied in the protein industry extraction. The genome sequence of Paenibacillus polymyxa Z6 that produces a protopectinases complex was partially determined. Two new genes, yxiA1 and yxiA3, were identified as uncharacterized protein in the P. polymyxa genome. And, they were classified as the member of the glycoside hydrolase family 43 (GH43) according to the primary protein sequence.
The two genes were cloned and expressed in Escherichia coli BL21 (DE3). And, the results indicated that the product of yxiA1 and yxiA3 were two endo-α-1,5-l-arabinanases. Thus, the two genes were renamed as abnZ2 (yxiA1) and abnZ3 (yxiA3). Recombinant AbnZ2 had optimal activity at pH 6.0 and 35°C. And, AbnZ3 had optimal activity at pH 6.0 and 30°C. However, unlike most reported endo-arabinanases, the specific activity of AbnZ3 remained 48.7% of maximum at 5°C, which meant AbnZ3 was an excellent cold-adapted enzyme.
This paper demonstrated that the gene yxiA1 and yxiA3 were two new endo-arabinanases, and renamed as abnZ2 and abnZ3. Moreover AbnZ3 was an excellent cold-adapted enzyme which could be attractive in fruit juice processing.
Erythromycin production often has concern with the consumption rate of amino nitrogen and phosphate, especially in the early fermentation phase. The dynamic regulation of nitrogen and phosphorus was put forward based on the comprehensive analysis of the contents of phosphorus and nitrogen in different nitrogen sources as well as the relations between nitrogen consumption and phosphorus consumption.
Firstly, the unstable nitrogen source, corn steep liquor, was substituted with the stable nitrogen source, yeast powder, with little effects on erythromycin production. Secondly, feeding phosphate in the early fermentation stage accelerated the consumption of amino nitrogen and ultimately increased erythromycin production by approximately 24% as compared with the control (without feeding potassium dihydrogen phosphate). Thirdly, feeding phosphate strategy successfully applied to 500 L fermenter with the final erythromycin concentration of 11839 U/mL, which was 17.3% higher than that of the control. Finally, the application of condensed soy protein (a cheap nitrogen source with low phosphorus content) combined with phosphate feed strategy led to a 13.0% increase of the erythromycin production as compared with the control (condensed soy protein, without feeding potassium dihydrogen phosphate).
Appropriately feeding phosphate combined with rational nitrogen regulation in the early fermentation phase was an effective way to improve erythromycin production.
An economical and integrated high-throughput primary screening strategy was developed for high-aerobic microbe Monascus purpureus cultivation. A novel and effective mixture culture method was proposed and used to realize the whole mutant library being high-throughput screened after mutagenesis.
The good correlation of fermentation results between differing-scale cultivations confirmed the feasibility of utilizing the 48-deep microtiter plates (MTPs) as a scale-down tool for culturing high-aerobic microbes. In addition, the fluid dynamics of 24-, 48-, and 96-deep MTPs and 500-mL shake flask were studied respectively using the computational fluid dynamic (CFD) tool ANSYS CFX 11.0 to get better understanding of their turbulent regimes.
The by-product citrinin production had no significant change while the pigment production had improved. As a result, the high-yield strain T33-6 was successfully screened out and the pigment was more than 50% higher than that of the parental strain in the shake flask.
Many short peptides have proved to exhibit potential anti-hypertensive activity through the inhibition of the Angiotensin I-converting enzyme (ACE) activity and the regulation of blood pressure. However, the traditional experimental screening method for ACE inhibitory peptides is time consuming and costly, accompanied with the limitations as incomplete hydrolysis and peptides loss during purification process. Virtual methods with the aid of computer can break such bottle-neck of experimental work. In this study, an attempt was made to establish a library of di- and tri-peptides derived from proteins of Phascolosoma esculenta, a kind of seafood, through BIOPEP (http://www.uwm.edu.pl/biochemia/index.php/pl/biopep), and to screen highly active ACE inhibitory peptides by molecular docking with the help of LibDock module of Discovery Studio 3.5 software.
Two hundred and eighty four (284) di- and tri-peptides, derived from P. esculenta proteins after a virtual hydrolysis with pepsin, trypsin and a mixture of pepsin and trypsin, were predicted to possess ACE inhibitory activity, among which there are 99 ACE inhibitory peptides with estimated IC50 less than 50 μM. Nine peptides were synthesized for the comparison between the estimated and the experimentally determined IC50. The results indicated that errors between the estimated and measured log(1/IC50) are all less than 1.0 unit.
Virtual method for peptide library construction and ACE inhibitory peptides screening efficiently demonstrated that P. esculenta proteins are prospect resource for food-origin ACE inhibitory peptide.
In vitro reconstitution of an artificial metabolic pathway has emerged as an alternative approach to conventional in vivo fermentation-based bioproduction. Particularly, employment of thermophilic and hyperthermophilic enzymes enables us a simple preparation of highly stable and selective biocatalytic modules and the construction of in vitro metabolic pathways with an excellent operational stability. In this study, we designed and constructed an artificial in vitro metabolic pathway consisting of nine (hyper)thermophilic enzymes and applied it to the conversion of glycerol to lactate. We also assessed the compatibility of the in vitro bioconversion system with methanol, which is a major impurity in crude glycerol released from biodiesel production processes.
The in vitro artificial pathway was designed to balance the intrapathway consumption and regeneration of energy and redox cofactors. All enzymes involved in the in vitro pathway exhibited an acceptable level of stability at high temperature (60°C), and their stability was not markedly affected by the co-existing of up to 100 mM methanol. The one-pot conversion of glycerol to lactate through the in vitro pathway could be achieved in an almost stoichiometric manner, and 14.7 mM lactate could be produced in 7 h. Furthermore, the in vitro bioconversion system exerted almost identical performance in the presence of methanol.
Many thermophilic enzymes exhibit higher stability not only at high temperatures but also in the presence of denaturants such as detergents and organic solvents than their mesophilic counterparts. In this study, compatibilities of thermophilic enzymes with methanol were demonstrated, indicating the potential applicability of in vitro bioconversion systems with thermophilic enzymes in the conversion of crude glycerol to value-added chemicals.
This paper deals with the production of biodiesel from the brown seaweed Sargassum myriocystum, a third-generation biodiesel from the Gulf of Mannar, Rameshwaram, India. The optimization of reaction parameters was done using Design-Expert software version 8.0.7.1. Algal oil was transesterified using methanol and sodium hydroxide. The effect of oil:alcohol ratio, catalyst amount, temperature, and time on biodiesel yield was investigated by response surface methodology using central composite design.
It was found that the maximum biodiesel yield was obtained at 60°C for 1:6 (v/v) oil:alcohol ratio, 0.4 (w/w) catalyst amount, and 120 min. The R 2, adjusted R 2, and predicted R 2 values are 0.9977, 0.9956, and 0.9923, respectively, which implies that experimental values are in good agreement with predicted values. The fatty acid profile of S. myriocystum biodiesel was determined using gas chromatography. Algal biodiesel was stored in dark and light conditions. Fuel properties like kinematic viscosity and acid value were determined. It was found that the samples exposed to light led to an increase in kinematic viscosity and acid value with some sediment formation.
The acid value and kinematic viscosity of the samples stored in the dark environment had only marginal increase in fuel properties which were within the range specified by the American Society of Testing Materials (ASTM D6751).
Chitooligosaccharides (COS) with degrees of polymerization (DP) 6 to 8 are degraded from chitosan, which possess excellent bioactivities. However, technologies that could purify them from hydrolysis mixtures in the narrow DP range (984 to 1,306 Da) are absent. The objective of this research is to purify DP 6 to 8 COS by nanofiltration on the basis of appropriate adjustments of the feed condition.
Syrup containing DP 6 to 8 COS at different concentrations (19.0 to 46.7 g/L) was prepared. A commercial membrane (QY-5-NF-1812) negatively charged was applied. Experiments were carried out in full recycle mode, so that the observed COS retentions were investigated at various transmembrane pressures (6.0 to 20.0 bar), temperatures (10°C to 50°C), and pHs (5.0 to 9.0). Then, the feasibility of separation of DP 6 to 8 COS was further studied by concentration ratio under optimum conditions.
The results indicate that the purification of DP 6 to 8 COS by nanofiltration NF is feasible. It was found that the permeate flux was 95.0 L/(m2 h) at 10.0 bar, while it reached to 140.0 L/(m2 h) at 20.0 bar, and it increased with feed temperature, but the membrane pores were also swelled by heating and led to an irreversible wastage of target oligomers. Additionally, the retention behaviors of chitooligosaccharides are significantly influenced by pH.
Although glucosamine and dimer were permeatable at low pH, their retention ratios were remarkably varied from 0.458 to 0.864 when pH was 9.0. With the interaction of hydrogen bonds, structural curling and overlapping of chitooligosaccharides were formed. Consequently, the rejection of chitooligosaccharides at various pHs is variable. Spray-dried products were finally characterized by the matrix-assisted laser desorption/ionization time-of-flight mass spectrum. The spectrum identified the distributions of hexamer, heptamer, and octamer. Combined with high-performance liquid chromatography profiles, the purity and yield of DP 6 to 8 chitooligosaccharides were up to 82.2% and 73.9%, respectively.
The melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) can induce apoptosis in a wide variety of tumor cell types, whereas it has no toxicity in normal cells. However, recombinant human mda-7/IL-24 is difficult to obtain from Escherichia coli because of its insolubility.
In this study, we improved the structure of inclusion bodies (IBs) by optimizing the induction temperature, pH, concentrations of inducer, and metal ion additives. Statistically designed experimental analyses of three metal ion factors were performed using the Box-Behnken design. Induction temperature of 30°C, pH 7.0, and 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) were selected, and the optimized levels for the factors predicted by the model comprised the following: Mg2+ (15.7 mM), Ca2+ (16.6 mM), and Mn2+ (3.0 mM). The optimized culture conditions improved the structure of the IBs, which was validated by scanning electron microscopy (SEM) and the increase of IBs solubility.
After optimization, IB solubility and renatured mda-7/IL-24 increased by 51% and 84%, respectively. This study also provided a simple purification method of specific IB washing steps. Manipulating the fermentation parameters to optimize the refolding and purification process is likely to be widely applicable to other proteins.
The energy crisis and climate change necessitate studying and discovering of new processes involved in the production of alternative and renewable energy sources. Very high gravity (VHG) fermentation is one such process improvement aimed at increasing both the rate of fermentation and ethanol concentration. The technology involves preparation and fermentation of media containing 300 g or more of dissolved solids per liter to get a high amount of ethanol.
Saccharomyces cerevisiae was inoculated to the very high gravity medium containing 30% to 40% w/v glucose with and without supplementation of three selected fruit pulps (mango, banana, and sapota). The fermentation experiments were carried out in batch mode. The effect of supplementation of 4% fruit pulp/puree on the metabolic behavior and viability of yeast was studied. Significant increase in ethanol yields up to 83.1% and dramatic decrease in glycerol up to 35% and trehalose production up to 100% were observed in the presence of fruit pulp. The fermentation rate was increased, and time to produce maximum ethanol was decreased from 5 to 3 days with increased viable cell count. The physical and chemical factors of fruit pulps may aid in reducing the osmotic stress of high gravity fermentation as well as enhanced ethanol yield.
It was found that fruit pulp supplementation not only reduced fermentation time but also enhanced ethanol production by better utilization of sugar. Production of high ethanol concentration by the supplementation of cheap materials in VHG sugar fermentation will eliminate the expensive steps in the conventional process and save time.
Cotton gin trash (CGT) is a lignocellulosic residue that can be used in the production of cellulosic ethanol. In a previous research, the sequential use of ultrasonication, liquid hot water, and ligninolytic enzymes was selected as pretreatment for the production of ethanol from CGT. However, an increment in the ethanol production is necessary. To accomplish that, this research evaluated the effect of pretreating CGT using alkaline ultrasonication before a liquid hot water and ligninolytic enzymes pretreatments for ethanol production. Three NaOH concentrations (5%, 10%, and 15%) were employed for the alkaline ultrasonication. Additionally, this work is one of the first applications of Fourier transform infrared (FT-IR) spectrum and principal component analysis (PCA) as fast methodology to identify the differences in the biomass after different types of pretreatments.
The three concentrations employed for the alkaline ultrasonication pretreatment produced ethanol yields and cellulose conversions higher than the experiment without NaOH. Furthermore, 15% NaOH concentration achieved twofold increment yield versus the treatment without NaOH. The FT-IR spectrum confirmed modifications in the CGT structure in the different pretreatments. PCA was helpful to determine differences between the pretreated and un-pretreated biomass and to evaluate how the CGT structure changed after each treatment.
The combination of alkali ultrasonication hydrolysis, liquid hot water, and ligninolytic enzymes using 15% of NaOH improved 35% the ethanol yield compared with the original treatment. Additionally, we demonstrated the use of PCA to identify the modifications in the biomass structure after different types of pretreatments and conditions.
Synthetic biology is a newly emerged research discipline that focuses on the engineering of novel cellular behaviors and functionalities through the creation of artificial gene circuits. One important class of synthetic circuits currently under active development concerns the programming of bacterial cellular communication and collective population-scale behaviors. Because of the ubiquity of cell-cell interactions within bacterial communities, having an ability of engineering these circuits is vital to programming robust cellular behaviors. Here, we highlight recent advances in communication-based synthetic gene circuits by first discussing natural communication systems and then surveying various functional engineered circuits, including those for population density control, temporal synchronization, spatial organization, and ecosystem formation. We conclude by summarizing recent advances, outlining existing challenges, and discussing potential applications and future opportunities.
Second-generation feedstocks such as lignocellulosic hydrolysates are more and more in the focus of sustainable biotechnological processes. Corynebacterium glutamicum, which is used in industrial amino acid production at a million-ton scale, has been engineered towards utilization of alternative carbon sources. As for other microorganisms, the focus has been set on the pentose sugars present in lignocellulosic hydrolysates. Utilization of the hexuronic acids D-galacturonic acid (abundant in pectin-rich waste streams such as peels and pulps) and D-glucuronic acid (a component of the side-chains of plant xylans) for growth and production with C. glutamicum has not yet been studied.
Neither aldohexuronic acid supported growth of C. glutamicum as sole or combined carbon source, although its genome encodes a putative uronate isomerase sharing 28% identical amino acids with UxaC from Escherichia coli. Heterologous expression of the genes for both uptake and catabolism of D-galacturonic acid and D-glucuronic acid was required to enable growth of C. glutamicum with either aldohexuronic acid as the sole carbon source. When present in mixtures with glucose, the recombinant C. glutamicum strains co-utilized D-galacturonate with glucose and D-glucuronate with glucose, respectively. When transformed with the plasmid for uptake and catabolism of the aldohexuronates, model producer strains were able to grow with and produce from D-galacturonate or D-glucuronate as sole carbon source.
An easily transferable metabolic engineering strategy for access of C. glutamicum to aldohexuronates was developed and applied to growth and production of the amino acids L-lysine and L-ornithine as well as the terpene lycopene from D-galacturonate or D-glucuronate.
The concept of high-rate vermicomposting was successfully used to achieve direct vermicomposting of the aquatic weed salvinia - without any precomposting or cow dung supplementation as previously reported processes for the vermicomposting of phytomass had necessitated.
Both the epigeic species of earthworms that were explored, Eudrilus eugeniae and Eisenia fetida, provided efficient vermicast production with no mortality, persistent gain in body mass, and good fecundity over the 270-day-long course of the reactor operation. In this period, all reactors were pulse-fed at the solid retention time of 15 days and were operated in the pseudo-discretized continuous operation protocol developed earlier by the authors. With this, it was possible to almost completely dampen the influence of natural biodegradation of the feed or grazing by the earthworm born in the vermireactors. This has made it possible to link vermicast production directly to the ability of the earthworm to feed upon, and digest, salvinia. In turn, this enables accurate process monitoring and provides clear pointers on how to improve process efficiency.
The paper establishes the capability of high-rate vermicomposting technology developed earlier by the authors in direct and efficient vermicomposting of salvinia without any precomposting or manure supplementation. The findings have very significant implications in improving process economics and consequently process utility. No previous report exists in primary literature on the vermicomposting of salvinia.
The high rate of propagation and easy availability of water hyacinth has made it a renewable carbon source for biofuel production. The present study was undertaken to screen the feasibility of using water hyacinth's hemicelluloses as a substrate for alcohol production by microbial fermentation using mono and co-cultures of Trichoderma reesei and Fusarium oxysporum with Pichia stipitis.
In separate hydrolysis and fermentation (SHF), the alkali pretreated water hyacinth biomass was saccharified by crude fungal enzymes of T. reesei, F. oxysporum and then fermented by P. stipitis. In simultaneous saccharification and fermentation (SSF), the saccharification and fermentation was carried out simultaneously at optimized conditions using mono and co-cultures of selected fungal strains. Finally, the ethanol production kinetics were analyzed by appropriate methods. The higher crystalline index (66.7%) and the Fourier transform infrared (FTIR) spectra showed that the lime pretreatment possibly increased the availability of cellulose and hemicelluloses for enzymatic conversion. In SSF, the co-culture fermentation using T. reesei and P. stipitis was found to be promising with a higher yield of ethanol (0.411 g g−1) at 60 h. The additional yield comparable with the monocultures was due to the xylanolytic activity of P. stipitis which ferments pentose sugars into ethanol. In SHF, the pretreatment followed by crude enzymatic hydrolysis and fermentation resulted in a significantly lesser yield of ethanol (0.344 g g−1) at 96 h.
It is evident from the study that the higher ethanol production was attained in a shorter period in the co-culture system containing T. reesei and the xylose fermenting yeast P. stipitis. SSF of pretreated water hyacinth biomass (WHB) with P. stipitis instead of traditional yeast is found to be an effective biofuel production process.
Biopalladium (bioPd(0)) nanoparticles on Klebsiella Pneumoniae ECU-15 were synthesized mainly on the microorganism's surface. Data suggest that the resistance of mass transfer around the cell surface region plays a critical role in bioPd(0) synthesis process. However, the mechanisms for its role remains elusive.
The experimental results indicated that 1) diffusion resistance existed around the microorganism's cell in reaction vessel and 2) fluid shear stress affected the mass transfer rates differently according to its strength and thus had varying effects on the bioPd(0) synthesis. More than 97.9 ± 1.5% Chromium(VI)(Cr(VI)) (384 μM) was reduced to Cr(III) within 20 min with 5% Pd/bioPd(0) as catalyst, which was generated by the K. Pneumoniae ECU-15, and the catalytic performance of Pd/bioPd(0) was stable over 6 months. The optimal condition of bioreduction of Pd(II) to Pd(0) was determined at the Kolmogorov eddy length of 7.33 ± 0.5 μm and lasted for 1 h in the extended reduction process after the usual adsorption and reduction process.
It is concluded that a high bioPd(0) catalytic activity can be achieved by controlling the fluid shear stress intensity in an extended reduction process in the bioreactor.
