Confusing parental information may hinder to dissect mechanisms of amino acid hyper-producing Corynebacterium glutamicum strains. Thus, an efficient method for genotyping of the C. glutamicum is heavily called.
Multi-locus sequence typing (MLST) is currently the most popular molecular typing technique. But currently this method is not available for C. glutamicum. In this study, a MLST scheme was established based on sequences of seven housekeeping genes, for genotyping of C. glutamicum. The MLST method performed an efficient discrimination of 17 strains and helps to understand the population structure of this bacterium.
This work has expanded the MLST method to C. glutamicum and developed an efficient technique to discriminate strains of uncertain origin.
Endophytic non-streptomycetes group of actinomycetes from Hibiscus rosasinensis leaves were screened for biosurfactant, polythene, plastic, and diesel biodegradation activities. Biosurfactant activity was evaluated by hemolysis, drop-collapsing test, lipase production, oil-spreading technique, bacterial adhesion to hydrocarbon (BATH) assay, penetration assay, and emulsification assay. Similarly, polythene, plastic, and diesel biodegradation activity were also carried out.
Among the five non-streptomycetes isolates, only one newly found actinomycete isolate named as A9 exhibited significant biosurfactant and biodegradation activity. Based on Bergey's manual of systematic bacteriology, the strain A9 was similar to Nocardiopsis sp. Molecular characterization and phylogenetic analysis support the classification of the isolate as a new strain which was named as Nocardiopsis sp.mrinalini9. The isolate was closely related to the type strain of Nocardiopsis synnemataformans sharing a 16S rRNA gene sequence similarity of 99%. The partial 16S rRNA sequence of the isolate (1061 bp) was deposited in Genebank under the accession number KF909126. Still, DNA-DNA hybridizations, phenotypic comparisons, and chemotaxonomic analysis need to be performed to confirm its novelty.
It is evident from the study that rare endphytes associated with medicinal plants has got excellent hydrocarbon biodegradation capacity.
Xylanases are important members of the hemicellulolytic enzyme system. Xylanase plays a vital role in the hydrolysis of major hemicellulosic component xylan and converts it into xylooligosaccharides and ultimately yields xylose. Cellulase-lacking or cellulase-poor xylanase with high temperature and pH stability has gained special attention, especially in paper and pulp industries. Most of the available literature highlighted the fungal xylanase production by optimizing environmental and cultural parameters. However, the importance of enzyme recovery from fermented biomass still needs attention. In this study, upstream and downstream process parameters were studied for enhancing xylanase production and extraction by a newly isolated Aspergillus tubingensis FDHN1 under solid-state fermentation using low-cost agro-residues.
In the present study, A. tubingensis FDHN1 was used for the xylanase, with very low level of cellulase, production under solid-state fermentation (SSF). Among various agro-residues, sorghum straw enhanced the xylanase production. Under optimized upstream conditions, the highest xylanase production 2,449 ± 23 U/g was observed. Upon characterization, crude xylanase showed stability over a broad range of pH 3.0 to 8.0 up to 24 h. The temperature stability revealed the nature of the xylanase to be thermostable. Native polyacrylamide gel electrophoresis (native PAGE) and zymogram analysis revealed the multiple forms of the xylanase. Due to the many industrially important characteristics of the xylanases, the study was elaborated for optimizing the downstream process parameters such as volume of extractant, extraction time, temperature and agitation speed to recover maximum xylanase from fermented sorghum straw. The highest amount of xylanase (4,105 ± 22 U/g) was recovered using 0.05 M sodium citrate buffer (pH 6.5) at 12:1 (v/w) extractant/solid ratio, 90-min extraction time, 150-rpm agitation speed and 40°C. Finally, detailed bioprocess optimization shows an overall 6.66-fold enhancement in the xylanase yield.
The present study consolidates the importance of upstream and downstream process optimization for the overall enhancement in the xylanase production. The xylanase from A. tubingensis FDHN1 shows the stability at different pH and temperature, and it was also active in the presence of organic solvents. These properties of xylanase are very much important from an industrial application point of view.
It becomes more and more important to develop appropriate models for the efficient design of the cell factory for microbial biofuels and biochemical productions, since the appropriate model can predict the effect of culture environment and/or the specific pathway genes knockout on the growth characteristics. Among various modeling approaches, kinetic modeling is promising in the sense of realizing the essential feature of metabolic regulation. A brief overview is given for the current status of the kinetic modeling of the cell metabolism from the point of view of metabolic regulation focusing on Escherichia coli (but not limited to E. coli). For the proper modeling, it is important to realize the systems behavior by integrating different levels of information to understand and unravel the underlying principles of the living organisms, namely, it is important to properly understand how the environmental stimuli are detected by the cell, how those are transduced, and how the cell metabolism is regulated, and to express these into the model. In particular, it is important to incorporate the enzymatic regulations of Pyk, Pfk, and Ppc by fructose-1,6-bisphosphate (FBP), phosphoenol pyruvate (PEP), and acetyl-coenzyme A (AcCoA) to realize the flux-sensing and homeostatic behavior. The proper modeling for phosphotransferase system (PTS) and the transcriptional regulation by cAMP-Crp and Cra is also important to simulate the main metabolism in relation to catabolite regulation. The coordinated regulation between catabolic and anabolic (nitrogen source-assimilation) metabolisms may be simulated by the behavior of keto acid such as α-ketoglutarate (αKG). The metabolism under micro-aerobic conditions may be made by incorporating the global regulators such as ArcA/B and Fnr. It is quite important to develop quantitative kinetic models, which incorporate enzyme level and gene level regulations from the biological science and metabolic engineering points of view.
Flavonoids of Hypericum perforatum are important secondary metabolites which have been widely utilized in medicine for a range of purposes. The use of methyl jasmonate (MeJA) elicitation for the enhancement of flavonoid production in cell suspension culture of H. perforatum would be an efficient alternative method for the flavonoid production.
MeJA influenced the cells growth and flavonoid production. The optimal elicitation strategy was treatment of the cell cultures with 100 μmol/L MeJA on day 15, which resulted in the highest flavonoid production (280 mg/L) and 2.7 times of control cultures. The activities of catalase (CAT) were inhibited after MeJA treatment in the cell cultures, while the activities of phenylalanine ammonia lyase (PAL) increased, which led to the enhancement of flavonoid production.
MeJA elicitation is a useful method for the enhancement of flavonoid production in cell suspension culture of H. perforatum.
Optically active ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is an important chiral building block for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. It is reported that microbial or enzymatic reduction of ethyl 2-oxo-4-phenyl-butyrate (OPBE) is an attractive way to produce optically active (R)-HPBE.
The asymmetric reduction of OPBE to synthesize optically active (R)-HPBE with a newly isolated Rhodotorula mucilaginosa CCZU-G5 as catalyst was investigated in an aqueous/organic solvent biphasic system. R. mucilaginosa CCZU-G5 showed a good tolerance (the metabolic activity retention >80%) in the biphasic system composed of aqueous buffer and organic solvent with a log P value over 4.6. Isooctane was found to be the most suitable organic phase solvent. In the biphasic system, the volumetric phase ratio, OPBE concentration, cell concentration, reaction temperature, and buffer pH were optimized. Under the optimum conditions (volumetric phase ratio: 1/1, OPBE concentration: 100 mM, cell concentration: 0.075 g/mL, pH 7.5, 35°C), the final yield and the optical purity of (R)-HPBE reached 98.3% and >99.0% enantiomeric excess (ee), respectively, after 12 h of reaction.
All the results suggested that the OPBE-reducing enzymes in a newly isolated R. mucilaginosa cells possess highly stable and excellent stereoselectivity by establishing an aqueous/organic biphasic system.
Production of cellulose-degrading enzymes from Aspergillus terreus D34 using different growth substrates was studied under solid-state cultivation. We have tested two lignocellulosic biomass residues viz., rice straw (RS) and sugarcane bagasse (BG), both separately and in combinations, and crystalline cellulose as a sole source of carbon for cellulase production. We also demonstrated different cellulase cocktail formulations and enzymatic saccharification studies on mild-alkali and dilute-acid pretreated RS- and BG-biomass residues.
Substrate-specific non-denaturing native gels showed two exoglucanases, four endoglucanases, three β-glucosidases, and four xylanases in the microbial culture extract of RS-grown cells. While in the BG-culture extract, two exoglucanases, five endoglucanases, three β-glucosidases, and four xylanases were detected. Similarly, in crystalline cellulose-grown culture extract, three exoglucanases, four endoglucanases, one β-glucosidase, and two xylanases were detected. However, the cellulase compositions were highly varied with the culture extracts obtained from the mixed biomass (RSBG) growth substrate. We found that few enzymes were specifically induced while others were repressed in RSBG-grown cultures. Enzymatic saccharification resulted in the production of maximum reducing sugars of 0.733 and 0.498 g g−1 with mild-alkali treated RS- and BG-biomass residues with saccharification yields reaching up to 82.8% ± 1.0% and 59.3% ± 1.7%, respectively.
The cellulase activities, namely FPase, CMCase, avicelase, β-glucosidase, and endoxylanase, were significantly higher in the BG-grown culture extract. Optimization of microbial growth carbon sources produced an efficient cellulase enzyme cocktail mixture with an approximately twofold higher total cellulase (FPase) activity that drastically reduced the required amount of enzyme (in terms of unit volumes) for enzymatic hydrolysis studies.
The combined effect of drying temperature and time was evaluated on residual water content, yield of oil extraction, total phenolic compounds and antioxidant activity of seed kernel from a Cameroonian local variety of mango (Local Ngaoundere). Response surface methodology (RSM) using central composite design (CCD) as tool, was used to develop, validate and optimize statistical models in order to establish the impact of the drying parameters (temperature and time) either alone or in combination.
It was shown that drying temperature individually in its first order (X1) contributed 30.81, 21.11, 41.28 and 33.24% while drying time individually in its first order (X2) contributed 39.91, 15.12, 29.92 and 25.87% for residual water content, yield of oil extraction, total phenolic components and antioxidant activity respectively. The increase of drying temperature increased antioxidant activity while the other physicochemical characteristics such as water content, yield of oil extraction and total phenolic components decreased. Concerning drying time, only water content was reduced with an increase of that factor. The synergetic effect of drying temperature and time was effective only for antioxidant activity. A compromise for optimization were then fixed for water content ≤ 10% w/w; oil content ≥ 9% w/w; total polyphenols ≥ 1 mg/g and antioxidant activity ≥ 1000 mg AAE/100 g DM. A simulation for optimization gave, for 60 H and 60°C for drying time and temperature respectively permitted to obtain 4.10% w/w, 9.53% w/w, 1340.28 mg AAE/100 g DM and 1.16 mg/g for water content, oil content, antioxidant activity and total polyphenols respectively.
The physicochemical characteristics studied was globally influenced by the chosen factors (drying time and temperature).
Corynebacterium glutamicum is widely used in glutamate fermentation. The fermentation feature of the strain varies sometimes. These variations may lead to the reduction in the ability of the strain to resist environmental changes and to synthesize glutamate, resulting in abnormal glutamate fermentations.
In the abnormal glutamate fermentations, glutamate accumulation stopped after glucose feeding and the final glutamate concentration was at a lower level (50 to 60 g/L). The rNAD +/rNADH ratio was lower than that in normal batch which was reflected by lower oxidation-reduction potential (ORP) value. The abnormal fermentation performance was improved when glucose was co-fed with sorbitol/glycerol at a weight ratio of 5:1 or adding 10 to 15 g/L of sorbitol/glycerol in the initial medium. Under these conditions, glutamate synthesis continued after substrate(s) feeding and final glutamate concentration was restored to normal levels (≥72 g/L). rNAD +/rNADH ratio, ORP, and pyruvate dehydrogenase (PDH), isocitrate dehydrogenase (ICDH), and cytochrome c oxidase (CcO) activities were maintained at higher levels.
Sorbitol and glycerol were not used as carbon sources for the fermentation. They were considered as effective protective agents to increase cells' resistance ability against environmental changes and maintain key enzymes activities.
Enzymatic kinetic resolution is proved as an efficient strategy of accessing chiral secondary alcohols in organic synthesis. Although several synthetic methods have been developed for the preparation of chiral acetylenic alcohol, biotransformation remains as a direct approach in the synthesis of polyacetylene lipids, which represent an intriguing class of marine natural products featuring interesting biological profiles.
Novozym 435, a commercial lipase immobilized on macroporous acrylic resin, is utilized in the kinetic resolution of 1-yn-3-ol-4-(E)-ene (alkenyl acetylenic alcohol), which exists as terminus in recently isolated isofulvinol (1) both from the mollusk Peltodoris atromaculata and the sponge Haliclona fulva. The kinetic resolution enabled by Novozym 435 resulted in acetylenic alcohol and the corresponding acetate both in excellent enantiomeric excess and high isolated yield. The optimized reaction conditions allow us to realize the reaction at room temperature in toluene. The reaction is readily scaled up for synthetic use.
The current investigation builds up a platform for future exploration on stereoisomers of isofulvinol, synthetic intermediates with different chain length, and natural product analogs.
In the view of depleting resources and ever-increasing price of crude oil, there is an urge for the development of alternative sources to solve the issue of fuel in the coming years. Lignocellulosic biomass is considered to be the most potential alternative resources for fossil fuel. Bioconversion of cellulosic and hemicellulosic components into fermentable sugars is the key step in producing fuel ethanol from lignocellulose. The enzymatic hydrolysis of lignocellulosic biomass needs a highly balanced composition of cellulases and hemicellulases. Commercial enzymes are usually poor in accessory hemicellulolytic enzymes like α-L-arabinofuranosidase. The addition of such accessory enzymes in combination with cellulase or hemicellulase plays a vital role in improving the total yield of fuel ethanol by enhancing the saccharification yield.
The newly isolated fungal strain Aspergillus niger ADH-11 produced a maximum of 22.14 U/g of α-L-arabinofuranosidase under solid-state fermentation using wheat bran as the substrate and modified Mandels-Weber medium at 30°C after 180 h of incubation. The optimization of various fermentation parameters was performed by response surface methodology employing Plackett-Burman design followed by Box-Behnken design. The yield of α-L-arabinofuranosidase was enhanced by 2.34-fold after executing statistical optimization of various fermentative parameters. Crude α-L-arabinofuranosidase was found to be highly stable for 3 h at its optimum temperature (55°C) and pH (4.0). The assessment of the crude enzyme extract in saccharification of alkali-treated maize stover revealed that the supplementation of crude α-L-arabinofuranosidase to commercial cellulase and crude xylanase mixture increased the saccharification yield up to 730 mg/g of maize stover.
The newly isolated A. niger ADH-11 was found to be a potential producer of α-L-arabinofuranosidase. The crude enzyme was active at low pH and high temperature which makes it suitable for various industrial applications such as enzymatic saccharification of lignocellulosic biomass. The supplementation of α-L-arabinofuranosidase enzyme to commercial cellulases and hemicellulases improves the bioconversion of lignocellulosic biomass to a greater extent.
Recently, cell tolerance toward environmental stresses has become the major problem in the development of industrial microbial fermentation. Acetoin is an important chemical that can be synthesized by microbes. Its toxicity was investigated using Candida glabrata as the model in this study.
A series of physiological and biochemical experiments demonstrated that the organic solvent acetoin can inhibit cell growth by increasing intracellular reactive oxygen species (ROS) production and inducing damage to mitochondria and cell apoptosis. Integrating RT-PCR experiments, the genes fzo1 and dnm1 were overexpressed to regulate the balance between mitochondrial fusion and fission. Enhancement of mitochondrial fusion was shown to significantly increase cell tolerance toward acetoin stress by inhibiting ROS production and increasing the intracellular adenosine triphosphate (ATP) supply, which was also demonstrated by the addition of citrate.
Regulating mitochondrial fusion-fission may be an alternative strategy for rationally improving the growth performance of eukaryotes under high environmental stress conditions, and also expands our knowledge of the mechanisms of cell tolerance through the processes of energy-related metabolic pathways.
A wide range of value-added products can potentially be produced by bioprocessing hardwood spent sulfite liquors (HSSLs) that are by-products of pulp and paper industry with a high pentose sugar content. However, besides sugars, HSSLs contain considerable amounts of sulfonated lignin derivatives and acetic acid that inhibit the metabolic activity of most microorganisms. Scheffersomyces stipitis is a yeast with high capacity to ferment the pentose sugar xylose under appropriate microaerophilic conditions but it has limited tolerance to HSSL inhibitors. In the present study, cultivations of suspended and immobilized S. stipitis were compared in terms of growth capacity and by-product formation using rich medium and HSSL to investigate whether the immobilization of cells in calcium alginate beads could be a protection against inhibitors while favoring the presence of microaerophilic conditions.
Whereas cell immobilization clearly favored the fermentative metabolism in rich medium, pH control was found to play a more important role than cell immobilization on the ethanol production efficiency from bio-detoxified HSSL (bdHSSL), leading to an improvement of 1.3-fold on the maximum ethanol productivity than using suspended cells. When immobilization and pH control were applied simultaneously, the ethanol yield improved by 1.3-fold with unchanged productivity, reaching 0.26 g ethanol.(g glucose + xylose)−1. Analysis of the immobilized beads inside revealed that the cells had grown in the opposite direction of the cortex.
Immobilization and pH control at 5.5, when applied simultaneously, have a positive impact on the fermentative metabolism of S. stipitis, improving the ethanol production efficiency. For the first time light microscopic analysis of the beads suggested that the nutrient and mass transfer limitations played a more important role in the fermentation than a possible protective role against inhibitors.
Pseudomonas sp. AKS2 can efficiently degrade low-density polyethylene (LDPE). It has been shown that this degradation of LDPE by AKS2 is correlated to its ability to form biofilm on the polymer surface. However, the underlying mechanism of this biofilm-mediated degradation remains unclear. Since bioremediation potential of an organism is related to its adaptability in a given environment, we hypothesized that AKS2 cells undergo successful adaptation in biofilm on LDPE, which leads to higher level of LDPE degradation. To verify this, the current study investigated a number of parameters of AKS2 cells in biofilm that are known to be involved in adaptation process.
Successful adaptation always develops a viable microbial population. So we examined the viability of AKS2 cells in biofilm. We observed the presence of viable population in the biofilm. To gain an insight, the growth of AKS2 cells in biofilm on LDPE at different time points was examined. Results showed a better reproductive competence and more colonization for AKS2 biofilm cells than planktonic cells, indicating the increased fitness of AKS2 biofilm cells than their planktonic counterpart. Towards understanding fitness, we determined the hydrolytic activity, different carbon source utilization potentials, functional diversity and homogeneity of AKS2 biofilm cells. Results showed increased hydrolytic activity (approximately 31%), higher metabolic potential, higher functional diversity (approximately 27%) and homogeneity for biofilm-harvested cells than planktonic cells. We also examined cellular surface hydrophobicity, which is important for cellular attachment to LDPE surface. Consistent with the above results, the cell surface hydrophobicity of biofilm-harvested AKS2 cells was found to be higher (approximately 26%) compared to that of their planktonic counterpart. All these results demonstrated the occurrence of physiological as well as structural adaptations of AKS2 cells in biofilm on LDPE surface that resulted in better attachment, better utilization of polymer and better growth of AKS2 cells, leading to the development of a stable colony on LDPE surface.
The present study shows that AKS2 cells in biofilm on LDPE surface undergo successful adaptation that leads to enhanced LDPE degradation, and thus, it helps us to understand the underlying mechanism of biofilm-mediated polymer degradation process by AKS2 cells.
Optically active (R)-2-hydroxy-4-phenylbutanoate esters ((R)-HPBE) are key precursors for the production of angiotension-converting enzyme (ACE) inhibitors, which are important prescriptive drugs for preventing the formation of angiotensin II and lowering the blood pressure. The biocatalytic asymmetric reduction of ethyl 2-oxo-4-phenylbutanoate (OPBE) to (R)-HPBE with carbonyl reductases has several advantageous attributes, including high enantioselectivity, mild reaction condition, high catalytic efficiency, and environmental benignity. An increasing number of OPBE reductases have been discovered owing to the drastic achievements in genomics, screening and evolution technologies, and process engineering. The potential of (R)-HPBE production process has also been intensively evaluated. This review covers recent progress on the bioreductive preparation of (R)-HPBE, especially on various screening approaches for the identification of OPBE reductases and their characterization.
Enzymatic cascades in metabolic pathways are spatially organized in such a way as to facilitate the flow of substrates. The construction of artificial cellulase complexes that mimic natural multienzyme assemblies can potentially enhance the capacity for cellulose hydrolysis. In this study, an artificial cellulase complex was constructed by tethering three cellulases to a synthetic protein scaffold.
Three pairs of interacting proteins were selected and characterized. The artificial protein scaffolds were constructed by fusing three interacting proteins. Cellulases were tethered to these synthetic scaffolds in different orders. The optimal assembly resulted in a 1.5-fold higher hydrolysis of cellulose than that achieved by unassembled cellulases.
A novel artificial protein scaffold was constructed and used to assemble three cellulases. The resultant increase in enzymatic activity suggests that this can be used as a strategy for enhancing the biocatalytic capacity of enzyme cascades.
Biofuel and biochemical production by photosynthetic microorganisms such as cyanobacteria and algae is attractive to improve energy security and to reduce CO2 emission, contributing to the environmental problems such as global warming. Although biofuel production by photosynthetic microorganisms is called as the third generation biofuels, and significant innovation is necessary for the feasibility in practice, these fuels are attractive due to renewable and potentially carbon neutral resources. Moreover, photosynthetic microorganisms are attractive since they can grow on non-arable land and utilize saline and wastewater streams. Highly versatile and genetically tractable photosynthetic microorganisms need to capture solar energy and convert atmospheric and waste CO2 to high-energy chemical products. Understanding of the metabolism and the efficient metabolic engineering of the photosynthetic organisms together with cultivation and separation processes as well as increased CO2 assimilation enables the enhancement of the feasibility of biofuel and biochemical production.
Silver nanoparticles (SNPs) are used extensively in areas such as medicine, catalysis, electronics, environmental science, and biotechnology. Therefore, facile synthesis of SNPs from an eco-friendly, inexpensive source is a prerequisite. In the present study, fabrication of SNPs from the leaf extract of Butea monosperma (Flame of Forest) has been performed. SNPs were synthesized from 1% leaf extract solution and characterized by ultraviolet-visible (UV-vis) spectroscopy and transmission electron microscopy (TEM). The mechanism of SNP formation was studied by Fourier transform infrared (FTIR), and anti-algal properties of SNPs on selected toxic cyanobacteria were evaluated.
TEM analysis indicated that size distribution of SNPs was under 5 to 30 nm. FTIR analysis indicated the role of amide I and II linkages present in protein in the reduction of silver ions. SNPs showed potent anti-algal properties on two cyanobacteria, namely, Anabaena spp. and Cylindrospermum spp. At a concentration of 800 μg/ml of SNPs, maximum anti-algal activity was observed in both cyanobacteria.
This study clearly demonstrates that small-sized, stable SNPs can be synthesized from the leaf extract of B. monosperma. SNPs can be effectively employed for removal of toxic cyanobacteria.
In general, silver nanoparticles (AgNPs) are particles of silver with a size less than 100 nm. In recent years, synthesis of nanoparticles using plant extract has gained much interest in nanobiotechnology. In this concern, this study investigates green synthesis of AgNPs from silver nitrate using Sinapis arvensis as a novel bioresource of cost-effective nonhazardous reducing and stabilizing compounds. A stock solution of silver nitrate (0.1 M) was prepared. Different concentrations of silver nitrate (1, 2.5, 4, and 5 mM) were prepared from the above solution, then added to 5 mL of S. arvensis seed exudates. The mixtures were kept in 25°C. The synthesis of AgNPs was confirmed by the change in mixtures color from light yellow to brown. The antifungal activity of synthesized AgNPs was investigated in vitro.
The resulting AgNPs were characterized by UV-vis spectroscopy, X-ray diffraction (XRD), transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy (FTIR). Formation of the AgNPs was confirmed by the change in mixture color from light yellow to brown and maximum absorption at 412 nm due to surface plasmon resonance of AgNPs. The role of different functional groups in the formation of AgNPs was shown by FTIR. X-ray diffraction was shown that the AgNPs formed in our experiments were in the form of nanocrystal, and TEM analysis showed spherical particles with an average size of 14 nm. Our measurements indicated that S. arvensis seed exudates can mediate facile and eco-friendly biosynthesis of colloidal-spherical AgNPs with a size range of 1 to 35 nm. The synthesized AgNPs showed significance antifungal activity against Neofusicoccum parvum cultures.
The AgNPs were synthesized using a biological source. This synthesis method is nontoxic, eco-friendly, and a low-cost technology for the large-scale production. The AgNPs can be used as a new generation of antifungal agents.
Edwardsiella tarda, the etiologic agent of edwardsiellosis, is a devastating fish pathogen prevailing in worldwide aquaculture industries and accounting for severe economic losses. There is a raising concern about E. tarda being a significant zoonotic pathogen, and it is urgent to develop a rapid detection of this pathogen. This is the first study to develop a test strip for rapid detection of E. tarda in turbot.
Mouse monoclonal antibodies (MAbs) and rabbit polyclonal antibody (PAb) against E. tarda were generated from immunization of mice and rabbits with a virulent isolate of E. tarda EIB202. Two MAbs specific to isolates of E. tarda were obtained, and one of them (25C1) was selected to conjugate with colloidal gold as the detector antibody. Rabbit PAb was used as the capture antibody. It was found the strip had no cross-reactivity with non-E. tarda bacterial microbes and the limit of detection (LOD) was 1 × 105 colony-forming units (CFU)/ml. The detection could be visually observed by the naked eye within 5 min. This test strip was verified with a similar detection limit and much less analysis time compared with a dot blot immunoassay (1 × 105 CFU/ml for LOD and 120 min for reaction time). When the samples were mixed with turbot tissue homogenates, strong immunoreactivity was observed over 105 CFU/ml, which suggested that the turbot tissue homogenates did not affect the detection of the strip. Pre-enrichment with homogenized turbot tissue for 12 h could increase the detection limit of the E. tarda present in the sample up to 1 to 10 CFU/ml. In practice, in detecting 20 turbot ascite samples infected by E. tarda, the immunochromatographic test strip showed a high accuracy (100% positive).
The immunochromatographic test strip offers great promise for a rapid, simple, and economical method of E. tarda on-site detection, and with different antibodies, it might be used to detect other aquatic pathogens.
Microalgae are photosynthetic microorganisms presenting a diversity of biotechnological applications. However, microalgal cultivation systems are not energetically and economically feasible. Possible strategies that can be applied to improve the feasibility of microalgal production include biofouling control in photobioreactors, the use of attached growth systems and bioflocculation. These processes are ruled by surface physicochemical properties. Accordingly, the surface physicochemical properties of Chlorella vulgaris, Pseudokirchneriella subcapitata, Synechocystis salina and Microcystis aeruginosa were determined through contact angle and zeta potential measurements. Additionally, mixed cultures of the selected microorganisms were performed. Sedimentation kinetics of the studied cultures was also evaluated to understand how surface physicochemical properties influence microalgal recovery.
All studied microorganisms, except S. salina, presented a hydrophilic surface. The co-culture of S. salina with the other studied microorganisms resulted in a more hydrophobic algal suspension. Regarding zeta potential determinations, all studied suspensions presented a negatively charged surface (approximately -40.8 ± 4.4 mV). Sedimentation experiments have shown that all microalgal suspensions presented low microalgal recovery efficiencies. However, a negative linear relationship between microalgal removal percentage and free energy of hydrophobic interaction was obtained.
The evidence of a relationship between microalgal removal percentage and free energy of hydrophobic interaction demonstrates the importance of surface physicochemical properties on microalgal settling. However, the low recovery efficiencies achieved, as well as the high net zeta potential values determined, indicate that another factor to consider in microalgal settling is the ionic strength of the culture medium, which play an important role in suspensions’ stability.
Bioconversion of cellulosic biomass into fuel ethanol involves several steps, among which enzymatic breakdown of cellulose into fermentable sugars play a significant role. The key enzymes involved in cellulosic breakdown are mainly endoglucanases and β-glucosidases. Even though the biochemical and molecular characterization of number of endoglucanases and β-glucosidases was extensively studied, still there is a demand for novel microbial cellulases for industrial applications. Among the group of actinomycetes, Streptomyces spp. are well known as a cellulase producer. The advantage of using actinomycetes is being that production process could be easily scaled-up to commercial levels. However, recent research studies have shown that the production of cellulases from actinomycetes could also be significantly improved by employing different types of strain improvement methods, thus achieving high yields of extracellular proteins. Besides this, highly thermostable and broad pH range cellulases are required for bioethanol application.
A lignocellulose degrading actinomycetes strain was newly isolated and identified as Streptomyces griseoaurantiacus. Strain improvement using UV mutagenesis developed two mutants (SGUV30 and SGUV5) with 57.4 % and 12.8 % higher endoglucanase and β-glucosidase activities. The cellulases (endoglucanases and β-glucosidases) were found to be highly thermostable with no loss in enzyme activities at 80 °C for 60 min and nearly 80 % of initial activity was retained at 90 °C. Enzyme assays in presence of additives showed that CoCl2, CaCl2, and FeSO4 increased β-glucosidase activity but showed negative effect on endoglucanase activity. However, both the enzyme activities were significantly enhanced by addition of PEG 8000, sodium azide and MnSO4.
Strain improvement of S. griseoaurantiacus was performed by UV mutagenesis where two mutant strains (SGUV30 and SGUV5) were developed with improved endoglucanase and β-glucosidase activities. Cellulase production in submerged fermentation was carried out using a cheap lignocellulosic biomass residue, rice straw as a sole source carbon. The results clearly show that the mutant strains produced high-efficient cellulases that are stable at a broad pH range at very high temperatures. Besides, the mutants also showed high extracellular protein secretions, which could be promising in reducing the overall cellulase production costs at large scale.
Ligninolytic fungi and enzymes (i.e., laccase, manganese peroxidase, and lignin peroxidase) have been applied recently in the production of second-generation biofuels. This review contains the analysis of ligninolytic enzymes and their applications in second-generation biofuels. In here, each of the ligninolytic enzymes was described analyzing their structures, catalysis, and reaction mechanism. Additionally, delignification and detoxification, the two most important applications of ligninolytic enzymes, were reviewed and analyzed. The analysis includes an evaluation of the biochemical process, feedstocks, and the ethanol production. This review describes the current situation of the ligninolytic enzymes technology and its future applications in bioethanol industry.
Termites, which are among the nature’s most effective scavengers and earthmovers, are distinguished by their ability to process lignin. In this report, arguably the first of its type, we present studies in which termites have been utilized for solid waste disposal. Twenty-five different types of solid wastes were positioned by us in in situ termireactors, at different locations and at varying distances away from the mounds of different termite species. The rate of consumption of the substrates was monitored as a function of time.
The results reveal that substrates such as cotton waste, coconut shells, and torn jute bags, which resist composting, vermicomposting, or anaerobic digestion, are successfully decomposed by termites. Different termite species were able to bypass some substrates kept nearer their mounds and go to some other substrates kept farther away, showing their preference for different wastes as well as their ability to discern one waste type from the other.
The work provides proof-of-concept that termites can be used for the assimilative disposal of MSW.
A major challenge in downstream processing is the separation and purification of a target biomolecule from the fermentation broth which is a cocktail of various biomolecules as impurities. Aqueous two phase system (ATPS) can address this issue to a great extent so that the separation and partial purification of a target biomolecule can be integrated into a single step. In the food industry, starch production is carried out using thermostable glucoamylase. Humicola grisea serves as an attractive source for extracellular production of glucoamylase.
In the present investigation, the possibility of using polyethylene glycol (PEG)/salt-based ATPS for the partitioning of glucoamylase from H. grisea was investigated for the first time. Experiments were conducted based on one variable at a time approach in which independent parameters like PEG molecular weight, type of phase-forming salt, tie line length, phase volume ratio, and neutral salt concentration were optimized. It has been found that the PEG 4000/potassium phosphate system was suitable for the extraction of glucoamylase from the fermentation broth. From the results, it was observed that, at a phase composition of 22 % w/w PEG 4000 and 12 % w/w phosphate in the presence of 2 % w/w NaCl and at pH 8, glucoamylase was partitioned into the salt-rich phase with a maximum yield of 85.81 %.
A range of parameters had a significant influence on aqueous two-phase extraction of glucoamylase from H. grisea. The feasibility of using aqueous two-phase extraction (ATPE) as a preliminary step for the partial purification of glucoamylase was clearly proven.
In biorefineries, various pretreatments traditionally employ hazardous chemicals (ammonia, sulfuric acid, sulfite, etc.) for opening the softwood structure and to facilitate easy accessibility of the cellulose for further downstream processing. The resultant lignin (known as technical lignin) after extraction of the carbohydrate fraction as sugars has been either burned as fuel or used in biochemical or biofuel production. It has been observed that the technical lignin after such biomass pretreatments is often more condensed and, hence, cannot be easily used to produce fine chemicals of high value. In this study, we examine lignin after wet explosion pretreatment where the biomass in subjected to oxygen to understand how these interactions will affect lignin utilization for biochemical production.
In this study, the structural transformations within the softwood lignin as a response to wet explosion (WEx) pretreatment of loblolly pine at different experimental conditions (165–175 °C, 18–30 min) were examined using GC/MS and NMR spectroscopy. The results showed that the H-type structures within the lignin molecule decreased while S-type structures increased after pretreatment. Since S-type lignin sub-units have a higher degree of methoxylation compared to H units, the potential of S-type lignin to undergo re-condensation at lower temperatures (after pretreatment), by forming bonds with other lignin sub-units, is lower due to stearic hindrance, resulting in the generation of lignin with a lower tendency to form new complex lignin bindings (high-quality biorefinery lignin).
The less condensed biorefinery lignin generated after WEx pretreatment was found to provide a platform for production of lignin polymer with more labile β-O-4 linkages. This type of lignin could potentially be superior for the production of high-value bio-products compared to re-condensed lignin after acidic and other types of chemical pretreatments.
Ursodeoxycholic acid (UDCA) is an important clinical drug in the treatment of liver disease. In previous work, ursodeoxycholic acid was prepared by traditional organic synthesis. The preparation of ursodeoxycholic acid through an electrochemical method with higher stereoselectivity and environmental friendliness is described herein.
Dimethyl sulfoxide (DMSO), dimethylformamide (DMF), and N-methyl-2-pyrrolidone (NMP) were used as stereoselectivity additives during electroreduction. With 107.5 mM DMSO in methanol containing potassium bromide and a continuous current of 20 mA, 936 Coulombs was passed into the electrolysis system, achieving 88.5 % conversion of 7-ketone lithocholic acid (7K-LCA), while the yield of UDCA reached 72.8 %. Cyclic voltammetry (CV) was used to explore the electrochemical behavior of the reaction, and the electrolysis results were consistent with the cyclic voltammograms.
Ursodeoxycholic acid can be prepared by electroreduction with high stereoselectivity. The method developed here offers a potential application for large-scale production of ursodeoxycholic acid and an interesting reference to asymmetric electrochemical reduction of the keto group.
Shewanella species belonging to dissimilatory metal bacteria were found to decolorize most textile dyes and had also attracted great interests in regard to bioremediation. However, studies have rarely been reported on Shewanella xiamenensis BC01, which was isolated as a biodecolorization and bioelectricity strain recently. In this study, the effect of cultivation conditions on S. xiamenensis BC01 was studied to explore how environmental conditions may influence S. xiamenensis growth and swarming motility.
Shewanella xiamenensis BC01 grew over a wide range of pH (5.0–9.0) and mild temperatures (25–42 °C). The optimal conditions for cell growth were using Luria-Bertani (LB) as medium with shaking at 150 rpm, 37 °C, and pH 8.0 which had been confirmed by shift pH and temperature. S. xiamenensis BC01 was able to resist 1 mM concentrations of various metal ions, i.e., Ca2+, Mg2+, Cu2+, Zn2+, Mn2+, Fe3+, and Al3+, respectively. As shown in scanning electron microscopy (SEM) analyses, cell morphologies were slightly changed under metal stress. Swarming motility showed that the velocity ranking at 80 μM and 1 mM of metal was Al > Cr > LB > Zn > Fe > Cu and Mg > Mn > Ca, respectively.
This study evaluates the impact of cultivation methods and metal ions on the activity of S. xiamenensis BC01 and provides an alternative to bioremediation of heavy metal-containing wastewaters by utilizing this strain.
Zymomonas mobilis is an efficient ethanol fermentation strain, but its narrow substrate range limits its fermentation in lignocellulose hydrolysate. As a potential consolidated bioprocessing (CBP) stain for bioethanol production, the ability of cellulose utilization was necessary. In this study, extracellular expression of β-glucosidase on Z. mobilis was studied as the first step for construction of a practical CBP strain to reduce the use of β-glucosidase in the cellulase components.
The heterologous β-glucosidase from Bacillus polymyxa was expressed in the ethanologenic strain Z. mobilis (ZM4) and secreted extracellularly by an endogenous signal peptide and a fusion protein. The signal peptide SP1086 of the endoglucanase gene ZMO1086 from Z. mobilis was identified and facilitated 12 % of the endoglucanase encoded by ZMO1086 from Z. mobilis ZM4 and 16 % of the β-glucosidase encoded by bglB gene secreted out of the membrane of Z. mobilis ZM4. Another method for enhancement of the β-glucosidase secretion is to fuse the β-glucosidase encoded by bglB with the levansucrase encoded by sacB from Z. mobilis ZM4 to achieve the secretive expression. Its expression level was enhanced two times but only showed a 2 % secretion ratio in this situation.
The SP1086 signal peptide showed an obviously secreting capacity of the β-glucosidase protein. The fusion protein with SacB also showed the secretion effect, but it was less efficient.
The fractionation of lignocellulosic materials can be applied to get each of the components (cellulose, hemicellulose and lignin) of biomass in its maximum purity and yield. The individual component can be further processed to high-value products such as fuels and biomaterials at existing or newly developing biorefineries. The steam-assisted and microwave (MW)-assisted processes were used to fractionate sweet sorghum bagasse into hemicellulosic sugars, cellulose-rich and high-density residue and solid lignin. The treatment temperature evaluated for the fractionation process was 121 °C for 30 to 120 min. The substrate was autohydrolysed to extract the hemicellulose, and the residue was delignified using lime solution to obtain cellulose-rich residue. The lignin and lime from the liquor obtained after the lime treatment of the autohydrolysed sweet sorghum bagasse (SSB) was precipitated using carbon dioxide gas.
Under optimum conditions, the steam-assisted autohydrolysis extracted 72.69 (±0.08) % by weight of the hemicelluloses while the MW-assisted autohydrolysis extracted 70.83 (±0.49) % of the hemicelluloses from the sweet sorghum bagasse. The steam-assisted lime treatment resulted in 69.67 (±1.26) % of the lignin extraction the MW-assisted lime treatment resulted in 68.27 (±1.19) % of the lignin extraction from the corresponding autohydrolysed sweet sorghum bagasse samples. The CO2 treatment precipitated 58.85 (±3.2) % of the lignin dissolved in the yellow liquor of the steam-assisted process while 60.26 (±2.11) % of the dissolved lignin was recovered from the yellow liquor of the MW-assisted process.
The two methods have not exhibited significant differences in overall recovery of the solids, hemicellulose extraction, delignification, residual concentration of cellulose and ash or in the recovery of lignin and lime. The difference was significant (p value <0.05) only in the concentration of total reducing sugars in the hydrolysate and the yellow liquor. The MW-assisted process increased the total crystallinity index (TCI) of the cellulose in the treated SSB and also increased the concentration of guaiacyl lignin content in the recovered lignin which was thermally more stable than the lignin produced in the steam-assisted process.
Concurrent advances in a number of fields have fostered the development of bioprocesses for biochemical production. Ideally, future bioprocesses will meet the demands of commercial chemical markets in an economical fashion while being sustainable through the use of renewable starting materials. A number of different renewable and abundant biopolymers (e.g., cellulose, hemicelluloses, lignin, and chitin) are potential starting material for sustainable bioprocesses, but a broad challenge remains on how to efficiently depolymerize these biopolymers to generate monomeric sugars that can be metabolized by industrial microorganisms or other useful building block chemicals. Indeed, a variety of specialty chemicals may be able to be generated from these various monomers. This review focuses on the biopolymer chitin and discusses research and knowledge relevant to chitin degradation and potential chemical products that can be made from chitin degradation products.
Fructose, a monosaccharide, has gained wide applications in food, pharmaceutical and medical industries because of its favourable properties and health benefits. Biocatalytic production of fructose from inulin employing inulinase is the most promising alternative for fructose production. For commercial production, use of immobilized inulinase is advantageous as it offers reutilization of enzyme and increase in stability. In order to meet the demand of concentrated fructose syrup, inulin hydrolysis at high substrate loading is essential.
Inulinase was immobilized on chitosan particles and employed for fructose production by inulin hydrolysis. Fourier transform infrared spectroscopy (FTIR) analysis confirmed linkage of inulinase with chitosan particles. Immobilized biocatalyst displayed significant increase in thermostability at 60 and 65 °C. Statistical model was proposed with an objective of optimizing enzymatic inulin hydrolytic process. At high substrate loading (17.5 % inulin), using 9.9 U/g immobilized inulinase at 60 °C in 12 h, maximum sugar yield was 171.1 ± 0.3 mg/ml and productivity was 14.25 g/l/h. Immobilized enzyme was reused for ten cycles. Raw inulin from chicory and asparagus was extracted and supplied in 17.5 % for enzymatic hydrolysis as a replacement of pure inulin. More than 70 % chicory inulin and 85 % asparagus inulin were hydrolyzed under optimized parameters at 60 °C. Results of high performance liquid chromatography confirmed the release of fructose after inulin hydrolysis.
The present findings prove potentiality of immobilized thermostable inulinase from Aspergillus tubingensis CR16 for efficient production of fructose syrup. Successful immobilization of inulinase on chitosan increased its stability and provided the benefit of enzyme reutilization. Box-Behnken design gave a significant model for inulin hydrolysis. Extraction of raw inulin from chicory and asparagus and their enzymatic hydrolysis using immobilized inulinase suggested that it can be a remarkable cost-effective process for large-scale fructose production.
Polygalacturonase is one kind of pectinases which hydrolyze the alpha-1,4 glycosidic bond between galacturonic acid residue. Polygalacturonase has been widely used in the fields of food, biofuel, and textile industries, in which thermostable polygalacturonase is often demanded at high temperatures of 50–60 °C. Herein, we reported a thermostable polygalacturonase producing from Aspergillus fumigatus isolated from the pile fermentation of Pu’er tea in China.
The thermophilic polygalacturonase-producing strain was identified as A. fumigatus L45 on basis of its morphology, physicochemical properties, and 18S rDNA analysis. The crucial fermentation parameters affecting polygalacturonase activity were optimized by response surface methodology (RSM); the optimum fermentation parameters were the following: inoculums concentration of 0.07 % (v/v), fermentation time of 36 h, pH of 5.0, and temperature of 45 °C. Under the optimized conditions, the highest polygalacturonase activity of 359.1 ± 10.1 U/mL was obtained. The polygalacturonase showed good thermostability and pH stability. The enzyme was activated by metal ions Zn2+ and Mg2+, but inhibited by K+. However, Na+ and Ca2+ showed little effects on its activity. Km and Vmax values were estimated to be 35.0 mg/mL and 7.69 μmol/mL/min, respectively.
A polygalacturonase from A. fumigatus L45 was preliminarily investigated, the crucial fermentation parameters were optimized by RSM, and the properties of polygalacturonase was examined. The polygalacturonase showed good thermostability and pH stability, which suggested the enzyme has potential applications in the biofuel and textile industries.
The article aims to clarify an existing misunderstanding by the users of fetal bovine serum (FBS), who assume that certain countries, like Australia and New Zealand, have fewer cattle disease viruses and pose less risk for the presence of viruses, than do the other FBS producing countries. The article reviews the 2013 information from the World Organization for Animal Health (OIE), regarding the presence and absence of the 14 viruses of concern for FBS in the cattle populations of the 30 major FBS producing countries of the world. United States Department of Agriculture (USDA) and European Union (EU) regulations have identified 8 adventitious viruses and 6 additional viruses of importation concern that need to be tested for or eliminated in FBS, viruses that can cross the placental barrier from the donor cow to the fetus. A comparison is made regarding the number of viruses of concern reported presently in each of the FBS producing countries. The results of the comparison reveal that four Scandinavian countries report the fewest number of viruses of concern for FBS (six in total), while Australia and the USA are among the countries reporting the highest numbers of viruses of concern for FBS (ten in total). FBS from Australia and the USA has thus no advantage over the other FBS producing countries, regarding the number of viruses needed to be tested for and eliminated.
Microbial production of cellulose-degrading enzymes could be significantly improved using traditional mutagenesis treatment. Development of high-titre cellulase producing mutants drastically reduces the costs involved in cellulase production and downstream processing in commercial-scale enzyme production. Here, we have evaluated the efficacy of different Aspergillus terreus D34 mutants for hyper-production of improved cellulase enzymes utilizing locally available lignocellulosic biomass residues as growth substrates in solid state fermentation conditions. Further, enzymatic hydrolysis of mild-alkali pre-treated rice straw was performed using the improved cellulases.
A 4.9-fold higher β-glucosidase activity was obtained from ethyl methyl sulphonate (EMS) treated mutant strain (EMS2) when grown on mixed rice straw/sugarcane bagasse (RSBG) biomass growth substrate. Similarly with the EMS2 mutant and BG-grown culture extract a 1.1-fold higher xylanase activity was observed. Irrespective of the growth substrates and the mutant strains, the maximum cellulase (FPase, carboxymethyl cellulase, avicelase, β-glucosidase) and xylanase activities (U mL−1) were 2.34, 39.8, 2.46, 19.9 and 655, respectively. Further, external supplementation of 20% bovine serum albumin (BSA), 3% tween 80 and 20% polyethylene glycol (PEG) 6000 to the crude enzyme extract increased the FPase activity nearly 4.0-, 2.8- and 2.2-fold. Addition of 0.05% sodium benzoate marginally increased the stability of cellulase enzyme and retained more than 60% of the initial activity after 96 h incubation at 37°C. While at 4°C, no loss in enzyme activity was observed even after prolonged incubation period (up to 90 days). Further, maximum reducing sugars of 0.842 g g−1 at a rate of 0.25 mM g−1 h−1 at 10% biomass loading of mild-alkali pretreated rice straw was produced using the BG-grown culture extract of EMS2 mutant strain.
The extracellular protein production and corresponding cellulase activities of A. terreus D34 were significantly enhanced after combined UV and chemical mutagenesis treatments. In the present study, besides accelerating the rate of cellulase production, we have also demonstrated production of high reducing sugars by enzymatic saccharification of pretreated lignocellulosic biomass using hyper-produced cellulase enzymes. Due to high enzyme activity of the cellulase enzymes produced from the mutant strains, the volume of enzyme loadings in enzymatic hydrolysis could be reduced up to 7-fold. These studies clearly show the potential of the developed hyper-cellulase producing mutants in decreasing the overall process economics of cellulosic ethanol technology.
Microalgae are a promising new source for biomass production. One of the major challenges in regards to cost effectiveness is the biomass harvest. High energy input is required for the separation of the small algal cells from a large volume of surrounding media. Electroflocculation is reported as a promising harvesting technique to improve cost effectiveness within the downstream process. In the present study, six electrode materials were tested for electroflocculation of Scenedesmus acuminatus. Besides the commonly used aluminum and iron electrodes, magnesium, copper, zinc and brass electrodes were tested for biomass harvest and compared. The influence of four different voltages (10, 20, 30 and 40 V) was investigated and evaluated.
Electroflocculation was applicable with all tested electrode materials. The highest flocculation efficiency was achieved using magnesium electrodes followed by Al, Zn, Cu, Fe and brass. Using magnesium, 90% of the suspension was clarified at 40, 30, 20, and 10 V after 9.2, 12.5, 18.5, and 43 min, respectively. All electrode materials showed the fastest flocculation at 40 V and the lowest at 10 V. The pH increased from 7.5 to values between 9.3 and 11.9 during the flocculation processes. Reuse of the supernatant showed no adverse effect on algal growth. The highest cell counts after 12 days of incubation were achieved with iron at 1.86 × 107 cells ml−1 and the lowest with copper at 1.23 × 107 cells ml−1.
Besides the commonly used iron and/or aluminum electrodes, other materials like magnesium, copper, zinc and brass can be successfully used for microalgal biomass harvest. For special biomass applications like food or feed additives, metals like magnesium have other advantages besides their high flocculation efficiency such as their low toxicity at high concentrations. Higher voltages increased the maximum flocculation efficiency but also increased the required energy input.
Trehalose has many advantages due to its inertness and the ability to stabilize biomolecules. Trehalose synthase can catalyze intramolecular rearrangement of the inexpensive maltose into trehalose in a single step, which represents a simple, fast, and low-cost method for the future industrial production of trehalose.
In this work, an intelligent visualization method for producing trehalose via trehalose synthase was for the first time established and optimized by corresponding enzymatic hydrolysis with kudzu root starch as the initial raw material. For the first step, kudzu root starch was liquefied by α-amylase at a certain dextrose equivalent value of 19–21, and β-amylase and pullulanase to saccharify for a certain time, a four-factor nine-level experimental design with nine experiments was performed according to the uniform design table U9 *(94) to optimize the yield of maltose. Then, optimization of trehalose conversion ratio from kudzu root starch hydrolysate was carried out with a four-factor 12-level experimental design to forecast the optimal process conditions. By comparing verification tests and predicted results, the optimized operating conditions were pH 7.1, 22 °C, 32.5 % (14,000 U/ml) loading amounts of TreS enzyme and after 24 h, along with a 70.6 % trehalose conversion rate.
The intelligent visualization method has succeeded in exploiting the conversion process of trehalose from kudzu root starch hydrolysate, which contributes significant benefits to the further commercial production of trehalose.
The adverse climatic conditions due to continuous use of fossil-derived fuels are the driving factors for the development of renewable sources of energy. Current biofuel research focuses mainly on lignocellulosic biomass (LCB) such as agricultural, industrial and municipal solid wastes due to their abundance and renewability. Although many mesophilic cellulolytic microorganisms have been reported, efficient and economical bioconversion to simple sugars is still a challenge. Thermostable cellulolytic enzymes play an indispensible role in degradation of the complex polymeric structure of LCB into fermentable sugar stream due to their higher flexibility with respect to process configurations and better specific activity than the mesophilic enzymes. In some anaerobic thermophilic/thermotolerant microorganisms, few cellulases are organized as unique multifunctional enzyme complex, called the cellulosome. The use of cellulosomal multienzyme complexes for saccharification seems to be a promising and cost-effective alternative for complete breakdown of cellulosic biomass. This paper aims to explore and review the important findings in cellulosomics and forward the path for new cutting-edge opportunities in the success of biorefineries. Herein, we summarize the protein structure, regulatory mechanisms and their expression in the host cells. Furthermore, we discuss the recent advances in specific strategies used to design new multifunctional cellulosomal enzymes, which can function as lignocellulosic biocatalysts and evaluate the roadblocks in the yield and stability of such designer thermozymes with overall progress in lignocellulose-based biorefinery.
The activity of organophosphorus hydrolase (OPH) that catalyzes the hydrolysis of neurotoxic organophosphates (OPs) was reported to evolve from lactonase.
In this study, a putative OPH from Acinetobacter sp. (AbOPH) exhibited high lactonase activity with latent OPH activity. Sequence alignment and phylogenetic tree analysis revealed the unique status of AbOPH in evolution. The crystal structure of AbOPH was determined at 2.0 Å resolution and a semi-rational design was performed to enhance the OPH activity of AbOPH through a consensus sequence approach. Compared with wild-type AbOPH, which exhibited undetectable activity toward methyl-parathion (MP), the best variant AbOPHI211A showed markedly improved catalytic efficiency (1.1 μmol min−1 mgprotein −1) toward MP. Docking studies suggested that the mutation Ile211Ala affects substrate recognition and stabilizes substrate conformation.
This result presents the emergence of new enzyme function by a simple mutation strategy and confirms the high possibility that OPH was evolved from its lactonase ancestor.
A significant fraction of short fibers commonly called “reject fines” is produced while recycling wastepaper at paper mills producing linerboard. These fines are usually rejected into the solid waste stream that further requires land filling and poses environmental problems. The major component of these rejects is cellulose that can be a potential source of fermentable sugars for biofuels, bioplastics or other products. Therefore, a feasible process for converting these reject fines into sugars can profit the paper mills by producing value for their waste products while simultaneously mitigating their adverse environmental impact by avoided solid waste. Additionally, the sugar feedstocks can be used to reduce fossil carbon contributing to the sustainability of the industry.
Enzymatic conversion of rejects fines from paper mills was achieved using commercial cellulases from Trichoderma reesei. The presence of mineral particles along with the cellulosic fines was found to have potent inhibitory effects on enzyme hydrolysis. The mineral particles are kaolin and calcium carbonate and originate from the fillers used in the wastepaper. The adsorption of the cellulase onto these mineral components was measured and quantified by the slope of the adsorption isotherm. The application of a nonionic surfactant Tween-80, decreased the adsorption of cellulase and this improved the hydrolysis yield of sugars.
Enzymatic hydrolysis of rejects from recycled paper mills is feasible and provides a source of sugars for biofuels and bioplastics. However, the presence of mineral particles can be detrimental to this bioconversion. Calcium carbonate which occurs as a filler in waste paper shows high adsorption affinity to the cellulase enzymes and thus reduces the available enzyme for cellulolysis. This can be remedied by the application of surfactants which preferentially occlude to the mineral surfaces and thus increase enzyme availability in solution. The non-ionic surfactant, Tween-80, shows the best hydrolysis enhancement at a dosage of 3 % based on the dry weight of the biomass.
The utilization of both C6 and C5 sugars is required for economical lignocellulosic bio-based processes. A co-culture system containing multiple strains of the same or different organisms holds promise for conversion of the sugar mixture available in different lignocellulosic feedstock into ethanol.
Herein a co-culture kinetic model has been developed which can describe the co-cultivation of S. stipitis and S. cerevisiae for ethanol fermentation in mixed C6/C5 sugars. The predicted fermentation kinetics and ethanol production performance agreed well with experimental results, thus validating the model. The co-culture kinetic model has been implemented to design the optimal cell ratio for efficient conversion of rice straw or sugarcane bagasse feedstock into ethanol. The results reveal that the optimal co-culture system could enhance ethanol titer by up to 26 %, and ethanol productivity by up to 29 % compared to a single-strain culture. The maximum ethanol titer and productivity reached by the optimized co-culture was 46 and 0.49 g/l h, respectively.
The co-culture model described here is a useful tool for rapid optimization of S. stipitis/S. cerevisiae co-culture for efficient and sustainable lignocellulosic ethanol production to meet the economic requirements of the lignocellulosic ethanol industry. The developed modeling tool also provides a systematic strategy for designing the optimal cell ratio of co-culture, leading to efficient fermentation of the C6/C5 sugars available in any biomass feedstock.
Malachite green (MG) is a triphenyl methane cationic dye which is used to color fabrics and is employed as food additive, food coloring agent and medical disinfectant. MG is found to be toxic to aquatic organisms, animals including humans. Copper is a commonly found metal in environment due to anthropogenic activities. Most of the microorganisms show sensitivity toward it. This adversely affects their growth and activity. In the present study, biodegradation of MG by a copper-tolerant bacterium has been investigated. Biodegradation was confirmed by UV–Vis and FTIR spectroscopy. The metabolites generated after degradation of MG were identified by LC/MS and a plausible pathway of MG degradation has been elucidated. Microbial and phyto toxicity of generated metabolites were also evaluated.
A strain belonging to Ochrobactrum pseudogrignonense strain GGUPV1 was discovered from copper mine waste water. This bacterium could tolerate as high as 50 mM copper sulfate in minimal medium. It was observed that this bacterium could degrade 400 mg/L of MG in minimal medium. Decolorization of MG was also observed in presence of copper sulfate in the medium. Degradation of MG was confirmed by UV–Vis and FTIR spectroscopy. GC/MS study indicated that metabolites generated after degradation of MG were nontoxic to Staphylococcus aureus.
This is the first report showing degradation of MG by Ochrobactrum pseudogrignonense. This strain can be successfully employed for degradation of MG.
Rapamycin is produced from Streptomyces hygroscopicus, and was initially identified as an antifungal antibiotic. More recently, rapamycin has been found to have various medical applications, including in relation to immunosuppression and anti-aging. Due to its complex structure, biological production is the major route for commercialized rapamycin production. The conventional fermentation process requires a large seed fermenter for the inoculation process (in general, the volume of the seed fermenter is equal to 5–10 % that of the production fermenter), which presents challenges with regard to scaling up production, due to the high investment costs of seed fermenters. This study explored different inoculation strategies for rapamycin production in a 15-L agitation fermenter.
The results indicated that solid-state fermentation (SSF) using barley as the substrate is a suitable method for the inoculation. The highest rapamycin concentration measured in the batch with SSF (barley) inoculated was about 520 mg/L, which was significantly higher than that of 400 mg/L obtained in the batch inoculated with 5 % liquid seed medium. Besides the higher rapamycin production, using SSF of barley as the inoculation method can greatly reduce both the labor and cost requirements.
The usage of mycelium-covered barley as the solid substrate for the inoculation of 15-L fermenter leading to a higher rapamycin production compared to that of conventional liquid seed medium. The solid-state inoculation method can avoid both the intensive labor requirement and costly seed fermenter needed with the latter approach. This inoculation method thus has the potential to be applied to the large-scale production of rapamycin.
Carbonic anhydrase (CA, EC 4.2.1.1), an ancient enzyme and the fastest among many enzymes, is a useful biocatalyst for carbon capture use and storage (CCUS). The use of alkaline buffers and high temperatures are favorable for biomineralization. Hence, the stability of CA under such harsh conditions is extremely important for its practical application.
Herein, we report a new thermostable and alkaline-tolerant α-CA (designated as LdCA), with only 26 % identity to bovine CA (BCA), which was identified by genome mining from Lactobacillus delbrueckii CGMCC 8137. It was overexpressed in Escherichia coli in a soluble form and purified to electrophoretic homogeneity by His-Trap affinity chromatography. The dimer protein had a subunit molecular weight of 23.8 kDa and showed extremely high stability at pH 6.0–11.0 and 30–60 °C. Its activity was maintained even after incubation at 90 °C for 15 min. The half-lives of the enzyme measured at 30, 40, and 50 °C were 630, 370, and 177 h, respectively. At pH 9.0, 10.0, and 11.0, its half-lives were 105, 65, and 41 min, respectively. LdCA was applied at 50 °C to accelerate the formation of calcium carbonate in a vaterite phase.
In summary, a new CA with high thermal and alkaline stability was identified from a general bacterium, demonstrating an effective strategy for discovering new and useful biocatalysts.
The increasing incidence of degenerative diseases has attracted the interest in the obtaining of bioactive compounds. Since seeds and skins from grapes are important sources of polyphenols which have been associated with cancer incidence decreasing, then, one of the pisco (alcoholic beverage made of grape) manufacturing byproduct such as lees, could be a potential source of polyphenols. Supercritical fluid extraction is an environmentally friendly technique that has been applied for obtaining polyphenols. Carbon dioxide is used as unique or main extraction solvent instead of organic solvents, most of them toxics and responsible for reducing the application fields of the extracts. For that reason, among others, supercritical fluid extraction is preferred over conventional techniques for obtaining bioactive compounds. The aim of this work was to study the supercritical fluid extraction of polyphenols from lees of pisco-making. Supercritical carbon dioxide with 10 % of ethanol (w/w) was used as extraction solvent. Overall extraction curves were determined at 20 and 35 MPa; and the experimental data were used to estimate the kinetic parameters. Conventional techniques using ethanol as extraction solvent were performed for comparative purposes. The extracts were analyzed by thin-layer and high-performance liquid chromatography.
Lower global yield was obtained by supercritical fluid extraction than conventional techniques. From the kinetic parameters, the mass transfer rate and the amount of the extract dissolved in supercritical phase were higher at 20 than 35 MPa. Phenolic acids (gallic, protocatechuic, vanillic, syringic, ferulic derivatives and p-coumaric derivatives) and flavonoids (quercetin and its derivatives) were identified in the extracts obtained by all extraction techniques. Polyphenols were rapidly extracted with supercritical fluid and more concentrated extracts were obtained at 20 MPa. However, for longer extraction times, the highest values of extracted polyphenols were obtained by conventional techniques.
Lees from pisco-making are a promising source for recovery polyphenols. Low global yields were obtained when elevated pressures were used. Although supercritical fluid extraction at 20 MPa was the most efficient technique on the extraction of polyphenols from lees of pisco-making due to highly concentrated polyphenols, extracts were rapidly obtained.
The aim of this study was to use traditional mutagenesis to generate hyper-cellulolytic mutants with emphasis on stable, non-spore formers, shorter enzyme producing times and higher saccharification efficiency at high solid loadings. An in-house isolated strain of Aspergillus terreus (At) was identified, fingerprinted and mutated. A sequential process of mutation followed by stringent selection generated mutant At9, which produced optimal cellulase at day 4 instead of day 7, was non-spore former with high stability and grew on a lower pH than parental strain. At9 cellulases were used successfully at high solid loads [up to 25 % (w/v)] in a modified system at 50 °C with reduced hydrolysis times compared to parent strain.
In current work ultra violet (UV) mutagenesis and intelligent screening design combined with growth on a cheap substrate for enzyme production was demonstrated. With this work we present a single organism enzyme system with substantially lower production time and decreased saccharification time at high solid loads.
In this century, the development of nanotechnology is projected to be the establishment of a technological evolutionary of this modern era. Recently, nanotechnology is one of the most active subjects of substantial research in modern material sciences and hence metal nanoparticles have a great scientific interest because of their unique optoelectronic and physicochemical properties with applications in diverse areas such as electronics, catalysis, drug delivery, or sensing. Nanotechnology provides an understanding on fundamental properties of objects at the atomic, molecular, and supramolecular levels. Besides, nanotechnology also leads an alternative technological pathway for the exploration and revolution of biological entities, whereas biology provides role models and biosynthetic constituents to nanotechnology. The findings of this review are important to provide an alternative for the green synthesis of silver nanoparticles. It showed more cost-effective and environmental friendly application as well as easier for large production, with relation to the properties of silver nanoparticles as antimicrobial, can be served well as an alternative antiseptic agent in various fields. Typically, silver nanoparticles are smaller than 100 nm and consist of about 20–15,000 silver atoms. Due to the attractive physical and chemical properties of silver at the nanoscale, the development of silver nanoparticles is expanding in recent years and is nowadays significant for consumer and medical products.
An exopolymer producing bacterial strain was identified as Halomonas sp. S19 by 16S rRNA gene sequencing isolated from Mandapam, Southeast coast of India. Strain S19 produces a significant amount of exopolymer (320 mg L−1) in a medium optimized with 2.5 % glucose, 0.6 % peptone, 7.5 % salt and pH 7.5 at 35 °C. The exopolymer consists of total sugars (65 %), proteins (4.07 %), uronic acids (8.08 %) and sulphur contents (6.39 %). FT-IR and 1H NMR analysis revealed the presence of functional groups corresponding to carbohydrates, proteins and sulphates. The exopolymer of Halomonas sp. S19 emulsifies different oils. However, 10 % exopolymer shows 55.18, 55.18, 49.81 and 24.62 % of emulsifying activity for sesame oil, coconut oil, paraffin and kerosene. The present study was focused on optimisation of exopolymer production using Box–Behnken experimental design and its possibility for potential emulsification index.
In recent year, aqueous two-phase system (ATPS) has become a proven tool used in separation and purification technology. The application of ATPSs in clarification, partitioning and partial purification of biomolecules and bioproducts had showed the rapid development. This method is able to give high recovery yield and high purity in a single step. The ATPS shows characteristics of high selectivity and is easily to scale up. Therefore, ATPS offers an attractive alternative that meets the requirements of the high demand in industry processes and it is also beneficial in terms of economic and environmental protection. In the past, a lot of works and researches have been done in order to develop feasible separation processes using different types of ATPSs and their applications in numerous product separations. This paper aims to review on the recent literature works in the development of different type of ATPSs and their applications in novel separations and purifications of biomaterials.
