Lignocellulosic feedstocks, which are currently under-exploited, can be used for the production of biofuels, such as ethanol, and for biorefinery applications to produce a variety of value-added products. Although bioconversion of lignocellulose by microbial or yeast fermentation have been reported, efficient and economical lignocellulosic fermentation process is still a challenge due to multiple process parameters involved for bioprocess design, optimization and scale-up. Bioprocess modelling strategies have been proven effective for achieving high-production process* efficiency in yield, productivity or titer of desired product. Several types of bioprocess modelling for lignocellulosic application have been developed and successfully validated as a promising alternative for rapid design, optimization and scaling up of biomass-based process. This review aims to summarize the important development of bioprocess modelling for lignocellulosic bioprocess applications towards the success of biorefineries and bio-based economy. In particular, we discuss modelling relevant to lignocellulosic bioprocess including cell modelling based on kinetics, stoichiometry and integrative approaches and fermentation kinetic modelling for process performance assessment. An overview of these modelling approaches and their application for systematic design of efficient and economical lignocellulose-based bioprocesses are given.
As nonionic surfactants derived from naturally renewable resources, sugar fatty acid esters (SFAEs) have been widely utilized in food, cosmetic, and pharmaceutical industries.
In this study, six enzymes were screened as catalyst for synthesis of glucose laurate. Aspergillus oryzae lipase (AOL) and Aspergillus niger lipase (ANL) yielded conversions comparable to the results obtained by commercial enzymes such as Novozyme 435 and Lipozyme TLIM. The productivity obtained by AOL catalysis in anhydrous 2–methyl–2–butanol (2M2B) (38.7 mmol/L/h and 461.0 μmol/h/g) was much higher than the other literature results. Factors affecting the synthetic reaction were investigated, including water content, enzyme amount, substrate concentrations and reaction temperature. The process was greatly improved by applying the Box-Behnken design of response surface methodology (RSM). Solubilities of glucose in 14 different organic solvents were determined, which were found to be closely associated with the polarity of the solvents.
Aspergillus oryzae lipase is a promising enzyme capable of efficiently catalyzing the synthesis of sugar fatty acid esters with excellent productivity.
Cross-linked enzyme aggregate (CLEA) is considered as an effective technique in the production of immobilized biocatalysts for its industrially attractive advantages. Simplicity, stability, low cost, time saving and reusability are proved to be some of CLEA’s main advantages.
In this study, an active, stable and recyclable CLEA-protease from the viscera of channel catfish Ictalurus punctatus has been prepared. Optimization of the preparation parameters is carried out with the help of Response Surface Methodology. This methodology helped in studying the interaction between the most contributing factors such as cross-linker, precipitant and the additive concentrations. The optimum specific activity for CLEA-protease of 4.512 U/mg protein has shown a high stability against the denaturation forces such as temperature and pH as compared to free protease. It is further found from the study that the highest activity was achieved at the pH of 6.8 and at the temperature of 45 °C. After six cycles, CLEA-protease maintained 28 % of its original activity. Additionally, Michaelis–Menten models were used to determine the kinetic parameters i.e. Km and Vmax that helped in showing a significant difference after immobilization as compared to free protease.
This work found that this novel CLEA-protease can be used as a very active biocatalyst in industrial applications.
In a conventional syngas fermentation process, gas was released into the fermentation broth through a single orifice or multiple orifices, except the hollow fiber membrane reactor. Consequently, a simplified bioreactor has been developed employing an innovative gas supply and effluent extraction systems.
A continuous stirred tank bioreactor (CSTBR) has been developed by incorporating an innovative gas supply and effluent extraction system to ferment syngas into ethanol. The working volume of the bioreactor was controlled to 2 L. The CO gas was fermented in the developed bioreactor by using a microorganism (Clostridium ljungdahlii) with different gas (5–15 mL/min), media, and effluent flow rates (0.25–0.75 mL/min) and stirrer speed (300–500 rpm). Gas was diffused into the fermenting broth through an aqueous aeration tube commonly used in the small household aquarium, placed at the bottom layer throughout the periphery. The effluent was extracted from the top layer of the broth by using a membrane separator. Ethanol and acetic acid concentrations were varied from 0.17–1.17 and 8.50–23.68 g/L-effluent, respectively.
It seems that the performance of CSTBR can be enhanced with an innovative gas supply system, which may reduce the gas bubble size and result in higher lateral velocity at the releasing point, especially, throughout the periphery instead of the center of the reactor through a single or multiple orifice.
Optimization of chemically defined medium has been a critical way to produce monoclonal antibody. Usually, amount of glutamine was added into the feed medium, but a half of asparagine was added. Our study found that asparagine was important in the antibody production phase. Increasing the ratio of asparagine to glutamine in feed medium for enhancement of antibody production in CHO-DHFR cell culture would be an efficient way.
We optimized the total amount and the ratio of the two vital amino acids in feed medium to increase antibody production. In this work, we have demonstrated that feeding medium of high ratio between asparagine and glutamine (FB-H) can enhance the cell density after reaching the stationary phase. Moreover, FB-H was shown to improve cell maintenance, and increased the antibody production. The metabolic flux analysis proved that ratio of asparagine to glutamine had little influence on glycolysis. Furthermore, the TCA cycle of FB-H was enhanced by 20 % compared to that of low ratio of asparagine to glutamine (FB-L). And the energy metabolism of FB-H was 22.6 % higher than that of FB-L. For the later, lactate can be less produced in FB-H.
We should improve the ratio between asparagine and glutamine in feed medium properly under the premise of no influence on cell growth to achieve high mAb producing goal.
Wastewaters from the textile industry are an environmental problem for the well-known Colombian textile industry. Ligninolytic fungi and their enzymes are an option for the treatment of these wastewaters; however, the Colombian biodiversity has not been deeply evaluated for fungal strains with ligninolytic activities. In this research, 92 Colombian fungal isolates were collected from four locations around the Aburrá valley, Antioquia, Colombia. Their decolorizing activities were evaluated using Novacron Red, Remazol Black and Turquoise Blue in solid and liquid media at different culture conditions. The best fungal isolate was evaluated in the bioremediation of two real effluents and its enzymatic extracts were used in the decolorization of the three dyes.
From 92 Colombian fungal isolates, Leptosphaerulina sp. exhibited the best decolorization percentage (>90 %) in solid and liquid cultures, and in agitated and un-agitated conditions. Leptosphaerulina sp. effectively decolorized the three dyes and two real effluents from textile industries. This decolorization was catalyzed by the production of significant quantities of laccase (650 U/L) and manganese peroxidase (100 U/L). Leptosphaerulina sp. enzymatic extracts exhibited decolorizing activity when ABTS as mediator was added. Leptosphaerulina sp. decolorized two real effluents from textile industries (>90 %) under conditions of low pH and glucose supplementation. Enzymatic degradation and decolorization products’ innocuity was demonstrated by cytotoxic and chromatographic analyses.
Leptosphaerulina sp. was the best Colombian isolate. This fungal strain achieved a decolorization above 90 % for the three dyes and two real effluents from a textile industry. This decolorization was performed by producing significant amounts of laccase and manganese peroxidase. Leptosphaerulina sp. is an interesting prospect to treat waters polluted with dyes without the production of compounds dangerous for the environment.
Copper is one of the heavy metals whose presence in aquatic environment in higher concentration poses a major threat to the environment. This is due to their toxic effects on the plants, animals and human health. Biosorption an innovative biotechnological technique with superior advantages was used for the remediation of Cu2+ from aqueous solution in this study.
Biosorbent was prepared from raw (RAWB) and oxalic acid modified (OAMB) Saccharum officinarum. They were characterized by scanning electron microscopy (SEM/EDAX), fourier transform infrared (FTIR), and X-ray diffraction (XRD) for surface morphological study. Experimental data obtained from batch equilibrium studies were subjected to two-parameters [Freundlich, Langmuir, Tempkin and Dubinin–Radushkevich (D–R)] and three-parameter [Redlich–Peterson (R–P), Sips, Hill and Toth] isotherm models. Kinetic data were analysed with pseudo first-order, pseudo second-order, Elovich and Avrami kinetic models.
The results of proximate analysis and characterization show that the oxalic acid modification affected the biosorbents content, surface modifications and the functional groups present. The experimental data from the equilibrium studies were best fitted to the isotherms with R 2 >0.9 for the OAMB and RAWB. The adsorption energies (E) from the D-R isotherms were found to be 0.36 and 0.06 kJ/mol for OAMB and RAWB respectively, which is indication of physisorption favoured processes. Pseudo second-order model best fitted the data with a coefficient of determination (R 2) of above 0.998 with an average relative and hybrid errors lower than 5 %. Intraparticulate diffusion model analysis showed that the adsorption process develops in two stages as rapid and slow phase. Changes in standard free energy (∆G°), enthalpy (∆H°) and entropy (∆S°) for the biosorption processes were estimated using the thermodynamic equilibrium model. The calculated thermodynamics parameters indicated that the process is spontaneous and endothermic in nature.
This study revealed the feasibility of Cu2+ removal through biosorption processes using S. officinarum biomass as biosorbent. The improved surface morphology for increase biosorption by oxalic acid modification is also favourable. The biosorption process for the removal of the Cu2+ is pH dependent as the efficiency increased from 36.4 to 65.3 % for raw biomass while the OAMB has increase efficiency from 57.5 to 88.6 as the pH increases from 2 to 6.
The fabrication of silver nanoparticles (Ag-NPs) through green chemistry is an emerging area in the field of medical nanotechnology. Ag-NPs were fabricated by enzymatic reduction of AgNO3 using two lignin-degrading fungus Aspergillus flavus (AfAg-NPs) and Emericella nidulans (EnAg-NPs). The prepared Ag-NPs were characterized by different spectroscopic techniques. Antibacterial activity of prepared Ag-NPs was demonstrated against selected Gram negative (Escherichia coli and Pseudomonas aeruginosa) and Gram positive (Staphylococcus aureus) bacteria in the term of minimum bactericidal concentration (MBC) and susceptibility constant (Z). The synergistic antibacterial activity of Ag-NPs with four conventional antibiotics was also determined by the fractional inhibitory concentration index (FICI) using the checkerboard microdilution method. The antibiofilm potential of Ag-NPs was also tested.
The plasmon surface resonance of biosynthesized Ag-NPs shows its characteristic peaks at UV and visible region (~450 and 280 nm). Fourier transform infrared spectrometer (FTIR) analysis confirms the nature of the capping agents as protein (enzyme) and indicates the role of protein (enzyme) in reduction of silver ions. The average particle size and charge of synthesized Ag-NPs was ~100 nm and ~−20 mV, respectively. X-ray diffraction (XRD) and TEM analysis confirmed the purity, shape, and size (quasi-spherical, hexagonal, and triangular) of Ag-NPs. Energy-dispersive X-ray spectroscopy (EDX) data validate the biological synthesis of Ag-NPs. Low MBC and high susceptibility constant indicate the high antimicrobial strength of biosynthesized Ag-NPs. The antibacterial analysis demonstrates the synergistic antimicrobial activity of Ag-NPs with antibiotics. This study also shows that biosynthesized Ag-NPs have ability to inhibit the biofilm formation by 80–90 %.
The Aspergillus flavus and Emericella nidulans-mediated biosynthesized Ag-NPs have significant antimicrobial activity and demonstrate synergistic effect in combination with antibiotics. It suggests that nanoparticles can be effectively used in combination with antibiotics to improve the efficacy of antibiotics against pathogenic microbes. The substantial antibiofilm efficiency of biosynthesized Ag-NPs would also be helpful against sensitive and multidrug-resistant strains.
The use of residues in industrial processing is expanding due to their low cost and abundant availability. Coconut shell is generated in large amounts in tropical areas due to the fruit processing and the coconut water consumption. In the present work, a new microbial strain was isolated from the coconut shell powder, molecularly identified as Melanoporia sp. CCT 7736 and applied for cellulase production in solid-state fermentation using the green coconut shell powder as substrate.
The complete production process was optimized. Fermentation time was only 24 h, and the enzyme produced presented maximal activity at neutral pH (6.5) and 60 °C. The maximal enzyme activity after extraction optimization was 7.5 IU/gds (international units of enzyme activity per gram of dry solid). For the enzyme extraction, the rotation velocity, the extraction time, the temperature, and the solvent volume (buffer) were optimized using response surface methodology (RSM). The best results for the enzyme extraction were obtained at 250 rpm (orbital shaker) at 30 °C using 13.79 mL of a sodium acetate buffer (200 mM) at pH 6.5 after 10 min. Delignification pretreatment was not necessary since this fungus strain was able to degrade the lignin.
To the best of our knowledge, this work is the first report of cellulase production by Melanoporia sp. CCT 7736. Good results were obtained without the need for expensive pretreatment usually applied to lignocellulosic residues because the strain was isolated from coconut shell powder, and it is well adapted to this kind of substrate. The enzyme presented maximum activity at neutral pH instead of acidic pH as reported for the majority of industrial cellulases. The use of lignified coconut shell and the optimal pH at neutral values are the main advantages of the enzyme produced by Melanoporia sp. CCT 7736. In addition, the enzyme showed good stability during storage even at the crude broth and without any cryoprotection.
Styrene monooxygenases (SMOs) can catalyze the asymmetric epoxidation of alkenes to obtain optically active epoxides. This account describes a series of work of our group on the isolation, application, and improvement of an SMO from Pseudomonas sp. LQ26. The strain was isolated from an active sludge sample based on indigo-forming capacity. Then the gene encoding SMO was expressed in Escherichia coli, and the whole cells were applied in biocatalytic reactions. The substrate spectrum of SMO was successfully expanded from conjugated styrene derivatives to non-conjugated alkenes, especially α-substituted secondary allylic alcohols, affording enantiopure epoxides. Most significantly, cascade reactions involving ketoreductase and SMO were designed, which resulted in glycidol derivatives or epoxy ketones with excellent enantio- and diastereo-selectivity using α,β-unsaturated ketones as the substrates. In addition, mutants of SMO with altered substrate preference and enhanced activity were constructed, which indicated great potential of SMO for further improvement.
In current times, enzyme-catalyzed reactions have gained importance for the development of new chemical processes. These require the production of large quantity of enzyme at low cost. Solid-state fermentation (SSF) is an efficient process because this bioprocess has a potential to convert agro-industrial residues into valuable compounds. Hence, the current study focuses on the optimization of process parameters for the higher production of laccase using a novel basidiomycete fungi Tricholoma giganteum AGHP under solid-state fermentation (SSF). Further, the purification of laccase using column chromatographic technique was performed.
Various physico-chemical parameters were evaluated and maximum production obtained was 2.69 × 105 U/g using wheat straw as a dry substrate. Optimum pH was found to be 5.0 and the temperature of 30 °C with 0.3 mM copper as an inducer. The enzyme was purified from the initial protein preparation by two-step column chromatography. A yield of 10.49 % with 3.33-fold purification was obtained using Sephadex G-75 gel permeation chromatography. Further increase in purification (total) was found to be 10.80-fold with a yield of 8.50 % using DEAE Sephadex A-50 ion exchange column chromatography. The purified enzyme was identified as a monomeric protein with a molecular weight of 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).
In view of the results obtained, we can conclude that the extracellular laccase production is governed by various cultural parameters such as pH, temperature, and the composition of culture medium. “One-factor-at-a-time” methodology was capable of establishing the optimum conditions that significantly increases the enzyme production several folds using lignocellulosic substrate. Therefore, laccase from T. giganteum AGHP has a potential in several industrial applications.
Water, a precious resource for all living organisms has become a diminishing source in the present world. Reuse of water is a way of combating the scarcity of water. The remedy for the polluted water through eco-friendly approaches is a major challenging task for the researchers. Nanotechnology is expected to provide a better solution than the existing methods. Graphene, a novel carbonaceous material widely used in the field of nanotechnology is a promising substance in the treatment of polluted water.
The present work deals with the bio-reduction of graphene oxide using the aqueous extract of Amaranthus polygonoides. The synthesized graphene was embedded with silver nanoparticles and Moringa oleifera pulverized seed powder. This modified graphene was used as adsorbent for simulated textile, tannery and paper mill effluents. The adsorption efficacy of graphene and modified graphene was compared with that of a commonly available adsorbent activated charcoal by analysing the water quality parameters before and after treatment.
The results revealed that the graphene loaded with the silver nanoparticles and M. oleifera seed powder possessed excellent adsorbent properties and showed good efficacy on reusability compared to conventional activated charcoal.
Feathers are the major byproducts of poultry industry and considered as waste. Feathers (composed of protein keratin) are metabolized by a number of microorganisms as a source of carbon and nitrogen. Degradation of feathers results in production of amino acids and peptides, which can be employed as precursors for plant growth-promoting metabolites such as indole acetic acid, ammonia and HCN. The aim of the present investigation was to assess the influence of these metabolites (termed as feather protein hydrolysate) on plant growth promotion activity of keratinolytic bacterial strain Bacillus subtilis PF1.
Strain PF1 exhibits potent keratinolytic activity and can efficiently degrade 10 g/l chicken feathers under submerged cultivation with 81.4 ± 4.40 U/ml keratinase activity. Different concentrations of feathers supported the production of indole acetic acid by strain PF1. Strain PF1 produces maximum indole acetic acid (46.2 ± 0.21 µg/ml) in the presence of 2.0 % feathers at 120 h of incubation. The indole acetic acid production was confirmed by thin-layer chromatography and Fourier transform infrared spectroscopic analysis. However, increased concentration of feathers exhibited negative effect on phosphate solubilization due to increased alkalinity. HCN production also exhibited positive correlation with concentration of feathers. Finally, plant growth of Vigna radiata in the presence of strain PF1 with chicken feathers in soil was investigated, which showed good plant growth promotion activity. Increased ratio of C/N in soil also supported the plant growth promotion activity of feathers.
Feather degradation property of B. subtilis PF1 could be efficiently utilized for feather waste management. The metabolites released by feather degradation along with strain PF1 could be successfully employed as an economic source of nitrogen fertilizers for plants.
Silver nanoparticles (SNPs) play important role in the field of optics and electronics and also as a novel antibacterial agents. Here, we report a simple and green method for the biosynthesis of SNPs using aqueous leaf extract of Origanum majorana and Citrus sinensis as a novel bio source of cost-effective, non-hazardous reducing, and stabilizing agents. A 3 mM solution of silver nitrate was prepared. Five milliliter aqueous leaf extract was slowly added to 20 ml silver salt solution (3 mM) with constant stirring. No noticeable color change was observed. The solution was then heated in domestic microwave for variable time intervals. The intense brown colored solution was obtained on 1 min heating with O. majorana and 5 min heating with C. sinensis extract. The intense brown color indicated the formation of SNPs. The antibacterial activity of synthesized SNPs was investigated.
SNPs were rapidly synthesized using aqueous leaf extract of O. majorana and C. sinensis on microwave irradiation. Formation of SNPs was confirmed by the change in color from yellowish green to brown and absorption maximum around ~420 and 410 nm due to surface plasmon resonance of SNPs. They were also characterized by other physical–chemical techniques like Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope coupled with X-ray energy dispersive spectroscopy, and high-resolution transmission electron microscopy. TEM analysis showed the presence of feather-shaped NPs in O. majorana and spherical as well as cubical-shaped NPs in C. sinensis-mediated SNPs. The synthesized SNPs showed significance antibacterial activity against two human pathogenic strains.
The SNPs were synthesized using leaf extract of plants. This synthesis method is nontoxic, eco-friendly, and a low-cost technology for the large-scale production. The SNPs can be used as a new generation of antibacterial agents.
Application of modern biomass pyrolysis methods for production of biofuels and biochar is potentially a significant approach to enable global carbon capture and sequestration. To realize this potential, it is essential to develop methods that produce biochar with the characteristics needed for effective soil amendment.
Biochar materials were produced from peanut hulls and pine wood with different pyrolysis conditions, then characterized by cation exchange (CEC) capacity assays, nitrogen adsorption–desorption isotherm measurements, micro/nanostructural imaging, infrared spectra and elemental analyses.
Under a standard assay condition of pH 8.5, the CEC values of the peanut hull-derived biochar materials, ranging from 6.22 to 66.56 cmol kg−1, are significantly higher than those of the southern yellow pine-derived biochar, which are near zero or negative. The biochar produced from peanut hulls with a steam activation process yielded the highest CEC value of 66.56 cmol kg−1, which is about 5 times higher than the cation exchange capacity (12.51 cmol kg−1) of a reference soil sample. Notably, biochar produced from peanut hulls with batch barrel retort pyrolysis also has a much higher CEC value (60.12 cmol kg−1) than that (12.45 cmol kg−1) from Eprida’s H2-producing continuous steam injection process. The CEC values were shown to correlate well with the ratios of oxygen atoms to carbon atoms (O:C ratios) in the biochar materials. The higher O:C ratio in a biochar material may indicate the presence of more hydroxyl, carboxylate, and carbonyl groups that contribute to a higher CEC value for the biochar product. In addition, the increase in surface area can also play a role in increasing the CEC value of biochar, as in the case of the steam activation char.
Comparison of characterization results indicated that CEC value is determined not only by the type of the source biomass materials but also by the pyrolysis conditions. Biochar with the desirable characteristics of extremely high surface area (700 m2/g) and cation exchange capacity (> 60 cmol kg) was created through steam activation.
Many methods are available for the concentration of proteins; however, most are not easily scalable due to costs, the need of specialized instruments and skilled workers or are very time-consuming. Three-phase partitioning (TPP) is a separation technique that has gained a lot of interest due to its rapid, simple and scalable use for concentration, isolation and decontamination of proteins from crude samples with high recovery yields. In the present work, the effect of various parameters of TPP was evaluated to optimize the concentration of proteins from Chlorella pyrenoidosa (CP), is green algae that increasingly being used as food supplements because of its positive impacts on human health.
Chlorella pyrenoidosa was cultivated in a closed system under controlled conditions. After reaching maximum growth, the microalgae was harvested, dried and powdered. Afterwards, TPP of CP cell lysate was done to concentrate protein content. To maximize protein concentration, various parameters were optimized such as solvent (t-butanol), ammonium sulphate concentration (40 % w/v), solid load (0.75 g/20 mL), pH (6), incubation time (20 min), slurry to butanol ratio (1:1.5) and enzymatic treatment (combination of Stargen™ and Carezyme™). Also, total starch, cellulose and carbohydrate content before and after the enzymatic treatment were determined to comprehend the impact of enzymatic treatment on protein concentration. Using these optimized parameters, 78.1 % w/w protein concentration was obtained in middle protein concentrate phase. This protein concentrate was characterizedfor proximate composition, colour analysis, water holding capacity, oil-holding capacity, foaming capacity, foam stability, amino acid composition, protein quality and thermal properties.
Various process parameters of TPP influence the protein concentration of middle protein concentrate phase. Enzymatically treated biomass also enhanced protein concentration in middle protein concentrate phase. Characterization of protein concentrate revealed the presence high-quality protein. Therefore, it is possible to implement TPP at an industrial scale for protein concentration.
Gaseous substrates such as O2 and CO2 are often required in fermentation processes. However, a simple methodology to compare different gas supply strategies using gaseous substrates from different sources is missing.
In this study, we present a methodology to identify and theoretically compare different configurations to supply mixtures of gaseous compounds to fermentations that consume these gases. For the different configurations that were identified, all gas flow rates can be calculated in terms of other process parameters such as optimal concentrations of the gaseous compounds in the liquid phase, top pressures of the fermentation, and consumption/production rates. The approach is demonstrated for fumaric acid fermentation with Rhizopus delemar, which consumes O2 and can theoretically produce or consume CO2. Three different gas supply configurations were identified: Air supplemented with O2, a mixture of O2 and CO2, and air supplemented with CO2. All three configurations lead to gas supply costs in the same order of magnitude. O2 and CO2 prices and consumption rates determine which configuration is best. However, the overall production costs will not be dominated by the gas costs, but by the glucose costs.
The presented methodology enables a simple way to identify and compare different gas supply strategies for fermentations that require more than one gaseous substrate. This includes the costs for compression of gases. Other substrate costs are easily added for overall process optimization.
Bioformulations are the preparations that contain beneficial microorganisms as active ingredients and they may represent a novel alternative to be used in crop protection because of their safety to humans and non-target organisms. Xenorhabdus sp. is an entomopathogenic bacterium that symbiotically associates with nematodes of the family Steinernematidae and has potential to be used in bioformulations due to its pesticide activities. The aim of this study was to determine the efficacy of bioformulations containing Xenorhabdusstockiae PB09 for controlling mushroom mites.
The results showed that different Xenorhabdus bioformulations, including wettable powder (WP), liquid cell pellet (LC), and liquid supernatant (LS) were shown to cause very high miticidal activities at 90.25, 86.50, and 92.78 %, respectively. When X. stockiae PB09 bioformulations were stored at room temperature (28 ± 2 °C) and 4 °C for up to 60 days, their viable cells and efficacy were found to decrease. However, storing at 4 °C could relatively maintain both viable cells and efficacy of the bioformulations, especially after 45 days of storage, whereby all the formulations that were kept at 4 °C had 5–10 % and 2–15 times higher miticidal activities and viable cells than that kept at room temperature, respectively. Storing at 4 °C was more appropriate than room temperature for maintaining both viable cells and miticidal activities of all X. stockiae PB09 bioformulations.
In conclusion, this study showed that WP and LC formulations were found to be effective and have potentials to be further developed as commercial products for controlling mushroom mites.
Palm oil mill effluent (POME) generated from the palm oil milling process contains high organic matter from which the molecule of interest for bioconversion such as fermentable sugar can be derived. This study examined the enzymatic hydrolysis of complex celluloses and polysaccharides present in POME with the aim to release the simple sugars as low-cost fermentable feedstock convertible into bioflocculant.
The maximum concentration of fermentable sugar produced after 24 h of hydrolysis was 30.5 g/L. The POME hydrolysate served as a carbon source for Bacillus marisflavi NA8 to produce bioflocculant. The optimum conditions obtained were an initial pH of 7.0 and a temperature of 37 °C with an inoculum size of 5 % (v/v), yielding 6.4 g/L or 32 kg/t bioflocculant from POME.
This finding indicated that POME could be utilized as a low-cost substrate to improve the feasibility of commercial production of bioflocculant.
Carboxypeptidases are exopeptidases that catalyze the release of amino acids from the C-terminus of peptides or proteins. The peptides consisting of hydrophobic amino acids taste bitter. Therefore, the hydrolytic capability of carboxypeptidase toward hydrophobic amino acids at the C-terminus of peptides is useful for the degradation of bitter peptides.
Using the genome data of Streptomyces cinnamoneus TH-2, we expressed and characterized a novel metallocarboxypeptidase (TH2-CP) in Streptomyces lividans. TH2-CP had a molecular mass of 37.7 kDa. As TH2-CP possesses a zinc-binding consensus motif (HXXE……H) and N-terminal prosegment residues, we suggest that TH2-CP could be classified into the M14A subfamily. In the presence of Z-Gly-Leu as the substrate, TH2-CP showed optimum activity at pHs 7 and 8 in potassium phosphate and Tris–HCl buffers, respectively. The optimum temperature for activity was 51 °C. Furthermore, 50 % activity was conserved after incubation at 38 °C for 30 min. TH2-CP showed broad substrate specificity, with a preference for hydrophobic amino acids, as demonstrated by casein hydrolysate breakdown.
A novel metallocarboxypeptidase, TH2-CP, from S. cinnamoneus TH-2 was characterized. TH2-CP preferred substrates with hydrophobic amino acids at the C-terminal position for casein peptides. This property indicates that TH2-CP can be used to decrease the bitterness of peptides in food industries.
Nowadays, biodiesel has become more attractive in the quest for a novel fuel source due to its environmental benefits and the biodegradable properties. Transesterification of Helianthus annuus (sunflower) seeds oil is one of methodsacci for producing biodiesel fuel. However, during this process, the impurities are frequently found in the synthesized biodiesel which degrades the quality of biodiesel. Therefore, a proper catalyst is required to obtain the high-quality biodiesel. In this study, a series of aminated polyethersulfone (PES-
Based on elemental analysis, lipase was successfully attached onto PES-
Biodiesel from sunflower seeds oil was successfully synthesized through a transesterification reaction catalyzed by Mucor miehei lipase immobilized onto the synthesized aminated PES membrane. The addition of glutaraldehyde as a cross-linker showed an excellent enzyme loading value and led to significantly increased biodiesel conversion efficiency. This method is good to be developed, since the immobilized enzyme stability could be increased, the separation process was easier, and a high yield with good quality of biodiesel could be obtained.
Biodiesel, one of the most important sources of renewable energy, is produced in large quantities around the world; however, its production generates different kinds of residues and by-products which raise economic and environmental concerns. This review presents a compilation of the data on current state of transformation of residues and by-products of biodiesel industry into products that are suitable for bio-refining. The review has analyzed glycerol, biodiesel washing wastewaters, and solid residues. The technologies were described and the most significant experimental results and variables were summarized to allow researchers an easy access to this information.
Bilobalide and ginkgolides are reported to be present only in Ginkgo biloba. However, only trace amounts of bilobalide and ginkgolides are contained in the ginkgo leaves. Nowadays, there has been considerable interest in plant cell cultures as a potential alternative to traditional agriculture for the industrial production of secondary metabolites. Much effort has been put into the use of in vitro cultures as one attractive biotechnological strategy for producing bilobalide and ginkgolides of commercial interest. The aim of this study was to enhance the production of bilobalide and ginkgolides A, B, and C in cell cultures of G. biloba using immobilized cell cultures and the process of elicitation.
Based on the effect of the immobilization components on the free cell suspension cultures, it was considered that the jute fiber acted as an elicitor and forced the cells to release the product into the culture media. The resulting biomass was approximately 1.4 times higher than in the cell suspension cultures, and the production of bilobalide and ginkgolides A, B, and C increased 5.0, 3.3, 6.1, and 4.1 times, respectively. Eliciting with methyl jasmonate (MJ) and salicylic acid (SA) in the immobilized cells enhanced bilobalide and ginkgolides A, B, and C, compared with the unelicited controls. The highest accumulation was observed using a combination of 0.1 mM MJ + 0.1 mM SA in the immobilized cells, which produced 1.78, 1.95, 2.05, and 2.95 times more bilobalide and ginkgolides A, B, and C, respectively, than the controls.
The positive effects of immobilized cell cultures using jute fiber and the synergism effect of SA and MJ on immobilized cells of G. biloba appear to be the optimal conditions for continuous in vitro production for commercial purposes. This is the first report on analyzing the effects of jute fiber as immobilized cell material on G. biloba cell cultures and the synergism of MJ and SA on bilobalide and ginkgolide production.
In this study, polyurethane (PU) films from palm kernel oil-based polyester (PKO-p) incorporated multi-walled carbon nanotubes (MWNTs) were prepared via evaporative casting method. Nanoparticle fluid dispersions containing 0.01, 0.04 and 0.08 % wt. of MWNTs were added into PKO-p-based resin and mixed by digital probe sonicator for 20 min followed by mixing with isocyanate to produce PU-MWNTs composite films. The mechanical properties, water resistance, water vapor transmission rates (WVTR), biocompatibility, and antibacterial activities of the PU-MWNTs composite films were examined.
Results show that PU containing 0.01 wt. % of MWNTs demonstrated optimum mechanical properties as it possessed high tensile strength, modulus, and good flexibility compared to PU film and other PU-MWNTs composite films. There are no significant difference in swelling values as well as water vapor transmission rates for PU film and PU-MWNTs composite films. All the films showed low swelling values (17–23 %) and WVTR values in the range 181–269 g m−2 d−1. Cell studies revealed that PU and PU-MWNTs composite films are non-cytotoxic to human skin fibroblast cells (CRL2522) and the cell proliferation was increased after incubation of 72 h. The in vitro qualitative antibacterial results showed both PU and PU-MWNTs composite films exhibited bactericidal effect against Gram-positive (Staphylococcus aureus and Bacillus cereus) and Gram-negative bacteria (Escherichia coli and Klebsiella pneumonia).
In summary, incorporation of MWNTs improved the mechanical properties of the polyurethane films with no cytotoxic effect against normal human skin fibroblast cells.
The production of bioethanol by co-culture of cellulolytic and xylanolytic bacteria isolated from agro-waste-impacted soil through simultaneous saccharification and co-fermentation (SSCF) of steam-exploded bagasse was investigated.
The cellulolytic (VCE-19) and xylanolytic (VXE-41) isolates were screened using the Congo Red Plate Method. The DNS method was used in the determination of reducing sugar content. Chemical analysis of the sugarcane bagasse was determined using standard methods. The bagasse was subjected to steam explosion to reduce lignin content and enhance cellulose availability.
Mean proximate composition analysis of the bagasse showed total carbohydrate and lignin content (% dry weight) of 70.3 ± 1.9 and 19.2 ± 1.2 before pretreatment and 85.4 ± 2.33 and 4.2 ± 0.44 after pretreatment, respectively. Phylogenetic analysis based on partial sequence of the 16S rRNA gene classified VCE-19 and VXE-41 as Bacillus cereus GBPS9 and Bacillus thuringiensis serovar kurstaki HD1, respectively. The sequences obtained from these isolates have been submitted to GenBank and accession numbers (KT350986.1 for VXE-41 and KT318371.1 for VCE-19) assigned to them. The result of the optimization of cultural conditions of the bacterial co-culture revealed optimum cellulase production at the following conditions: temperature, 40 °C; pH, 7; substrate concentration, 4.0 % (w/v); inoculum concentration, 4 % (v/v) and when yeast extract was used as nitrogen source. The gas chromatography–mass spectrometry (GC–MS) analysis of the fermentation broth detected the following components: acetone (3.49 g/L), ethylacetate (8.75 g/L), ethanol (19.08 g/L), N-propanol (4.96 g/L), isobutanol (3.73 g/L) and acetic acid (6.53 g/L).
This study has demonstrated the production of significant quantity of ethanol by a co-culture of B. cereus GBPS9 and B. thuringiensis serovar kurstaki HD1 through SSCF of steam-exploded bagasse. Efficient bioethanol production from bagasse can help solve the need for alternative source of energy and the crisis that results from bioethanol production from food and feed crops.
The aim of the present investigation was to develop Acacia lignin-gelatin (LG) blended films using glycerol as plasticizer and to establish correlation between lignin contents and structure, thermal and mechanical properties of the film. Acacia lignin extracted by alkali method was used for the preparation of LG blended films by solution casting method.
Solubility and swelling tests of the films concluded that the lignin incorporation reduced water affinity of film. Lignin incorporation produces a noticeable plasticizing effect on the blended film, showing optimum values for film incorporated with 20 and 30 % (w/v) lignin, as deduced from their mechanical and thermal properties. Lignin blended film had lower glass transition temperatures (Tg) as compared to control gelatin. Infrared spectroscopy (FTIR) analysis of films suggested that lignin interacts with gelatin by hydrogen bonding and hydrophobic interaction consequently creating conformational changes. Atomic force microscopic (AFM) study displays smooth surface of synthesized films. Light barrier properties of film revealed that the lignin addition improved barrier properties against UV light in the range of 280–350 nm. Furthermore, the lowest scavenging activity was observed in LG-E (111.10 µg/ml) trailed by LG-D (249.29 µg/ml) and LG-C (259.53 µg/ml).
The LG films showed improved light barrier and antioxidant properties with low cytotoxicity, displaying great potential in food packaging and coating for preventing ultraviolet induced lipid oxidation with an extended biomedical applications.
Cow dung, an excreta of bovine animal, is a cheap and easily available bioresource on our planet. Many traditional uses of cow dung such as burning as fuel, mosquito repellent and as cleansing agent are already known in India. Cow dung harbours a diverse group of microorganisms that may be beneficial to humans due to their ability to produce a range of metabolites. Along with the production of novel chemicals, many cow dung microorganisms have shown natural ability to increase soil fertility through phosphate solubilisation. Nowadays, there is an increasing research interest in developing the applications of cow dung microorganisms for biofuel production and management of environmental pollutants. This review focuses on recent findings being made on cow dung that could be harnessed for usage in different areas such as medicine, agriculture and industry.
Rhizopus fungi is suitable for the production of lactic acid, which is the backbone material of polylactic acid used as green plastic from lignocellulosic biomass, since it can grow and ferment in simple medium with various carbon sources such as starch and cellulose. Although paper sludge (PS) contains a lot of cellulosic fibers and in general was incinerated for volume reduction and heat recovery, other efficient utilizations have hardly been developed. In effective production of lactic acid from PS, the research of the extraction of cellulosic fiber from raw PS to obtain effectively fermentable sugars by cellulase and the selection of lactic acid microorganism are necessary. In this study, the PS pretreatment method with NaOH and HCl and the optimization of cellulase reagent were achieved, and also a desirable thermotolerant Rhizopus was selected. Finally, the production of lactic acid from the treated PS at 40 °C by simultaneous saccharification and fermentation (SSF) with the strain and an optimized cellulase cocktail was investigated.
Rhizopus oryzae NBRC 5384 was selected for thermotolerant lactic acid production from Rhizopus library because of its heat tolerance up to 40 °C and high lactic acid production of 80 g/L. The strain can ferment to lactic acid from hexose, pentose, sugar alcohol, disaccharide and starch. The soaking of raw PS in NaOH and HCl was able to reduce effectively inorganic materials and other reagents for repulping, and the content of Al and Ca per PS dry matter was mainly decreased from 32.9 and 30.8 to 14.1 and 1.66 %, respectively. SSF of the treated PS of 50 g/L with optimized cellulase cocktail and 5384 at 40 °C resulted in lactic acid production of 9.33 g/L for 96 h.
The thermotolerant Rhizopus fungus was found based on its high performance in lactic acid production at high temperature from not only glucose, but also other various carbon sources including polysaccharides and the secretion of amylases and cellulases. The treatment of raw PS by NaOH and subsequent HCl was able to remove a large amount of inorganic materials with decrease of hydrophobicity. In SSF of the treated PS with the strain and the optimized cellulase cocktail, lactic acid was able to be produced. However, the increase of initial PS concentration in SSF led to the decrease of the yield with ethanol production, because of limited aeration due to increase of density. An appropriate oxygen supply to the strain is necessary to improve lactic acid production.
Present study aims to isolate and optimize fermentation conditions of milk-clotting enzyme producing rhizobacteria Bacillus amyloliquefaciens SP1.
A bacterium producing an extracellular milk-clotting enzyme (MCE) was isolated from the rhizosphere of the planted population of apple trees growing at Distt. Chamba of Himachal Pradesh, India. According to morphological, physiological, biochemical, and molecular characterization, isolate was identified as B. amyloliquefaciens. Single-factor testing was used to study the optimum physical conditions and nutritional parameters for production of the MCE per proteolysis activity which insures its usefulness as new source of milk coagulant for cheese making. The optimum conditions for production of the milk-clotting enzyme were: temperature, 30 °C; inoculum size, 1 %; and initial pH of the medium, 6.0. The maximum milk-clotting activity and milk-clotting activity per proteolysis activity were found using soyabean as nitrogen source and sucrose as carbon source.
These optimized conditions resulted in a 1.9-fold increase in production of the milk-clotting enzyme. This study reported a plant growth promoting rhizobacteria, i.e., B. amyloliquefaciens as source of milk-clotting enzyme which has potential as a calf rennet substitute.
Biodegradable carboxymethylated lignin–tetra ethoxysilane (TEOS) nanocomposites (CML–T) were synthesized using lignin extracted from rice straw (RS) followed by surface modification through carboxymethylation. Composites were characterized by UV-spectroscopy, Fourier transform infrared (FT-IR), scanning electron microscope (SEM), X-ray diffraction pattern (XRD), atomic absorption spectroscopy (AAS) and particle size distribution (PSD). The average diameter (D50) of the CML–T composite particles was observed in the range of 160–560 nm. XRD spectra and SEM micrographs confirmed the high degree of crystallinity (peaks located at lower angle, 2θ = 12 and 22.0°) and porous nature of nanocomposites with increasing concentrations of TEOS. The composite exhibited nickel (Ni2+) and cadmium (Cd2+) adsorption up to 70.72 and 81.79 %, respectively in AAS analysis. The CML–T composite was investigated to assess their future applications as wound dressings and antimicrobial and packaging agents. Based on the antimicrobial properties and potential to remediate toxic heavy metals, the composites are proposed to be used for wastewater treatments, as packaging materials and for preparation of biofilters for environmental protection.
The FadR subfamily of regulators plays essential roles in the regulation of diverse metabolic pathways in bacteria. LldR, an FadR-type regulator, regulates lactate utilization in Pseudomonas aeruginosa.
Sequence network analysis of the LldR proteins from different bacterial species showed that LldR proteins from Pseudomonas sp. and Escherichia coli were separated into different clusters, suggesting that LldRs are derived from two ancestors that functionally diverged. Then, the recombinant PLldR protein (LldR of P. aeruginosa) was expressed, purified, and crystallized. Preliminary X-ray diffraction analysis of LldR protein crystals was performed. The PLldR crystal diffracted to 2.55 Å resolution and belonged to the trigonal space group P3, with unit-cell parameters a = 68.5 Å, b = 68.5 Å, and c = 237.0 Å.
These results will facilitate further understanding of the regulatory mechanism and the adaptation to sensing of both l -lactate and d -lactate of LldR proteins from Pseudomonas sp. in lactate metabolism.
This study investigated the effect of aeration rate and light intensity on biomass production and total fatty acids (TFA) accumulation by Porphyridium purpureum. The red microalgae is also known to accumulate considerable amount of arachidonic acid (ARA).
In artificial seawater medium, the highest yield of TFA (473.44 mg/L) was obtained with the aeration rate of 3 L/min and light intensity of 165 µmol/m2s, whilst the highest yield of ARA (115.47 mg/L) was achieved with the aeration rate of 3 L/min and light intensity of 110 µmol/m2s. It was found that higher aeration rate led to more biomass and TFA/ARA production. However, higher light intensity could contribute to biomass accumulation, but it was adverse for TFA and ARA biosynthesis.
By optimizing two operating factors (i.e., light intensity and aeration rate), TFA and ARA production by P. purpureum was significantly improved. This research provides a potential alternative means for producing ARA.
Glucosamine hydrochloride (GAH) and N-acetyl glucosamine (NAG) are chitin derivatives. Owing to their excellent biological activity, they have long been used as pharmaceuticals and nutraceuticals. However, both of them exist simultaneously in chitin hydrolyzate or fermentation production. The aim of this study is to identify the feasibility of separating GAH and NAG by nanofiltration on the basis of appropriate adjustments of physical conditions.
One commercial spiral nanofiltration membrane (QY-5-NF-1812) was used. Experiments were carried out in full recycle mode and the membrane separation performance was investigated at various mass ratios (mass ratios of GAH to NAG were from 1:14 to 1:2), pressures (4–22 bar), temperatures (15–35 °C), and electrolytes (NaCl, MgSO4, and MgCl2). The influence of temperature on molecular characteristics that play an important role in the separation process was also studied.
Owing to the steric-hindrance effect, electrostatic effect, and different solute permeability, the GAH separation factor increased with increasing GAH concentration. Furthermore, upon temperature increasing, the permeability difference between GAH and NAG decreased, thus decreasing the GAH separation factor. Simultaneously, with increasing temperature, the polarities and calculated molecular diameters for both GAH and NAG increased evidently. The calculated reflection coefficients for both GAH and NAG can be well fitted by the steric-hindrance pore (SHP) model, suggesting that steric-hindrance effect played an important role on the separation process. Furthermore, owing to Donnan repulsion and solute diffusion effects, three electrolytes had noticeable effects on nanofiltration separation efficiency.
The nanofiltration separation efficiency of GAH and NAG was significantly affected by their physical properties in this system, and the mechanisms for GAH and NAG separation were elucidated. The current study could provide a certain basis for the nanofiltration separation of GAH and NAG on an industrial scale.
This paper reports on the growth condition of Nannochloropsis sp. in an annular column-type photobioreactor (PBR) using light-emitting diode as an internal illumination.
The microalgae growth in the 20-L batch culture mode under mixed blue (450 nm) and red (660 nm) light-emitting diode (LED) in various conditions such as photoperiod and light intensity (controlled by supplied current) was monitored. Compact-type 5-W LED module with narrow beam angle (radiation pattern) was installed in the PBR so as to obtain higher intensity and deeper penetration to the culture.
Based on the PBR dimension with optical path length 120 mm, the minimum light intensity required at the PBR tank inner surface at initial stage of cultivation was approximately 350–370 mol m−2s−1, while mean light intensity derived was 140–160 mol m−2s−1. Photoperiod ratio of light:dark at 18:6 h provided better results compared to 12:12 in terms of final cell density achieved. Efficiency of light utilization was calculated to be 9.0 × 109 cell/mol photon (0.49 g/mol photon), while biomass volumetric productivity was 0.04 g L−1day−1.
The usage of narrow beam angle LED was feasible to be used but with further improvement is necessary.
Porcine circovirus type 2 (PCV2) virus-like particles (VLPs) are an effective vaccine against post-weaning multisystemic wasting syndrome. Burst sequence (BS) and the chicken 5′-HS4 β-globin (HS4) insulator were used to improve the production of PCV2 VLPs in insect cells.
BS was used to modify the polyhedron (polh) promoter, and the chicken HS4 insulator was inserted downstream of the target gene expression cassette. This enhanced effect was detected by EGFP, a generally used protein as reporter. The transcription-level analysis demonstrated that the double BS and the chicken HS4 insulator improved the transcription levels of the target genes. At the same time, the two regulatory elements also improved the PCV2 VLPs’ expression. PCV2 VLPs’ expression levels increased by 14 and 24 % at 72 h p.i. Further research observed an additive effect when the double BS and the chicken HS4 insulator simultaneously acted on the expression of PCV2 VLPs, with the PCV2 VLPs’ expression level increasing by 35 % at 72 h p.i.
BS and HS4 insulator can be used to improve the production of PCV2 VLPs in insect cells.
Today we are witnessing a global energy crisis due to huge energy demands and limited resources. Non-renewable energy sources are depleting and renewable energy sources are not properly utilized. There is an immediate need for search of alternate routes for energy generation. Microbial fuel cell (MFC) technology, which uses microorganisms to transform chemical energy of organic compounds into electricity is considered a promising alternative. Extensive studies have corroborated new insights into MFC, which show that a wide array of carbon sources including wastes can be employed using a variety of microbes. Consequently, microbial transformation of wastes using novel bioremediation strategies such as MFC for energy generation is considered as an efficient and environmentally benign approach. This paper deals with critical review of different classes of xenobiotics and wastes that can be employed for bioenergy generation, microorganisms involved, power output, major benefits, challenges and pit holes of MFC technology.
Cellulase adsorption of lignocellulosic materials is the key link during enzymatic hydrolysis. Hot-washing process (above lignin glass transition temperature) was used to change the physical structure of lignin, decrease covalent connection between cellulose and lignin, reduce the concentration of inhibitor, and explore the feasibility of enzymatic hydrolysis. The general objective of the paper was conducted to determine whether the hot-washing process has the potential to change the mechanism of lignin on enzyme hydrolysis.
Hot-washing was carried out at 151 °C for 20 min. The ratio of acid insoluble lignin to acid soluble lignin was increased, while the formation of spherical lignin droplets on the cell wall surface was decreased. Enzymatic digestibility of hot-washed filter cakes showed enhanced digestibility over the control samples. The concentration of fermentation inhibitor (acetic acid, formic acid, furfural and 5-hydroxymethylfurfural) obviously decreased after hot-washing process.
Hot-washing process significantly increased the adsorption ability of cellulase on the substrates and digestibility of biomass without removing much of the insoluble lignin content. Lignin distribution and/or physical property composition play a role.
Considerable attention has been given to the use of biosurfactants in recent times because of their potential industrial and environmental applications and ecological friendliness. Hydrocarbon-polluted soils have been major sources of biosurfactant-producing bacteria; resultantly, this study had been aimed at isolating and characterizing biosurfactant produced by Klebsiella pneumoniae strain IVN51 isolated from hydrocarbon-polluted soil in Ogoniland, Nigeria.
The biosurfactant screening techniques employed were emulsification assay, emulsification index (E24), lipase activity, haemolytic assay, oil spreading, and tilted glass slide. The bacterial isolate was identified based on phenotypic, biochemical, and molecular means. Thin-layer chromatography (TLC) and gas chromatography mass spectrometry (GC–MS) analyses were used in the classification and characterization of the biosurfactant produced. The biosurfactant produced was applied on selected hydrocarbons to determine its emulsifying capacity.
The phylogenetic tree analysis of the 16S rRNA gene classified the isolate as K. pneumoniae strain IVN51. The sequence obtained from the isolate has been deposited in GenBank under the accession number KT254060.1. The result obtained from the study revealed high biosurfactant activity with a maximum E24 of 60 % compared to E24 of 70 % by sodium dodecyl sulphate (SDS). In addition, the biosurfactant showed emulsifying activity against the following hydrocarbons: petrol, kerosene, xylene, toluene, and diesel. The optimum cultural conditions (temperature, pH, carbon, nitrogen, hydrocarbon, inoculum concentration, and incubation time) for growth and biosurfactant production by K. pneumoniae IVN51 were determined. The biosurfactant was characterized as a phospholipid using TLC, while the GC–MS analysis identified the phospholipid as phosphatidylethanolamine.
This study has demonstrated the capacity of K. pneumoniae strain IVN51 isolated from hydrocarbon-polluted soil to produce biosurfactant and the effectiveness of the produced biosurfactant in emulsifying different hydrocarbons. Furthermore, the biosurfactant produced was found to belong to the class, phospholipids based on the TLC and GC–MS analyses.
Being biodegradable and renewable, polyhydroxyalkanoates (PHAs), a green polymer, attract much attentions as potential alternative for conventional plastics due to increased concern towards environmental issue and resource depletion. However, PHAs not only have suffered some economic disadvantages on the market, and its environmental-friendliness has also been questioned as well. Therefore, there is a growing demand to improve both economic and environmental performances of PHAs production, especially at earlier stage of the process where there are plenty of opportunities and the modification cost is cheap. Therefore, a preliminary integrated assessment is introduced to provide a rapid evaluation for PHAs biosynthesis at R&D stage by coupling material cost analysis together with life-cycle assessment. Using fuzzy approach multi-objective optimization, crude glycerol is the most optimum substrate for biopolymer productions from Cupriavidus necator. The insight from sensitivity analysis has showed that the integrated assessment is sensitive to fluctuation in price and yield of substrate, while maintaining its robustness as similar result is obtained when using different multi-objective optimization tools. Providing some novel insights on PHAs biosynthesis like performance and site selection influencing factor, the integrated assessment can be used to facilitate screening for large-scale production of PHAs.
Biosurfactants (BSs) are amphipathic, surface active molecules produced by microorganisms and can reduce the surface tension and interfacial tension. The present study emphasizes the isolation and structural characterization of the BS produced by Pseudomonas otitidis P4.
An efficient BS producing bacterial strain isolated from the unexplored coal mining site of Chirimiri, India was identified as P. otitidis P4 based on morphological, biochemical and 16S rRNA gene sequence analysis. The surface tension of the culture medium was reduced from 71.18 to 33.4 mN/m. The surface tension and emulsification activity of the BS remained stable over a wide range of temperature, pH and salt concentrations indicating its scope of application in bioremediation, food, cosmetics, and pharmaceutical industries. Structural attributes of BS were determined by biochemical tests, thin layer chromatography (TLC), Fourier transform infrared (FTIR) and nuclear magnetic resonance (1H NMR) spectroscopy analyses, which confirmed the glycolipid nature of BS. Lipid and sugar fractions were the main constituents of the extracted BS. Thermogravimetric (TG) and Differential scanning calorimetry (DSC) analyses showed the thermostable nature of BS. As determined from TGA graph, the degradation temperature of biosurfactant was found to be 280 °C while complete weight loss was observed after 450 °C.
The BS isolated from P. otitidis P4 was identified as glycolipid and showed high emulsification activity and stability in a wide range of temperature, pH and salinity which makes it suitable for various industrial and environmental applications.
The organic nitrogen source is one of the key factors affecting Mortierella alpina cell growth as well as arachidonic acid (ARA) production. The aim of the present work is to achieve an optimized recipe of organic nitrogen source for ARA production by M. alpina by testing four organic nitrogen sources.
In the flasks, the results showed yeast extract or corn steep liquor were the most suitable sole nitrogen sources for biomass and ARA yield. In the bioreactor, a biomass of 17.5 g L−1 and an ARA yield of 2.7 g L−1 were achieved when using sole yeast extract, while cell autolysis was induced when using sole corn steep liquor; When using the combined nitrogen source with corn steep liquor and yeast extract at a 3:7 weight ratio, the biomass and ARA yield were significantly improved to 37 and 7.8 g L−1, respectively.
This work evaluated whether using a mix of organic nitrogen sources could improve ARA production when scaling up from a flask to a bioreactor culture. The 3:7 ratio of corn steep liquor to yeast extract was quite favourable for large-scale ARA production, and as a result, this combination has great potential for improving fungal cultures.
Recent methodology development in directed evolution of stereoselective enzymes has shown that various mutagenesis strategies based on saturation mutagenesis at sites lining the binding pocket enable the generation of small and smart mutant libraries requiring minimal screening.
In this endeavor, limonene epoxide hydrolase (LEH) has served as an experimental platform, the hydrolytic desymmetrization of cyclohexene oxide being the model reaction with formation of (R,R)- and (S,S)-cyclohexane-1,2-diol. This system has now been employed for testing reduced amino acid alphabets based on the Hecht concept of binary patterning, with and without additional hydrophobic amino acids.
It turns out that in binary pattern based saturation mutagenesis as applied to LEH, polar amino acids are seldom introduced. When applying binary patterning in combination with additional hydrophobic amino acids as building blocks in iterative saturation mutagenesis, excellent LEH variants were evolved for the production of both (R,R)- and (S,S)-diols (80–97 % ee), but again the introduction of polar amino acids occurs rarely. Docking computations explain the source of enhanced and inverted stereoselectivity. Some of the best variants are also excellent catalysts in the hydrolytic desymmetrization of other meso-epoxides, although both enantiomeric diols are not always accessible.
Chaetominine (CHA) is a novel alkaloid with excellent medicinal activities produced by Aspergillus fumigatus CY018. However, its further application has been severely restricted by the low production yield. In this work, the fermentation titer of CHA was investigated by medium composition optimization and amino-acid addition strategies.
Under the optimized conditions of sucrose 115.03 g/L, ammonium acetate 3.98 g/L, d-tryptophan 3.84 g/L, KH2PO4 1.5 g/L, FeSO4·7H2O 0.02 g/L, MgSO4·7H2O 0.7 g/L, sodium glutamate 3 g/L, sodium tartrate 1.5 g/L, and CaCl2 0.045 g/L), a CHA production yield of 55.92 mg/L was obtained, which increased significantly (3.99-fold) as compared with the unoptimized basal medium. Scale-up fermentation was carried out in a 5-L bioreactor based on the shake-flask fermentation results, maximum CHA yield of 48.53 mg/L was obtained at an air flow rate of 2.0 ± 0.1 VVM and an agitation rate of 400 rpm.
These results demonstrated that medium composition optimization and amino-acid addition were useful strategies for improving CHA production via biotechnological process. The methods in this work would be useful for the biotechnological production of CHA from A. fumigatus.
Streptomyces aureofaciens strain A6-9, obtained with traditional mutagenesis, produces elevated levels of 6-DCT. The increased formation of 6-DCT may be attributable to the changes in the expression of some proteins in the 6-DCT biosynthetic pathway. For this reason, we explored the differences in protein expression between A6-9 and wild-type (WT) strains of Streptomyces aureofaciens, and based on the differences (CtcH and CtcJ were overexpressed in A6-9), investigated the roles of CtcH and CtcJ in biosynthesis.
Two-dimensional gel electrophoresis and a Mascot search indicated that some enzymes (including CtcH and CtcJ) involved in the primary and secondary metabolism were more strongly expressed in the high-6-DCT-yielding strain A6-9 than in the WT strain DT1. To examine the roles of CtcH and CtcJ in 6-DCT biosynthesis, ctcH-deleted, ctcJ-deleted, ctcH-overexpressing, and ctcJ-overexpressing mutants and a mutant overexpressing both ctcH and ctcJ were constructed. Compared with WT, 6-DCT production was 50 and 37 % higher in the ctcH-overexpressing and ctcJ-overexpressing strains, respectively, and increased by 60 % in the ctcH–ctcJ-overexpressing strain. The ctcH-deleted and ctcJ-deleted strains produced almost no 6-DCT. Analysis of the metabolic flux distribution indicated that ctcH encodes a hydroxyacyl-CoA dehydrogenase and ctcJ encodes a monooxygenase that are essential for 6-DCT biosynthesis.
Protein expression differs between high-6-DCT-yielding and WT strains, and the enzymes increased in the high-6-DCT-yielding strain explain the increased 6-DCT production. ctcH encodes a hydroxyacyl-CoA dehydrogenase and ctcJ encodes a monooxygenase that are essential for 6-DCT biosynthesis.
Pichia pastoris is a popular recombinant protein expression system for its accessibility of efficient gene manipulation and high protein production. Sufficient supply of precursors, energy, and redox cofactors is crucial for high recombinant protein production. In our present work, we found that the addition of glutamate improved the recombinant β-galactosidase (β-gal) production by P. pastoris G1HL.
To elucidate the impacts of glutamate on the central metabolism in detail, a combined 13C-assisted metabolomics and 13C metabolic flux analysis was conducted based on LC–MS/MS and GC–MS data.
The pool sizes of intracellular amino acids were obviously higher on glucose/glutamate (Glc/Glu). The fluxes in EMP entry reaction and in downstream TCA cycle were 50 and 67% higher on Glc/Glu than on Glc, respectively. While the fluxes in upstream TCA cycle kept almost unaltered, the fluxes in PPP oxidative branch decreased.
The addition of glutamate leads to a remarkable change on the central metabolism of high β-galactosidase-producing P. pastoris G1HL. To meet the increased demands of redox cofactors and energy for higher β-galactosidase production on Glc/Glu, P. pastoris G1HL redistributes the fluxes in central metabolism through the inhibitions and/or activation of the enzymes in key nodes together with the energy and redox status.
Succinic acid is a valuable product due to its wide-ranging utilities. To improve succinate production and reduce by-products formation, Escherichia coli NZN111 was constructed by insertional inactivation of lactate dehydrogenase (LDH) and pyruvate formate lyase (PFL) encoded by the genes ldhA and pflB, respectively. However, this double-deletion mutant is incapable of anaerobically growing on glucose in rich or minimal medium even with acetate supplementation. A widespread hold view is that the inactivation of NADH-dependent LDH limits the regeneration of NAD+ and consequently disables proper growth under anaerobic conditions.
In this study, genome-scale metabolic core model of E. coli was reconstructed and employed to perform all simulations in silico according to the reconstruction of engineered strain E. coli NZN111. Non-optimized artificial centering hit-and-run (ACHR) method and metabolite flux-sum analysis were utilized to evaluate metabolic characteristics of strains. Thus, metabolic characteristics of the strains wild-type E. coli, ldhA mutant, pflB mutant, and NZN111 under anaerobic conditions were successfully unraveled.
We found a viewpoint contrary to the widespread realization that an NADH/NAD+ in NZN111 mainly resulted from the inactivation of PFL rather than the inactivation of LDH. In addition, the two alternative anaerobic fermentation pathways, lactate and ethanol production pathways, were blocked owing to the disruption of ldhA and pflB, resulting in insufficient NAD+ regeneration to oxidize or metabolize glucose for cell growth. Furthermore, we speculated reaction NADH16, the conversion of ubiquinone-8 (q8) to ubiquinol-8 (q8h2), as a potential amplification target for anaerobically improving cell growth and succinate production in NZN111.
Enzymatic approaches have become promising alternatives to chemical methods for the production of semi-synthetic β-lactam antibiotics. In this work, enzymatic synthesis of N-bromoacetyl-7-aminocephalosporanic acid (N-bromoacetyl-7-ACA), the key intermediate for the production of cefathiamidine, was reported for the first time.
Of the immobilized penicillin acylases (PAs) tested, PGA-750 was the best biocatalyst. Optimization of the biocatalytic process was conducted. The optimal acyl donor, molar ratio of acyl donor to 7-ACA, pH, temperature, 7-ACA concentration, and enzyme dosage were methyl bromoacetate, 3, 7.5, 20 °C, 50 mmol/L and 4 U/mL, respectively. Under the optimal conditions, enzymatic N-acylation of 7-ACA with methyl bromoacetate afforded the desired product with the yield of 85% in 2 h, where the synthesis/hydrolysis (S/H) ratio was approximately 1.5. The immobilized enzyme PGA-750 exhibited good operational stability, and the relative yields of approximately 90% and 63% were achieved, respectively, when it was reused in 7th and 11th batch.
An enzymatic approach to N-bromoacetyl-7-ACA, the key intermediate for the industrial production of cefathiamidine, has been developed successfully in a fully aqueous medium. The present work may open up a novel opportunity for the production of cefathiamidine through a simple and green process.
Rice bran is a by-product of the rice milling process and mostly discarded in Japan. Although many studies have shown that microbial fuel cells (MFCs) are able to generate electricity from organic wastes, limited studies have examined MFCs for generating electricity from rice bran.
Laboratory-scale single-chamber MFCs were inoculated with paddy field soil and supplied with rice bran for examining electricity generation. Power outputs and microbiome compositions were compared between MFCs containing pure water as the liquid phase (MFC-W) and those containing mineral solution (MFC-M). Polarization analyses showed that both MFCs successfully generated electricity with the maximum power densities of 360 and 520 mW m−2 (based on the projected area of anode) for MFC-W and MFC-M, respectively. Amplicon-sequencing analyses revealed that Trichococcus and Geobacter specifically occurred in anode biofilms in MFC-W and MFC-M, respectively.
The results suggest that rice bran is a feasible fuel by itself for generating electricity in MFCs.
The cost-effective production of second-generation bioethanol, which is made from lignocellulosic materials, has to face the following two problems: co-fermenting xylose with glucose and enhancing the strain’s tolerance to lignocellulosic inhibitors. Based on our previous study, the wild-type diploid Saccharomyces cerevisiae strain BSIF with robustness and good xylose metabolism genetic background was used as a chassis for constructing efficient xylose-fermenting industrial strains. The performance of the resulting strains in the fermentation of media with sugars and hydrolysates was investigated.
The following two novel heterologous genes were integrated into the genome of the chassis cell: the mutant MGT05196 N360F, which encodes a xylose-specific, glucose-insensitive transporter and is derived from the Meyerozyma guilliermondii transporter gene MGT05196, and Ru-xylA (where Ru represents the rumen), which encodes a xylose isomerase (XI) with higher activity in S. cerevisiae. Additionally, endogenous modifications were also performed, including the overproduction of the xylulokinase Xks1p and the non-oxidative PPP (pentose phosphate pathway), and the inactivation of the aldose reductase Gre3p and the alkaline phosphatase Pho13p. These rationally designed genetic modifications, combined with alternating adaptive evolutions in xylose and SECS liquor (the leach liquor of steam-exploding corn stover), resulted in a final strain, LF1, with excellent xylose fermentation and enhanced inhibitor resistance. The specific xylose consumption rate of LF1 reached as high as 1.089 g g−1 h−1 with xylose as the sole carbon source. Moreover, its highly synchronized utilization of xylose and glucose was particularly significant; 77.6% of xylose was consumed along with glucose within 12 h, and the ethanol yield was 0.475 g g−1, which is more than 93% of the theoretical yield. Additionally, LF1 performed well in fermentations with two different lignocellulosic hydrolysates.
The strain LF1 co-ferments glucose and xylose efficiently and synchronously. This result highlights the great potential of LF1 for the practical production of second-generation bioethanol.
Optically pure acetoin (AC) is an important platform chemical which has been widely used to synthesize novel optically active α-hydroxyketone derivatives and liquid crystal composites.
In this study, slaC and gldA encoding meso-2,3-butanediol dehydrogenase (meso-2,3-BDH) and glycerol dehydrogenase (GDH), respectively, in S. marcescens MG1 were knocked out to block the conversion from AC to 2,3-butanediol (2,3-BD). The resulting strain MG14 was found to produce a large amount of optically pure (3R)-AC with a little 2,3-BD, indicating that another enzyme responsible for 2,3-BD formation except meso-2,3-BDH and GDH existed in the strain MG1. Furthermore, SlaR protein, a transcriptional activator of AC cluster, was overexpressed using PC promoter in the strain MG14, leading to enhancement of the (3R)-AC yield by 29.91%. The recombinant strain with overexpression of SlaR, designated as S. marcescens MG15, was used to perform medium optimization for improving (3R)-AC production.
Under the optimized conditions, 39.91 ± 1.35 g/l (3R)-AC was produced by strain MG15 with the productivity of 1.11 g/l h and the conversion rate of 80.13%.
This study was carried out to isolate and characterize the bacterial strains from lindane-contaminated soil and they were also assessed for their lindane-degrading potential.
In this study the enrichment culture method was used for isolation of lindane degrading bacterial isolates, in which the mineral salt medium (MSM) supplemented with different concentrations of lindane was used. Further, the screening for the potential lindane degrading isolates was done using the spray plate method and colorimetric dechlorinase enzyme assay. The selected isolates were also studied for their growth response under varying range of temperature, pH, and NaCl. The finally selected isolates DAB-1Y and DAB-1W showing best lindane degradation activity was further subjected to biochemical characterization, microscopy, degradation/kinetic study, and 16S rDNA sequencing. The strain identification were performed using the biochemical characterization, microscopy and the species identifies by 16S rDNA sequence of the two isolates using the standard 16S primers, the 16 S rRNA partial sequence was analyzed through BLAST analysis and phylogenetic tree was generated based on UGPMA clustering method using MEGA7 software. This shows the phylogenetic relationship with the related strains. The two isolates of this study were finally characterized as Kocuria sp. DAB-1Y and Staphylococcus sp. DAB-1W, and their 16S rRNA sequence was submitted to GenBank database with accession numbers, KJ811539 and KX986577, respectively.
Out of the 20 isolates, the isolates DAB-1Y and DAB-1W exhibited best lindane-degrading activity of 94 and 98%, respectively, recorded after 8 days of incubation. The optimum growth was observed at temperature 30 °C, pH 7, and 5% NaCl observed for both isolates. Of the four isomers of hexachlorocyclohexane, isomer α and γ were the fastest degrading isomers, which were degraded up to 86 and 94% by isolates DAB-1Y and up to 93 and 98% by DAB-1W, respectively, reported after 8 days incubation. Isomer β was highly recalcitrant in which maximum 35 and 32% lindane degradation was observed even after 28 days incubation by isolates, DAB-1Y and DAB-1W, respectively. At lower lindane concentrations (1–10 mg/L), specific growth rate increased with increase in lindane concentration, maximum being 0.008 and 0.006/day for DAB-1Y and DAB-1W, respectively. The 16 S rRNA partial sequence of isolate DAB-1Y showed similarity with Kocuria sp. by BLAST analysis and was named as Kocuria sp. DAB-1Y and DAB-IW with Staphylococcus sp. DAB-1W. The 16S rDNA sequence of isolate DAB-1Y and DAB-1W was submitted to online at National Centre of Biotechnology Information (NCBI) with GenBank accession numbers, KJ811539 and KX986577, respectively.
This study has demonstrated that Kocuria sp. DAB-1Y and Staphylococcus sp. DAB-1W were found efficient in bioremediation of gamma-HCH and can be utilized further for biodegradation of environmental contamination of lindane and can be utilized in bioremediation program.
