Centrifugal Washing Research Articles

Micro-flow cell washing technique combined with single-cell Raman spectroscopy for rapid and automatic antimicrobial susceptibility test of pathogen in urine

Facing the rapid spread of antimicrobial resistance, methods based on single-cell Raman spectroscopy have proven their advances in reducing the turn-around time (TAT) of antimicrobial susceptibility tests (AST). However, the Raman-based methods are still hindered by the prolonged centrifugal cell washing procedure, which may require complex labor operation and induce high mechanical stress, resulting in a pretreatment time of over 1 h as well as a high cell-loss probability. In this study, we developed a micro-flow cell washing device and corresponding Raman-compatible washing chips, which were able to automatically remove the impurities in the samples, retain the bacterial cell and perform Raman spectra acquisition in situ. Results of washing the 5- and 10-μm polymethyl methacrylate (PMMA) microspheres showed that the novel technique achieved a successful removal of 99 % impurity and an 80 % particle retention rate after 6 to 10 cycles of washing. The micro-flow cell washing technique could complete the pretreatment for urine samples in a 96-well plate within 10 min, only taking 15 % of the handling time required by centrifugation. The AST profiles of urine sample spiked with E. coli 25922, E. faecalis 29212, and S. aureus 29213 obtained by the proposed Raman-based approach were found to be 100 % consistent with the results from broth micro-dilution while reducing the TAT to 3 h from several days which is required by the latter. Our study has demonstrated the micro-flow cell washing technique is a reliable, fast and compatible approach to replace centrifuge washing for sample pretreatment of Raman-AST and could be readily applied in clinical scenarios.

Performance and mechanisms of alkaline solid waste in CO2 mineralization and utilization

CO2 mineral sequestration using alkaline solid waste (ASW) is a promising strategy for synergistically reducing CO2 emissions and reusing industrial waste. However, improvement the carbonation degree still remains challenges due to the sluggish leaching rate of Ca/Mg ion at low pH. To the issues, this study proposed an amine-mediated CO2 absorption and mineralization process with six common ASWs, as well an ecological utilization route of CO2-ASW productions. Experimental results indicated that calcium carbide slag (CS) had greater CO2 mineralization capacity (86.2 g-CO2/kg-CS) than other ASWs, while stirring rate and particle size played a more important role during CO2 capture. Amine-mediated CO2 capture was verified to be more excellent with steel slag (SS) as mineral medium. When the MEA concentration was increased to 2 mol/L, the extraction efficiency of Ca2+ was increased by 35 %, leaded to the CO2 removal efficiency significantly promoted from 49 % to 92 %. The characterization of structural morphology referred spherical aragonite or needle-bar calcite was dominant for the porous mineralization products (30.6 m2/g). High germination index of pea seed (112.1 % at a dose of 10 g/L) inferred the negligible toxicological effects of tiny MEA residue over SS mineralization products, after centrifugally washing treatment. Pea seeds cultivated with mineralized products after centrifugal washing can achieve a growth rate of about 4 mm/d. Overall, this work provides a feasible route to apply the porous CO2-ASWs production into water conservation in arid and sandy land.

Reduced bacterial load in stallion semen by modified single layer centrifugation or sperm washing

The presence of bacteria poses a significant challenge to the quality of stallion semen used in artificial insemination. The bacterial content of insemination doses arises from various sources, such as the healthy stallion, environment, and collection equipment, and is implicated in fertility problems as well as reduced sperm quality during storage. The conventional approach of adding antibiotics to semen extenders raises concerns about antimicrobial resistance and potential negative effects on sperm characteristics, and may not be effective in inhibiting all bacteria. The objective of this study was to determine whether an innovative alternative to antibiotic usage – centrifugation through a single layer of a low density colloid (SLC) – could reduce the bacterial load in stallion semen, and to compare sperm characteristics in samples arising from this procedure, or simple extension of the ejaculate in semen extender, or from sperm washing, i.e. adding extender and then centrifuging the sample to allow the removal of most of the seminal plasma and extender. Eighteen semen samples were collected from six stallions. The semen samples were split and extended prior to washing or SLC, or received no further treatment other than extension. After preparation aliquots from each type of sample were sent for bacteriological examination; the remaining samples were stored for up to 72 h, with daily checks on sperm quality. The low density colloid SLC outperformed sperm washing or extension for bacterial reduction, effectively removing several bacterial species. The bacterial load in the samples was as follows: extended semen, 16 ± 6.7 × 105; washed, 5.8 ± 2.0 × 105; SLC, 2.3 ± 0.88 × 105, p < 0.0001. In addition, SLC completely removed some bacterial species, such as Staphylococcus xylosus. Although there is no selection for robust spermatozoa with the low density colloid, sperm motility, membrane integrity, and DNA fragmentation were not different to washed sperm samples. These findings suggest that SLC with a low density colloid offers a promising method for reducing bacterial contamination in stallion semen without resorting to antibiotics.

COMPARISON BETWEEN CENTRIFUGATION, COARSE FILTRATION AND MICROFILTRATION TECHNIQUES TO PREPARE AUTOLOGOUS BLOOD TRANSFUSION

In case of a bleeding patient, centrifugal cell salvage washing technique for autologous blood preparation is a preferred medical treatment in order to retain lost RBCs without contaminants. Although highly efficient in collection and washing shed blood, centrifugal cell salvage technology is costly and often unpractical or unavailable, especially in middle or low income countries, creating a need for more cost-effective and practical technologies. This study assessed two innovative filter devices as an alternative for the centrifugal cell salvage technology; a coarse collection filter device (Hemafuse) and a microfiltration device (HemoClear). Both devices were recently introduced in the Kenyan and European markets, respectively. Their accessible design could make these devices valuable additions to the current cell salvage technologies. We compared the effectiveness of these filtration technologies to remove plasma constituents and recover and concentrate the cellular components with a centrifugal device (autoLog®). Diluted whole blood was processed with each technology according to their manufacturer's instructions. The centrifugal technology confirmed its efficacy to remove potentially harmful solutes and capture red blood cells. The microfiltration technology (HemoClear) reached comparable levels of removal of solutes as the centrifugal technology, with a potential advantage over centrifugal technology in the ability to also recover platelets. The coarse filtration technology (Hemafuse) had no washing capacity but like the microfiltration technology, has the advantage of recovering platelets.

Comparison of Washing Efficiency and Recovery of Blood Cells Between Centrifugation, Coarse Filtration and Microfiltration Techniques to Prepare Autologous Blood for Transfusion.

PurposeCell salvage is the process by which blood lost in surgery is collected and washed or filtered to produce autologous blood for re-transfusion to the patient. Cell salvage aims to reduce the need for donor blood. Centrifugal cell salvage washing technique is a preferred medical treatment in order to retain lost red blood cells (RBCs) without contaminants. Although this technology very efficiently collects and washes shed blood, it is costly and often impractical or unavailable, especially in middle- or low-income countries. This study assessed two innovative filter devices as an alternative to centrifugal cell salvage technology: a coarse collection filter device (Hemafuse) and a microfiltration device (HemoClear). In contrast to centrifugal technology, both filter devices do not require electricity, nor costly equipment and extensive training. We compared the effectiveness of these filtration technologies to remove plasma constituents and recover and concentrate the cellular components with centrifugal technology (autoLog® device).MethodsWhole blood was processed with each technology according to the device manufacturer’s instructions. Before and after processing, the blood products were analyzed for supernatant solutes and cellular composition.ResultsThe centrifugal technology confirmed its efficacy to remove potentially harmful solutes and capture red blood cells. The microfiltration technology (HemoClear) reached comparable levels of removal of solutes, with a potential advantage over centrifugal technology in the ability to also recover platelets. The coarse filtration technology (Hemafuse) had no washing capacity but, like the microfiltration technology, has the advantage of recovering platelets.ConclusionInnovative filtration devices represent an alternative to centrifugal technology in the preparation of autologous blood for reinfusion. The HemoClear technology for the first time enables the recovery of washed platelets and red blood cells. Clinical trials will have to be performed to investigate the clinical value of this new autologous blood product.

Cell radiolabeling with acoustophoresis cell washing

Labeling immune cells with zirconium-89 (89Zr)-oxine has become a viable method to track cells in vivo by PET in various pre-clinical animal models and in clinical applications. Currently, 89Zr-oxine cell labeling is performed manually, which requires a highly trained specialist and is prone to human error. As the first phase in developing a fully automated radiolabeling system to address this problem, we assess the use of acoustophoresis cell washing to replace the centrifugal cell washing used in the current 89Zr-oxine cell radiolabeling procedure. To accomplish this, a cell radiolabeling procedure was developed in which two steps requiring a centrifuge to wash cells were replaced using acoustophoresis cell washing methods. The process was tested using murine EL4 lymphoma and T cells. The centrifuge cell labeling procedure was used as a control to compare the acoustophoresis cell washing procedure. The acoustophoresis method produced radiolabeled cells with similar properties to the centrifugal method when comparing labeling efficiency, labeled specific activity, efficacy of removing unbound 89Zr-oxine from the suspension, cell viability measured using annexin V/propidium iodide staining and activation function. This suggests that acoustophoresis cell washing can be used in the design of an automated benchtop, good manufacture practice-qualified acoustophoresis cell radiolabeling device.

NMR spectroscopy of live human asthenozoospermic and normozoospermic sperm metabolism.

Sperm motility varies between ejaculates from different men and from individual men. We studied normozoospermic and asthenozoospermic ejaculates after density-gradient centrifugation washing (DCG, 80/40%) and compared high- (80%) and low (40%)-motility sperm populations within the same sample. Our objective was to identify differences in endogenous metabolomes and energy metabolism in relation to sperm motility. 1H-Nuclear Magnetic Resonance spectroscopy (NMR) measured the endogenous metabolome of live human sperm. Incubating sperm with 13C-labelled substrates detected energy metabolism by 13C-NMR. The study examined 850 ejaculates and diagnosed asthenozoospermia in 6.1%. DGC was used to wash 160 normozoospermic (N) and 52 asthenozoospermic (A) ejaculates to recover high-motility sperm from the pellet (80N/80A) and low motility from the interface (40N/40A). 1H-NMR spectra, 45(N) and 15(A), were binned and the integrals normalised by sperm concentration. Sperm from 126(N) and 36(A) ejaculates were incubated with either 13C-glucose, 13C-fructose or 13C-pyruvate. 13C-NMR lactate and bicarbonate integrals were normalised by motile or vital sperm concentrations. 1H-NMR spectra choline integrals from the 80A population were significantly lower than the 80N, P < 0.0001. 13C-substrate conversion to lactate was significantly higher for 40A sperm than 80A sperm when normalised by motile sperm concentration. Bicarbonate integrals were sporadically observed. Sperm from asthenozoospermic ejaculates had similar glycolytic requirements to normozoospermic ones, with larger differences observed between 40 and 80% sperm populations. Higher lactate levels produced by 40% sperm may indicate that impaired sperm motility is due to dysregulated energy metabolism. The alteration in choline metabolism provides opportunities to understand the aetiology of asthenozoospermia.Lay summaryHow well sperm swim (motility) varies between ejaculates from different men? Normal sperm motility is beneficial to conception and some men diagnosed with infertility have low sperm motility. Sperm metabolise molecules to produce the energy required for motility. We measured concentrations of molecules within sperm and metabolism of molecules given to sperm and related these to the proportion of motile sperm. The study examined 850 sperm samples and found low motility in 6.1%. Metabolism of molecules given to sperm was similar between low and normal motility sperm samples. However, when the most motile sperm were separated from the rest, they were more efficient in metabolising these molecules to achieve motility. Lower concentrations of a molecule called choline were found in low-motility sperm samples compared to normal samples. Choline is associated with cell membranes, energy metabolism and oxidative stress, which may give opportunities to understand the causes of low motility.

Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain

Carrier-Free Deferoxamine Nanoparticles against Iron Overload in Brain

Feasibility and mechanism of an amine-looping process for efficient CO2 mineralization using alkaline ashes

• Amines significantly improved the CO 2 sequestration capacity of ashes. • Amines enhanced Ca 2+ leaching from alkaline ashes. • The phytotoxicity of alkaline ashes were significantly reduced after carbonation. Amine-looping-based CO 2 mineralization is a promising technology for simultaneous CO 2 absorption, mineralization, and carbonate crystallization in a single step. This paper performed a detailed investigation of the feasibility and underlying mechanism of the amine-looping process using industrial alkaline solid wastes, including one Biomass ash (BA) and two coal-fired fly ashes named FA1 and FA2. The CO 2 sequestration capacity and CO 2 removal efficiency of selected ashes were investigated in five typical amine solutions, including monoethanolamine (MEA), diethanolamine (DEA), triethanolamine (TEA), 2-amino-2-methy-1-propanol (AMP), and piperazine (PZ). The physicochemical property of ashes before and after carbonation and the dissolution of alkaline minerals in various amine solutions were systematically determined to explore the underlying mechanism involved in the amine-looping process. Results show that greater improvement in CO 2 removal efficiencies and CO 2 sequestration capacities were obtained by selected ashes in amine solutions compared to the traditional CO 2 mineralization in the water-ash-CO 2 system. It also revealed that amines played important roles in promoting CO 2 mass transfer, enhancing Ca 2+ leaching, and producing small-sized CaCO 3 . The largest CO 2 sequestration capacity (102.9 g/kg) was achieved by FA1 in PZ solution which was suggested as the preferred solvent for the amine-looping process. In addition, the environmental risk of carbonated ashes for agricultural application in terms of amine loss and phytotoxicity was evaluated. Results implied that the phytotoxicity of carbonated BA could be neglected when a simple centrifugal wash was used to remove the absorbed amine on the surface of carbonated BA whilst the phytotoxicity of selected ashes can be significantly reduced after carbonation reactions.

Methods to isolate adipose tissue-derived stem cells.

The use of adipose tissue has seen increasing interest in recent years for treating plastic surgery defects and for regenerative medicine applications. Adipose tissue is considered an optimal source of stem cells, as it contains more multipotent cells than bone marrow for the same volume. The adipose tissue-derived stem cells (ADSCs), isolated from the heterogeneous stromal vascular fraction (SVF), possess self-renewal properties and multilineage differentiation potential. In addition, adipose tissue can be obtained with less invasive procedures and patient morbidity than other tissue. For these reasons, numerous enzymatic, and non-enzymatic isolation methods have been developed over the years. The traditional method for isolation and culture of primary ADSCs from adipose tissue relies on enzymatic digestion with collagenase, followed by multiple steps of centrifugation. Alternative non-enzymatic isolation methods are based closed, sterile, and safe isolation processes that differ from each other for parameters such as the centrifugation force, pressure, filtration, and washing. Despite the existence of this multitude of systems, the best isolation method has not been identified to date. Therefore, the great challenge remains the achievement of the standardization of cellular products to allow the comparability between clinical studies and trials.

IGF-I and NGFβ enhance in vitro progressive motility and vitality of human spermatozoa.

PurposeProgressive motility (PM) and vitality are positively associated with fertilization ability of spermatozoa. Here, the effects of IGF‐I and NGFβ on PM and vitality of human spermatozoa were investigated.MethodsForty‐three volunteers gave semen samples after 2‐3 days of sexual abstinence. Each sample was processed with density gradient centrifugation and sperm washing. The pellet was divided into 3 aliquots. An aliquot containing one million of progressively motile spermatozoa was incubated for an hour (37°C) in standard culture medium (control group), and two aliquots with the same number of progressively motile spermatozoa were incubated in medium supplemented with IGF‐I or NGFβ. Two concentrations of IGF‐I (100 ng/ml and 1000 ng/ml) and NGFβ (0,5 ng/ml and 5 ng/ml) were tested.ResultsBoth growth factors significantly increased PM and vitality in comparison with control either at the low or the high concentration. IGF‐I seemed to be more effective than NGFβ. The effects did not seem to be dose dependent with the exception of the effect of IGF‐I on vitality.ConclusionsThe enhancement of PM and vitality of human spermatozoa by IGF‐I and NGFβ opens new ways for the improvement of sperm processing. Further research is needed to determine the most effective concentrations.

Strategy for DNA extraction and detection from insect pests in stored home grain samples RESEARCH

Stored grains are subjected to infestations with more than 60 species of insects, that responsible for millions of dollars’ loss and cause several health problems including allergies and gastrointestinal disorders. Traditional detection techniques are laborious, expensive and not sensitive to detect insect contamination at the egg and larvae stages. Therefore, alternative methods are needed for rapid and sensitive detection. One obvious approach is to develop a molecular approach utilizing genetic information of the potential insect species that infest grains for amplification of specific target gene fragment utilizing polymerase chain reaction [PCR]. In the present study, a number of known infested grain samples were used in standardizing a method to isolate larvae and adult insects that were based on centrifugation washing method and a filtration washing method. The isolated insects were subjected to DNA extraction and PCR amplification of defined regions of cytochrome oxidase I (COI) gene followed by sequencing to identify the di"erent pest species. For PCR amplification new primers were designed and for this purpose the obtained COI sequences from di"erent insects were aligned to design two sets of primers (named: COI-PCR4 and COI-PCR5) specific for the indicated insect mitochondrial COI gene. The designed primers were tested for their specificity and sensitivity. The suitability of PCR primers and DNA extraction methods were evaluated on eleven samples of commercial grains utilizing each primer set with the two extraction methods.

Stored Stallion Sperm Quality Depends on Sperm Preparation Method in INRA82 or INRA96

Removal of seminal plasma facilitates stallion sperm survival during storage, but washing may damage sperm chromatin. Therefore, sperm quality was compared in samples following single-layer centrifugation (SLC) or sperm washing and controls (extension only) in two extenders, INRA82 and INRA96. Ejaculates from six stallions were split among six treatments: SLC, sperm washing, and controls, in INRA82 and INRA96. Sperm motility and acrosome status were evaluated at 0, 24, 48, 72 and 96 hours; morphology at 0, 24, 48, 72 hours and chromatin integrity at 0 and 96 hours, with storage at 6°C. Sperm samples in INRA96 had better motility, acrosome status, and normal morphology than samples in INRA82. The SLC samples had higher motility and fewer reacted acrosomes than controls, and lower fragmented chromatin than washed samples. Fewer spermatozoa with tail defects were observed after SLC than after sperm washing; spermatozoa washed in INRA82 had fewer tail defects than those washed in INRA96. In conclusion, sperm quality (except for morphology) was better in INRA96 than in INRA82 and was better in SLC samples than in washed samples or controls. The SLC method is a useful adjunct to stallion sperm preparation, especially for storage before artificial insemination.

Synergistic Effects during Physical Refining of Palm Oil to Mitigate the Formation of Monochloropropanediols

AbstractThe aim of this study is to assess the synergy of mitigating main chlorine sources in palm oil and the oil refining conditions in a lab‐scale physical refining process. Mitigation concepts including centrifugation and washing steps are integrated into the physical refining protocol and the MCPD levels in the fully refined oils are determined. The endogenous chlorine content of the crude oil and that of the bleaching clay is confirmed to predominantly impact MCPD formation in this refining process. Residual sediments are shown to induce up to 1 ppm MCPD in the fully refined oils. Residual chlorine content of bleaching clay is shown to increase MCPD content of the oil twofold. t‐Test statistical analysis confirms significant effect of centrifugation, phosphoric acid content, clay type and clay washing on the formation of MCPD. Centrifugation is shown to effectively reduce the chlorine source coming from the sediments, while clay washing is shown to effectively reduce the chlorine source coming from the bleaching clay. These mitigation effects are confirmed both in sealed ampoule tests and in deodorizer experiments.Practical Applications: A combined application of strong centrifugation and clay washing is recommended to potentially improve the MCPD content of physically refined palm oil.

Optimization of Dean flow microfluidic chip for sperm preparation for intrauterine insemination

Sperm preparation is a necessary step in all methods of assisted reproductive technology. Here, we demonstrate the utility of a microfluidic spiral channel device for preparation of sperm for intrauterine insemination (IUI) directly from semen samples. After optimization of operating conditions, we use data from 130 washed semen samples to show that the microfluidic chip can retain 89% of all sperm cells and 90% of motile sperm cells, while removing 40% of the fluid volume and 82% of white blood cells. Then, we show that, when combined with a centrifugal wash, the microfluidic chip can be used in a protocol for sperm preparation. This protocol is capable of selecting 40–70% of motile sperm in 15 min. Compared to the clinical gold standard, density gradient centrifugation, this offers a higher sperm recovery, a gentler approach, and reduced reagent costs.

Mathematical Modeling of the Process of Centrifugal Washing of Sludge

In the chemical, food and other industries, in many cases it is necessary to clean crystalline powdery substances to remove surface impurities. For this, at the final stage of the treatment process, they are washed with water or other liquids. In this article, this process is investigated on the example of quantitative analysis of the dissolution of sodium chloride crystals in water by pressure filtration of the solution through the sludge of the processed product on the basis of filtration on centrifugal equipment. To this end, a physico-mathematical problem of the flow of substances in the interstitial capillary gap is formulated in terms of molecular mass by modeling a cylindrical channel. Based on the proposed model and operating parameters of the process of washing NaCl crystals with water from sludge on a centrifugal machine, numerical analysis of the process simulating cleaning the surface of this product to remove impurities was carried out.

Characterisation of polyphosphate coated aluminium-doped titania nanoparticles during milling

This paper investigates the characterisation of alumina-doped titania nanoparticles, milled under high-shear over time, in the presence of sodium hexametaphosphate (SHMP) dispersant. Transmission electron microscopy (TEM) indicated that prolonged milling times led to the formation of 10 nm particle fines which were electrostatically attracted to larger particles, where no change in the crystal structure was observed. Primary particle sizes measured by dynamic light scattering (DLS) and TEM were in agreement and showed no change in primary particle size (∼250 nm) with respect to milling time, however, there was a clear reduction in the magnitude of the slow mode decay associated to aggregates. The TiO2 was found to have an isoelectric point (iep) in the range of pH 3–4.5, where an increase in milling time led to a lower pHiep, indicative of an increase in SHMP coverage, which was further supported by an intensification in phosphorus content measured by X-ray fluorescence (XRF). Phosphorus content and zeta potential analysis before and after centrifugal washing showed that SHMP was partially removed or hydrolysed for the longer milled pigment samples, whereas no change was observed for shorter milled samples. Relaxation NMR was also performed, where enhanced relaxation rates at longer milling times were associated partially to increases in surface area and exposure of Al sites, as well as physicochemical changes to SHMP density and structure. It is thought that extended milling times may lead to hydrolysis or other structural changes of the dispersant from the high energy milling conditions, allowing easier removal after washing for longer milled pigments.

P156 Optimization of a rapid plate flow cytometry crossmatch assay on the cytoflex platform: The baylor protocol

Aim The Flow Cytometric Crossmatch (FCXM) assay has become the standard method for immunologic risk assesment of donor/recipient pairs. There is growing interest in streamlining the assay to expedite results and reduce performance costs without compromising assay accuracy or sensitivity. The introduction of powerful benchtop instruments, such as the Beckman Coulter CytoFLEX system, combined with the previously published Halifaster FCXM by Liwski et al., allowed our group to optimize and simplify the crossmatch procedure. Methods Instrument analytical performance was defined to validate use of the CytoFLEX, a research use only platform, for high complexity clinical testing. The instrument was evaluated for precision, stability, sensitivity, carryover, and accuracy. Performance characteristics were defined by Median Fluorescence Intensity (MFI), counts, and percentages, and compared to the current cytometer platform. The FCXM assay was optimized for time reduction (centrifugation cycles, incubation times, plating and washing), reduction of sample and reagent volumes, and minimization of cell loss during processing (plate type, pronase treatment). Results Instrument Performance: The instrument analytical performance is summarized in the following table. The CytoFLEX showed increased MFI precision and sensitivity when compared with current instrumentation. Assay Optimization: Each optimized component of the Baylor Protocol FCXM is summarized in the following table. The resulting process reduces assay completion time by 2 1/2 h with optimal cell recovery. Conclusions In this report we describe a model for step-by-step performance characterization of a RUO insturment and optimization of every component of a clinical test. We provide a method for CytoFLEX standardization, which may be reproducible with minimal variation across labs. We also demonstrate that the Baylor Protocol FCXM is a sensitive, fast, and cost effective method for use in clinical transplant labs. Download : Download high-res image (892KB) Download : Download full-size image

Acceleration and simplification of separation by addition of inorganic acid in biodiesel production

Abstract This paper describes a new approach to transesterification stopping and separation improving by the addition of inorganic acid, hydrochloric and phosphoric, to the whole reaction mixture after the reaction. The transesterification of oil was carried out by methanol and potassium hydroxide as the catalyst. The inorganic acid neutralises methoxide ions (formed from methanol and potassium hydroxide) and transforms soaps (formed by oil saponification) to fatty acids, which increases the acid number. The principle of stopping is the neutralization of the methoxide ions only. Best results are achieved by concentrated phosphoric acid with a ratio of 0.25 wt% to oil. The advantage is that separation is relatively fast (5 h) and the biodiesel conforms to EN 14214 without any other purification processes such as centrifugation, wet and dry washing and drying of the ester phase. Moreover, the content of glycerol in the formed glycerol phase is approximately 75%. This method of biodiesel production, which uses neutralization of methoxide ions after the transesterification followed by separation, saves energy because no purification processes are needed and no waste is formed. The biodiesel is more environmentally friendly because less energy is consumed.

Enhanced Purification Efficiency and Thermal Tolerance of Thermoanaerobacterium aotearoense β-Xylosidase through Aggregation Triggered by Short Peptides.

To simplify purification and improve heat tolerance of a thermostable β-xylosidase (ThXylC), a short ELK16 peptide was attached to its C-terminus, which is designated as ThXylC-ELK. Wild-type ThXylC was normally expressed in soluble form. However, ThXylC-ELK assembled into aggregates with 98.6% of total β-xylosidase activity. After simple centrifugation and buffer washing, the ThXylC-ELK particles were collected with 92.57% activity recovery and 95% purity, respectively. Meanwhile, the wild-type ThXylC recovery yield was less than 55% after heat inactivation, affinity and desalting chromatography followed by HRV 3C protease cleavage purification. Catalytic efficiency ( Kcat/ Km) was increased from 21.31 mM-1 s-1 for ThXylC to 32.19 mM-1 s-1 for ThXylC-ELK accompanied by a small increase in Km value. Heat tolerance of ThXylC-ELK at high temperatures was also increased. The ELK16 peptide attachment resulted in 6.2-fold increase of half-life at 65 °C. Released reducing sugars were raised 1.3-fold during sugar cane bagasse hydrolysis when ThXylC-ELK was supplemented into the combination of XynAΔSLH and Cellic CTec2.