Modeling and simulation of enzymatic gluconic acid production using immobilized enzyme and CSTR–PFTR circulation reaction system

Objectives

Production of gluconic acid by using immobilized enzyme and continuous stirred tank reactor-plug flow tubular reactor (CSTR–PFTR) circulation reaction system.

Results

A production system is constructed for gluconic acid production, which consists of a continuous stirred tank reactor (CSTR) for pH control and liquid storage and a plug flow tubular reactor (PFTR) filled with immobilized glucose oxidase (GOD) for gluconic acid production. Mathematical model is developed for this production system and simulation is made for the enzymatic reaction process. The pH inhibition effect on GOD is modeled by using a bell-type curve.

Conclusions

Gluconic acid can be efficiently produced by using the reaction system and the mathematical model developed for this system can simulate and predict the process well.

     

Biodegradation of saline phenolic wastewater in a biological contact oxidation reactor with immobilized cells of <Emphasis Type="Italic">Oceanimonas</Emphasis> sp.

Objective

To develop a method to treat saline phenolic wastewater in a biological contact oxidation reactor (BCOR) with immobilized cells of a marine microorganism, Oceanimonas sp., isolated from seawater.

Results

Cells were immobilized on fibre carriers in the BCOR. Saline wastewater with phenol at 1.5 g/l and NaCl at 6 % (w/v) was treated. In continuous assays, 99 % removal of phenol was achieved and a kinetic model for the phenol degradation is presented based on Monod’s equation.

Conclusion

The BOCR system using immobilized cells of Oceanimonas efficiently treats saline phenolic wastewaters without having decrease the salinity of the wastewater.

     

Enhancing the biomethane potential of liquid dairy cow manure by addition of solid manure fractions

Objectives

To assess the effect of adding solid manure fractions on the biomethane potential (BMP) of liquid dairy cow manure and on the biokinetic parameters of the process.

Results

The methanogenic potential of liquid dairy cow manure was strongly effected by adding a solid manure fraction. The 90/10 % (w/w) liquid/solid manure fraction mixture was the best substrate for CH₄ production. This substrate mixture improved by 50 % the final CH₄ production per g substrate and decreased the lag time by 220 % relative to the reference BMP test without the addition. Moreover, the addition of 20 % solid manure fraction adversely affected both the final CH₄ production and the maximum methane production rate, while increased the lag time by 400 % compared to the reference BMP test without addition.

Conclusions

Liquid dairy cow manure should be supplemented with no more than 10 % of solid manure fraction in order to improve the biomethane potential of this important agro-industrial residue.

     

Small temperature differences can improve the performance of mesophilic sludge-based digesters

Objective

To assess the effect of small temperature increases in mesophilic sludge-based digesters in order to develop and evaluate strategies for improving the biogas production in full-scale digesters.

Results

Methane production was strongly affected by small temperature differences, and this result was consistent across samples from 15 full-scale digesters. The specific methane yield varied between 42 and 97.5 ml g VS^?1 after 15 days of incubation at 35 °C, and improved when increasing the digester temperature to 39 °C. Only a limited quantity of additional gas was required to balance out the cost of heating and a positive energy balance was obtained. Further increases in temperature, in some cases, negatively affected the production when operated at 42 °C compared to 39 °C.

Conclusions

Small temperature increases should be applied to mesophilic sludge-based digesters to optimize the biogas production and is applicable to digesters operated in the lower mesophilic temperature range.

     

Quantitative analysis of tetrahydrofolate metabolites from <Emphasis Type="Italic">clostridium autoethanogenum</Emphasis>

Introduction

Quantification of tetrahydrofolates (THFs), important metabolites in the Wood–Ljungdahl pathway (WLP) of acetogens, is challenging given their sensitivity to oxygen.

Objective

To develop a simple anaerobic protocol to enable reliable THFs quantification from bioreactors.

Methods

Anaerobic cultures were mixed with anaerobic acetonitrile for extraction. Targeted LC–MS/MS was used for quantification.

Results

Tetrahydrofolates can only be quantified if sampled anaerobically. THF levels showed a strong correlation to acetyl-CoA, the end product of the WLP.

Conclusion

Our method is useful for relative quantification of THFs across different growth conditions. Absolute quantification of THFs requires the use of labelled standards.

     

NMR-based metabolic characterization of chicken tissues and biofluids: a model for avian research

Introduction

Poultry is one of the most consumed meat in the world and its related industry is always looking for ways to improve animal welfare and productivity. It is therefore essential to understand the metabolic response of the chicken to new feed formulas, various supplements, infections and treatments.

Objectives

As a basis for future research investigating the impact of diet and infections on chicken’s metabolism, we established a high-resolution proton nuclear magnetic resonance (NMR)-based metabolic atlas of the healthy chicken (Gallus gallus).

Methods

Metabolic extractions were performed prior to ¹H-NMR and 2D NMR spectra acquisition on twelve biological matrices: liver, kidney, spleen, plasma, egg yolk and white, colon, caecum, faecal water, ileum, pectoral muscle and brain of 6 chickens. Metabolic profiles were then exhaustively characterized.

Results

Nearly 80 metabolites were identified. A cross-comparison of these matrices was performed to determine metabolic variations between and within each section and highlighted that only eight core metabolites were systematically found in every matrice.

Conclusion

This work constitutes a database for future NMR-based metabolomic investigations in relation to avian production and health.

     

Optimization of fecal sample preparation for untargeted LC-HRMS based metabolomics

Introduction

Collecting feces is easy. It offers direct outcome to endogenous and microbial metabolites.

Objectives

In a context of lack of consensus about fecal sample preparation, especially in animal species, we developed a robust protocol allowing untargeted LC-HRMS fingerprinting.

Methods

The conditions of extraction (quantity, preparation, solvents, dilutions) were investigated in bovine feces.

Results

A rapid and simple protocol involving feces extraction with methanol (1/3, M/V) followed by centrifugation and a step filtration (10 kDa) was developed.

Conclusion

The workflow generated repeatable and informative fingerprints for robust metabolome characterization.

     

Corneal Collagen Cross-Linking for the Management of Mycotic Keratitis

Purpose

To evaluate the efficiency of corneal collagen cross-linking (CXL) in addition to topical voriconazole in cases with mycotic keratitis.

Design

Retrospective case series in a tertiary university hospital.

Participants

CXL was performed on 13 patients with mycotic keratitis who presented poor or no response to topical voriconazole treatment.

Methods

The clinical features, symptoms, treatment results and complications were recorded retrospectively. The corneal infection was graded according to the depth of infection into the stroma (from grade 1 to grade 3). The visual analogue scale was used to calculate the pain score before and 2 days after surgery.

Main Outcome Measures

Grade of the corneal infection.

Results

Mean age of 13 patients (6 female and 7 male) was 42.4 ± 17.7 years (20–74 years). Fungus was demonstrated in culture (eight patients) or cytological examination (five patients). Seven of the 13 patients (54%) were healed with topical voriconazole and CXL adjuvant treatment in 26 ± 10 days (15–40 days). The remaining six patients did not respond to CXL treatment; they initially presented with higher grade ulcers. Pre- and post-operative pain score values were 8 ± 0.8 and 3.5 ± 1, respectively (p < 0.05).

Conclusions

The current study suggests that adjunctive CXL treatment is effective in patients with small and superficial mycotic ulcers. These observations require further research by large randomized clinical trials.

     

Pranlukast,a novel binding ligand of human Raf1 kinase inhibitory protein

Objectives

To study the binding of pranlukast to hRKIP and its regulatory role in the Raf1/MEK/ERK signal pathway.

Results

NMR and fluorescence experiments demonstrated hRKIP could bind pranlukast with a binding constant of 1016 mM^?1. Residues (Y81, S109 and Y181) on the conserved ligand-binding pocket of hRKIP played a crucial role in binding pranlukast, and their mutations reduced the binding affinity more than 85 %. Furthermore, 25 μM pranlukast could up-regulate the ERK phosphorylation by about 17 %.

Conclusion

Pranlukast may be used as a potential drug precursor for treating hRKIP involved diseases.

     

Maximizing power generation from dark fermentation effluents in microbial fuel cell by selective enrichment of exoelectrogens and optimization of anodic operational parameters

Objective

To selectively enrich an electrogenic mixed consortium capable of utilizing dark fermentative effluents as substrates in microbial fuel cells and to further enhance the power outputs by optimization of influential anodic operational parameters.

Results

A maximum power density of 1.4 W/m³ was obtained by an enriched mixed electrogenic consortium in microbial fuel cells using acetate as substrate. This was further increased to 5.43 W/m³ by optimization of influential anodic parameters. By utilizing dark fermentative effluents as substrates, the maximum power densities ranged from 5.2 to 6.2 W/m³ with an average COD removal efficiency of 75% and a columbic efficiency of 10.6%.

Conclusion

A simple strategy is provided for selective enrichment of electrogenic bacteria that can be used in microbial fuel cells for generating power from various dark fermentative effluents.

     

Fungal fermentation on anaerobic digestate for lipid-based biofuel production

Background

Anaerobic digestate is the effluent from anaerobic digestion of organic wastes. It contains a significant amount of nutrients and lignocellulosic materials, even though anaerobic digestion consumed a large portion of organic matters in the wastes. Utilizing the nutrients and lignocellulosic materials in the digestate is critical to significantly improve efficiency of anaerobic digestion technology and generate value-added chemical and fuel products from the organic wastes. Therefore, this study focused on developing an integrated process that uses biogas energy to power fungal fermentation and converts remaining carbon sources, nutrients, and water in the digestate into biofuel precursor-lipid.

Results

The process contains two unit operations of anaerobic digestion and digestate utilization. The digestate utilization includes alkali treatment of the mixture feed of solid and liquid digestates, enzymatic hydrolysis for mono-sugar release, overliming detoxification, and fungal fermentation for lipid accumulation. The experimental results conclude that 5 h and 30 °C were the preferred conditions for the overliming detoxification regarding lipid accumulation of the following fungal cultivation. The repeated-batch fungal fermentation enhanced lipid accumulation, which led to a final lipid concentration of 3.16 g/L on the digestate with 10% dry matter. The mass and energy balance analysis further indicates that the digestate had enough water for the process uses and the biogas energy was able to balance the needs of individual unit operations.

Conclusions

A fresh-water-free and energy-positive process of lipid production from anaerobic digestate was achieved by integrating anaerobic digestion and fungal fermentation. The integration addresses the issues that both biofuel industry and waste management encounter—high water and energy demand of biofuel precursor production and few digestate utilization approaches of organic waste treatment.

     

Bioprocess optimization for pectinase production using <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a submerged cultivation system

Background

Pectinase enzymes present a high priced category of microbial enzymes with many potential applications in various food and oil industries and an estimated market share of $ 41.4 billion by 2020.

Results

The production medium was first optimized using a statistical optimization approach to increase pectinase production. A maximal enzyme concentration of 76.35 U/mL (a 2.8-fold increase compared with the initial medium) was produced in a medium composed of (g/L): pectin, 32.22; (NH₄)₂SO₄, 4.33; K₂HPO₄, 1.36; MgSO₄.5H₂O, 0.05; KCl, 0.05; and FeSO₄.5H₂O, 0.10. The cultivations were then carried out in a 16-L stirred tank bioreactor in both batch and fed-batch modes to improve enzyme production, which is an important step for bioprocess industrialization. Controlling the pH at 5.5 during cultivation yielded a pectinase production of 109.63 U/mL, which was about 10% higher than the uncontrolled pH culture. Furthermore, fed-batch cultivation using sucrose as a feeding substrate with a rate of 2 g/L/h increased the enzyme production up to 450 U/mL after 126 h.

Conclusions

Statistical medium optimization improved volumetric pectinase productivity by about 2.8 folds. Scaling-up the production process in 16-L semi-industrial stirred tank bioreactor under controlled pH further enhanced pectinase production by about 4-folds. Finally, bioreactor fed-batch cultivation using constant carbon source feeding increased maximal volumetric enzyme production by about 16.5-folds from the initial starting conditions.

     

Minimal variation of the plasma lipidome after delayed processing of neonatal cord blood

Background

Cord blood lipids are potential disease biomarkers. We aimed to determine if their concentrations were affected by delayed blood processing.

Method

Refrigerated cord blood from six healthy newborns was centrifuged every 12 h for 4 days. Plasma lipids were analysed by liquid chromatography/mass spectroscopy.

Results

Of 262 lipids identified, only eight varied significantly over time. These comprised three dihexosylceramides, two phosphatidylserines and two phosphatidylethanolamines whose relative concentrations increased and one sphingomyelin that decreased.

Conclusion

Delay in separation of plasma from refrigerated cord blood has minimal effect overall on the plasma lipidome.

     

Synthesis and physicochemical characterization of the one-carbon carrier 10-formyltetrahydrofolate; a reference standard for metabolomics

Introduction

Metabolomics analysis depends on the identification and validation of specific metabolites. This task is significantly hampered by the absence of well-characterized reference standards. The one-carbon carrier 10-formyltetrahydrofolate acts as a donor of formyl groups in anabolism, where it is a substrate in formyltransferase reactions in purine biosynthesis. It has been reported as an unstable substance and is currently unavailable as a reference standard for metabolomics analysis.

Objectives

The current study was undertaken to provide the metabolomics community thoroughly characterized 10-formyltetrahydrofolate along with analytical methodology and guidelines for its storage and handling.

Methods

Anaerobic base treatment of 5,10-methenyltetrahydrofolate chloride in the presence of antioxidant was utilized to prepare 10-formyltetrahydrofolate.

Results

Pure 10-formyltetrahydrofolate has been prepared and physicochemically characterized. Conditions toward maintaining the stability of a solution of the dipotassium salt of 10-formyltetrahydrofolate have been determined.

Conclusion

This study describes the facile preparation of pure (>90%) 10-formyltetrahydrofolate, its qualitative physicochemical characterization, as well as conditions to enable its use as a reference standard in physiologic samples.

     

Selective oxidation of UDP-glucose to UDP-glucuronic acid using permeabilized <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> expressing human UDP-glucose 6-dehydrogenase

Objectives

To use permeabilized cells of the fission yeast, Schizosaccharomyces pombe, that expresses human UDP-glucose 6-dehydrogenase (UGDH, EC 1.1.1.22), for the production of UDP-glucuronic acid from UDP-glucose.

Results

In cell extracts no activity was detected. Therefore, cells were permeabilized with 0.3 % (v/v) Triton X-100. After washing away all low molecular weight metabolites, the permeabilized cells were directly used as whole cell biocatalyst. Substrates were 5 mM UDP-glucose and 10 mM NAD⁺. Divalent cations were not added to the reaction medium as they promoted UDP-glucose hydrolysis. With this reaction system 5 mM UDP-glucose were converted into 5 mM UDP-glucuronic acid within 3 h.

Conclusions

Recombinant permeabilized cells of S. pombe can be used to synthesize UDP-glucuronic acid with 100 % yield and selectivity.

     

Does centrifugation matter? Centrifugal force and spinning time alter the plasma metabolome

Background

Centrifugation is an indispensable procedure for plasma sample preparation, but applied conditions can vary between labs.

Aim

Determine whether routinely used plasma centrifugation protocols (1500×g 10 min; 3000×g 5 min) influence non-targeted metabolomic analyses.

Methods

Nuclear magnetic resonance spectroscopy (NMR) and High Resolution Mass Spectrometry (HRMS) data were evaluated with sparse partial least squares discriminant analyses and compared with cell count measurements.

Results

Besides significant differences in platelet count, we identified substantial alterations in NMR and HRMS data related to the different centrifugation protocols.

Conclusion

Already minor differences in plasma centrifugation can significantly influence metabolomic patterns and potentially bias metabolomics studies.

     

Reengineering substrate specificity of <Emphasis Type="Italic">E. coli</Emphasis> glutamate dehydrogenase using a position-based prediction method

Objective

To re-engineer the active site of proteins for non-natural substrates using a position-based prediction method (PBPM).

Results

The approach has been applied to re-engineer the E. coli glutamate dehydrogenase to alter its substrate from glutamate to homoserine for a de novo 1,3-propanediol biosynthetic pathway. After identification of key residues that determine the substrate specificity, residue K92 was selected as a candidate site for mutation. Among the three mutations (K92V, K92C, and K92M) suggested by PBPM, the specific activity of the best mutant (K92 V) was increased from 171 ± 35 to 1328 ± 71 μU mg^?1.

Conclusion

The PBPM approach has a high efficiency for re-engineering the substrate specificity of natural enzymes for new substrates.

     

Regular enzyme recovery enhances cellulase production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis> in fed-batch culture

Objective

To protect the enzymes during fed-batch cellulase production by means of partial enzyme recovery at regular intervals.

Results

Extracellular enzymes were partially recovered at the intervals of 1, 2, or 3 days. Mycelia were also removed to avoid contamination. Increases in the total harvested cellulase (24–62%) and β-glucosidase (22–76%) were achieved. In fermentor cultivation when the enzymes were recovered every day with 15% culture broth. The total harvested cellulase and β-glucosidase activity increased by 43 and 58%, respectively, with fungal cell concentration maintained at 3.5–4.5 g l^?1.

Conclusion

Enzyme recovery at regular intervals during fed-batch cellulase cultivation could protect the enzyme in the culture broth and enhance the enzyme production when the fungal cell concentration is maintained in a reasonable range.

     

NMRProcFlow: a graphical and interactive tool dedicated to 1D spectra processing for NMR-based metabolomics

Introduction

Concerning NMR-based metabolomics, 1D spectra processing often requires an expert eye for disentangling the intertwined peaks.

Objectives

The objective of NMRProcFlow is to assist the expert in this task in the best way without requirement of programming skills.

Methods

NMRProcFlow was developed to be a graphical and interactive 1D NMR (¹H & ¹³C) spectra processing tool.

Results

NMRProcFlow (http://nmrprocflow.org), dedicated to metabolic fingerprinting and targeted metabolomics, covers all spectra processing steps including baseline correction, chemical shift calibration and alignment.

Conclusion

Biologists and NMR spectroscopists can easily interact and develop synergies by visualizing the NMR spectra along with their corresponding experimental-factor levels, thus setting a bridge between experimental design and subsequent statistical analyses.

     

A novel method to harvest <Emphasis Type="Italic">Chlorella</Emphasis> sp. by co-flocculation/air flotation

Objectives

To develop a more effective dissolved air flotation process for harvesting microalgae biomass, a co-flocculation/air flotation (CAF) system was developed that uses an ejector followed by a helix tube flocculation reactor (HTFR) as a co-flocculation device to harvest Chlorella sp. 64.01.

Results

The optimal size distribution of micro-bubbles and an air release efficiency of 96 % were obtained when the flow ratio of inlet fluid (raw water) to motive fluid (saturated water) of the ejector was 0.14. With a reaction time of 24 s in the HTFR, microalgae cells and micro-bubbles were well flocculated, and these aerated flocs caused a fast rising velocity (96 m/h) and high harvesting efficiency (94 %).

Conclusions

In a CAF process, micro-bubbles can be encapsulated into microalgae flocs, which makes aerated flocs more stable. CAF is an effective approach to harvesting microalgae.