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The catalytic function of lytic polysaccharide monooxygenases (LPMOs) to cleave and decrystallize recalcitrant polysaccharides put these enzymes in the spotlight of fundamental and applied research. Here we demonstrate that the demand of LPMO for an electron donor and an oxygen species as cosubstrate can be fulfilled by a single auxiliary enzyme: an engineered fungal cellobiose dehydrogenase (CDH) with increased oxidase activity. The engineered CDH was about 30 times more efficient in driving the LPMO reaction due to its 27 time increased production of H2O2 acting as a cosubstrate for LPMO. Transient kinetic measurements confirmed that intra‐ and intermolecular electron transfer rates of the engineered CDH were similar to the wild‐type CDH, meaning that the mutations had not compromised CDH’s role as an electron donor. These results support the notion of H2O2‐driven LPMO activity and shed new light on the role of CDH in activating LPMOs. Importantly, the results also demonstrate that the use of the engineered CDH results in fast and steady LPMO reactions with CDH‐generated H2O2 as a cosubstrate, which may provide new opportunities to employ LPMOs in biomass hydrolysis to generate fuels and chemicals., Lytic polysaccharide monooxygenases (LPMOs) require an electron donor and an oxygen species as a cosubstrate. We show that a single auxiliary enzyme, an engineered cellobiose dehydrogenase (CDHoxy+), provides both. CDHoxy+ produces about 30 times more H2O2 than the wild‐type CDH and greatly boosts the activity of LPMO. These results demonstrate H2O2‐driven LPMO activity and provide new opportunities for the application of CDH in industrial biomass utilization.

Additional file 1: Figure S1. Incubation of 3 µM LPMO and 0.8 mg mL−1 PASC with CDH at concentrations of 0.5 µM (red) 1 µM (blue) or 3 µM (green) in absence of cellobiose. Black line: 3 µM CDH and 10 mM cellobiose in absence of LPMO. All reactions were carried out under constant stirring at 30 °C in 50 mM sodium phosphate buffer, pH 6.0. Figure S2. Titration of oxidized LPMO (3 µM) and 0.8 mg mL−1 PASC with 20 µM (green), 40 µM (red) or 80 µM (blue) H2O2 (solid lines). Dashed, coloured lines show the titration of 2 mM ascorbate and 0.8 mg mL−1 PASC with 20 µM (green), 40 µM (red) or 80 µM (blue) H2O2. The vertical dashed lines indicate the addition of H2O2. The arrow indicates the addition of LPMO (solid lines) or 2 mM ascorbate (dashed lines). All reactions were carried out under constant stirring at 30 °C in 50 mM sodium phosphate buffer, pH 6.0. Figure S3. Titration of LPMO (3 µM) and 0.8 mg mL−1 PASC with 40 µM H2O2. The vertical dashed lines indicate the addition of H2O2. The arrow indicates the addition of fresh PASC which was either added alone (green line) or simultaneously with 1 mM ascorbate (AscA, black line). The blue line indicates the addition of ascorbate (1 mM). All reactions were carried out under constant stirring at 30 °C in 50 mM sodium phosphate buffer, pH 6.0.

The tobacco-related plant species Nicotiana benthamiana has recently emerged as a versatile expression platform for the rapid generation of recombinant biopharmaceuticals, but product yield and quality frequently suffer from unintended proteolysis. Previous studies have highlighted that recombinant protein fragmentation in plants involves papain-like cysteine proteinases (PLCPs). For this reason, we have now characterized two major N. benthamiana PLCPs in detail: aleurain-like protease (NbALP) and cathepsin B (NbCathB). As typical for PLCPs, the precursor of NbCathB readily undergoes autocatalytic activation when incubated at low pH. On the contrary, maturation of NbALP requires the presence of a cathepsin L-like PLCP as processing enzyme. While the catalytic features of NbALP closely resemble those of its mammalian homologue cathepsin H, NbCathB displays remarkable differences to human cathepsin B. In particular, NbCathB appears to be a far less efficient peptidyldipeptidase (removing C-terminal dipeptides) than its human counterpart, suggesting that it functions primarily as an endopeptidase. Importantly, NbCathB was far more efficient than NbALP in processing the human anti-HIV-1 antibody 2F5 into fragments observed during its production in N. benthamiana. This suggests that targeted down-regulation of NbCathB could improve the performance of this plant-based expression platform.

The flavocytochrome cellobiose dehydrogenase (CDH) is secreted by wood-decomposing fungi, and is the only known extracellular enzyme with the characteristics of an electron transfer protein. Its proposed function is reduction of lytic polysaccharide mono-oxygenase for subsequent cellulose depolymerization. Electrons are transferred from FADH2 in the catalytic flavodehydrogenase domain of CDH to haem b in a mobile cytochrome domain, which acts as a mediator and transfers electrons towards the active site of lytic polysaccharide mono-oxygenase to activate oxygen. This vital role of the cytochrome domain is little understood, e.g. why do CDHs exhibit different pH optima and rates for inter-domain electron transfer (IET)? This study uses kinetic techniques and docking to assess the interaction of both domains and the resulting IET with regard to pH and ions. The results show that the reported elimination of IET at neutral or alkaline pH is caused by electrostatic repulsion, which prevents adoption of the closed conformation of CDH. Divalent alkali earth metal cations are shown to exert a bridging effect between the domains at concentrations of > 3 mm, thereby neutralizing electrostatic repulsion and increasing IET rates. The necessary high ion concentration, together with the docking results, show that this effect is not caused by specific cation binding sites, but by various clusters of Asp, Glu, Asn, Gln and the haem b propionate group at the domain interface. The results show that a closed conformation of both CDH domains is necessary for IET, but the closed conformation also increases the FAD reduction rate by an electron pulling effect.

Lignocellulose is a renewable resource that can be used for the sustainable production of platform chemicals or fuels. However, the recalcitrance of cellulose to hydrolytic depolymerization is a barrier to microbial and industrial utilization of lignocellulosic biomass. Cellulose is broken down to glucose by endo- and exo-acting glycosyl hydrolases which process the glucan chains in cellulose to soluble cellobiose moieties. In a process known as oxidative cellulose degradation, copper-dependent lytic polysaccharide monooxygenases (LPMO) cleave polymeric substrates, including crystalline cellulose, hemicelluloses and starch. It remains difficult to find how LPMOs orchestrate the complex reaction between copper reduction, co-substrate activation and substrate binding. To test the hypothesis that copper reduction increases binding of LPMO to cellulose, we studied the binding of oxidized and reduced LPMO to amorphous cellulose (PASC) and to microcrystalline cellulose (MC). Also, we aimed to quantify the substrate binding by measuring binding isotherms. The data demonstrate that copper reduction is a driver for substrate binding.

Sugar beet pulp (SBP) is a promising feedstock for the production of 2nd generation biofuels, but efficient enzymatic hydrolysis remains a key challenge ; therefore, new process designs and/or bioreactor designs are crucial to overcome this hurdle. In this regard, horizontal rotating tubular bioreactors (HRTB) offer the advantage of high substrate loadings while minimizing the space and energy demand compared with conventional stirred tank reactors. Here, a statistical approach is used to optimize the hydrolysis of sugar beet pulp in laboratory experiments, and it is shown that such a process can be implemented in a HRTB. Using the design of experiments (DOE) method, the reaction conditions of four commercial enzyme mixtures (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase) was optimized for the degradation of SBP in small-scale experiments. Using Ultrazym AFP-L as the most efficient mixture, a 10 L scale conversion was performed in a HRTB. At a substrate loading of 135 g L−1 and optimized conversion parameters (enzyme load, pH and rotating speed of the reactor), 0.525 W dm−3 were needed to achieve solubilisation of 30% of the total mass of initial SBP after 24 h. DOE was found to be an easy-to-apply method that allowed optimizing the conditions for enzymatic hydrolysis of SBP, resulting in a higher sugar yield. The results could be transferred to an HRTB, which is a suitable system for enzymatic conversion and efficient saccharification of semi-solid or solid substrates with relatively low energy consumption.

The depolymerisation of complex biomass such as lignocellulose by organisms depends on a network of enzymatic and chemical reactions. Fungi that can grow on lignocellulosic materials are equipped with a complex enzymatic degradation system, including a broad variety of hydrolases and oxidoreductases. In 2010, a new class of enzymes was discovered, which carry out oxidative cleavage of polysaccharides. These enzymes, today known as lytic polysaccharide monooxygenases (LPMOs, CAZy family AA9) are copper-dependent oxidoreductases that can hydroxylate the C1 or C4 positions of glucose and thereby cleave the glycosidic bonds in cellulose. These “cellulase activity boosters” provide hydrolytic enzymes access to crystalline cellulose by introducing chain breaks. LPMOs, despite their importance in biotechnology, are difficult to investigate because of their complex heterogeneous reaction mechanism. An open question is how LPMOs orchestrate the complex reaction between copper reduction, co-substrate activation and substrate binding. It is generally accepted that the first reaction step of LPMO is the reduction of the active-site copper by small molecule reductants or by other enzymes (CDH, GDH). In this work, we address the substrate binding of oxidized and reduced LPMO-02916 from Neurospora crassa to phosphoric acid swollen cellulose (PASC) and microcrystalline cellulose (MC) to investigate the role of the copper centre for substrate binding.

Lytic polysaccharide monooxygenases (LPMOs) represent a recent addition to the carbohydrate-active enzymes and are classified as auxiliary activity (AA) families 9, 10, 11, and 13. LPMOs are crucial for effective degradation of recalcitrant polysaccharides like cellulose or chitin. These enzymes are copper-dependent and utilize a redox mechanism to cleave glycosidic bonds that is dependent on molecular oxygen and an external electron donor. The electrons can be provided by various sources, such as chemical compounds (e.g., ascorbate) or by enzymes (e.g., cellobiose dehydrogenases, CDHs, from fungi). Here, we demonstrate that a fungal CDH from Myriococcum thermophilum (MtCDH), can act as an electron donor for bacterial family AA10 LPMOs. We show that employing an enzyme as electron donor is advantageous since this enables a kinetically controlled supply of electrons to the LPMO. The rate of chitin oxidation by CBP21 was equal to that of cosubstrate (lactose) oxidation by MtCDH, verifying the usage of two electrons in the LPMO catalytic mechanism. Furthermore, since lactose oxidation correlates directly with the rate of LPMO catalysis, a method for indirect determination of LPMO activity is implicated. Finally, the one electron reduction of the CBP21 active site copper by MtCDH was determined to be substantially faster than chitin oxidation by the LPMO. Overall, MtCDH seems to be a universal electron donor for both bacterial and fungal LPMOs, indicating that their electron transfer mechanisms are similar.

Additional file 1. Production, purification and characterization of lytic polysaccharide monooxygenases secreted by Pestalotiopsis sp. NCi6.

submitted by Daniel Kracher Zusammenfassung in deutscher Sprache Universität für Bodenkultur Wien, Dissertation, 2016 OeBB

The textile industry is one of the biggest polluters of the environment, with significant consumption of water resources, energy and chemicals. Moreover, the processing of wastewaters from the textile industry is very complex, and the costs of these processes are very high. The main objective of this work was to select the most effective enzymes for wastewater treatment, as well as to detect dyes from different chemical groups which are mostly suitable for the enzymatic treatment and decolorization in the wastewaters of textile industry. 186 textile dyes were tested in reactions with six oxidative reductive enzymes (two laccases, lytic polysaccharide monooxygenase, cellobiose dehydrogenase, horseradish peroxidase and glucose oxidase), under different reaction conditions. The kinetics of pre-selected dyes and enzymes, as well as the toxicological properties of those dyes, were determined in batch reactions which were optimized by statistical method of the design of the experiment with regard to the concentrations of dyes, enzymes, temperature and pH values, and then scaled down to the microreactor levels. With the goal to perform scaling down to the level of micro-bioreactor, we have designed three prototypes of microreactors: i) microreactors produced in the workshop from two different materials and ii) printed by 3D printer. Results of reactions in microreactors were compared with the results achieved in the tubular and batch microreactor systems. All microreactors' prototypes were tested in the laboratory, using two enzymes and four different dyes. Studies have shown that the concentration of adsorbed oxygen measured in a microreactor system showed a linear increase with increasing flow rate. Decolorization of the Direct blue 78 dye in microreactors with and without aeration using laccase caused a 50% decrease in the decolorization speed, while in the tubular microreactor with aeration, the decolorization speed exponentially decreased by 80%. Those results should serve as a basis for the development of the wastewater treatment processes as well as for other enzymatic applications which are advantageous from an environmental and economical point of view.

The UDP-sulfoquinovose synthase Agl3 from Sulfolobus acidocaldarius converts UDP-d-glucose and sulfite to UDP-sulfoquinovose, the activated form of sulfoquinovose required for its incorporation into glycoconjugates. Based on the amino acid sequence, Agl3 belongs to the short-chain dehydrogenase/reductase enzyme superfamily, together with SQD1 from Arabidopsis thaliana, the only UDP-sulfoquinovose synthase with known crystal structure. By comparison of sequence and structure of Agl3 and SQD1, putative catalytic amino acids of Agl3 were selected for mutational analysis. The obtained data suggest for Agl3 a modified dehydratase reaction mechanism. We propose that in vitro biosynthesis of UDP-sulfoquinovose occurs through an NAD+-dependent oxidation/dehydration/enolization/sulfite addition process. In the absence of a sulfur donor, UDP-d-glucose is converted via UDP-4-keto-d-glucose to UDP-d-glucose-5,6-ene, the structure of which was determined by 1H and 13C-NMR spectroscopy. During the redox reaction the cofactor remains tightly bound to Agl3 and participates in the reaction in a concentration-dependent manner. For the first time, the rapid initial electron transfer between UDP-d-glucose and NAD+ could be monitored in a UDP-sulfoquinovose synthase. Deuterium labeling confirmed that dehydration of UDP-d-glucose occurs only from the enol form of UDP-4-keto-glucose. The obtained functional data are compared with those from other UDP-sulfoquinovose synthases. A divergent evolution of Agl3 from S. acidocaldarius is suggested., Extremophiles, 19 (2)

Synthetic dyes used in the textile- or pulp- and paper industry are often harmful or toxic compounds and represent a worldwide ecological problem. In the presented work we aim to develop an eco-friendly, enzymatic approach for dye removal. We investigated 6 different oxidoreductases for their potential to specifically decolorize and detoxify 207 industrially relevant dyes including anthraquinone-, azo-, trimethylmethane-, and indigo chromophores. Utilized enzymes were cellobiose dehydrogenase from the plant-pathogenic fungus Sclerotium rolfsii, laccases from the ascomycete Botrytis aclada and the basidiomycete Trametes pubescens, glucose oxidase from Aspergillus niger, glucose dehydrogenase from Glomerella cingulata and horseradish peroxidase. Decolorization was studied at alkaline, neutral and acidic pH using UV/vis spectroscopy in the 96-well plate format. Absorbances of most of the investigated dyes could be reduced by at least 50% within 24 hours of incubation at room temperature. Highest rates of decolorization were observed at pH 4 for most of the enzymes. Horseradish peroxidase efficiently degraded 79 of the 175 dyes diluted in water (45%) followed by laccase from T. pubescens (27%), laccase from B. aclada (27%), cellobiose dehydrogenase (15%) and glucose dehydrogenase (5.7%). Glucose oxidase (4%) was least successful, indicating that peroxide has little effect on the chromophores. Toxicity of three dyes was investigated using freshwater algae Selenastrum capricornutum. Ongoing research aims to assess the toxicity and chemical structure of the reaction products. Elucidation of the kinetic parameters of the reactions might help to define enzyme mixtures for efficient dye decolorization.

Synthetic dyes used in the textile- or pulp- and paper industry are often harmful or toxic compounds and represent a worldwide ecological problem. In the presented work we investigate an eco-friendly, enzymatic approach for dye removal. We evaluated seven different oxidoreductases for their potential to specifically decolorize and detoxify 175 industrially relevant dyes including anthraquinone-, azo-, trimethylmethane-, and indigo chromophores. Utilized enzymes were cellobiose dehydrogenase from the plant-pathogenic fungus Sclerotium rolfsii, laccases from the ascomycete Botrytis aclada and the basidiomycete Trametes pubescens, glucose oxidase from Aspergillus niger, horseradish peroxidase, manganese peroxidase from Phanerochaete chrysosporium and lignin peroxidase. Decolorization was studied at alkaline, neutral and acidic pH using a high-troughput spectroscopic assay. Absorbances of most of the investigated dyes could be reduced by at least 50% within 24 hours of incubation at room temperature. Highest rates of decolorization were observed at acidic pH for most of the enzymes. Laccase from T. pubescens efficiently degraded 85 of the 175 dyes (49%) followed by horseradish peroxidase (45%), laccase from B. aclada (23%) and cellobiose dehydrogenase (13%). Glucose oxidase (4%) was least successful, indicating that hydrogen peroxide production has little effect on the chromophores. Our results show that with a limited number of enzymes a multitude of dyes can be effectively decolorized. Further research was performed to study the chemical structure and toxic effects of the dyes and their degradation products to assess their environmental impact. Catalytic parameters of the enzymatic reactions were elucidated to define optimal application conditions for efficient dye degradation.

The recalcitrance of lignocellulose makes enzymatic hydrolysis of plant biomass for the production of second generation biofuels a major challenge. This work investigates an efficient and economic approach for the enzymatic hydrolysis of sugar beet pulp (SBP), which is a difficult to degrade, hemicellulose-rich by-product of the table sugar industry. Three fungal strains were grown on different substrates and the production of various extracellular hydrolytic and oxidative enzymes involved in pectin, hemicellulose, and cellulose breakdown were monitored. In a second step, the ability of the culture supernatants to hydrolyze thermally pretreated SBP was tested in batch experiments. The supernatant of Sclerotium rolfsii, a soil-borne facultative plant pathogen, was found to have the highest hydrolytic activity on SBP and was selected for further hydrolyzation experiments. A low enzyme load of 0.2 mg g(-1) protein from the culture supernatant was sufficient to hydrolyze a large fraction of the pectin and hemicelluloses present in SBP. The addition of Trichoderma reesei cellulase (1-17.5 mg g(-1) SBP) resulted in almost complete hydrolyzation of cellulose. It was found that the combination of pectinolytic, hemicellulolytic, and cellulolytic activities works synergistically on the complex SBP composite, and a combination of these hydrolytic enzymes is required to achieve a high degree of enzymatic SBP hydrolysis with a low enzyme load.

A crisis of energy storing molecules (fuels) which are currently produced from crude mineral oil is expected in the future due to limited resources. One strategy to compensate a part of the oil deficiency is the production of biofules from lignocelluloses waste materials from agriculture and wood waste biomass. This work investigates and efficient and economical approach for the integrated enzymatic hydrolisation and fermentation of lignocellulosess (sugar beet pulp, corn cob and beech bark) to bioethanol through a new aspect of bioethanol production processes by investigating sustainability of the design of experiment to optimize the effectiveness of different enzymes (cellulase, pectinase, Ultrazym AFP.L and Viscozym L).

Design of Experiment (DOE) is one of the most efficient tools that enables improving the efficiency of experimentation, reducing costs and obtaining many relevant information on the desired system [1]. In addition, it allows gaining knowledge on investigated parameters and their interaction with minimal number of experiments [2]. In order to investigate which design of experiment is the most suitable for investigation and optimization of the enzyme efficiency, in this work we have compared several different designs (General, Two level fractorial, Box Behnken, and D-optimal designs) for the optimization of model enzyme sample (Pectinase). During the optimization we have varied temperature (in ranges from 20 to 50 ºC), pH (in ranges from 3 to 7) and the volume of the enzyme (in ranges from 20 to 80 μL) in the system in order to estimate their individual and mutual effects on the enzymatic efficiency. The function of the optimization was “reducing sugars” function which was measured with the 3, 5-dinitrosalicylic acid (DNSA) assay procedure after 24 hours as the amount of galacturonic acid. The highest concentration of liberated galacturonic acid was reached at 45°C on a thermo-shaker with 80 μL of enzyme solution in citrate buffer pH 4. This experimental optimum was compared to data obtained after calculation with different designs of experiment, and a very good agreement between those data was achieved. Moreover, our results have shown that D-optimal design was the most suitable design for enzymatic research. In contrast, Box Behnken design was suitable only for preliminary testing of desired system and predicting the optimal working area. Therefore we showed that DOE is the methodology that enables comprehensive analysis of complex systems with highly efficiency, reduced costs and increased value of the quality of the obtained information.

Sugar beet pulp (SBP) was hydrolyzed with combinations of commercially available fungal enzymes (Ultrazym AFP-L, Viscozyme L, Pectinase and Cellulase) and wood-rotting fungi (Trametes multicolor, Sclerotium rolfsii and Neurospora crassa) lignocellulolytic enzymes. Among the tested enzymes, the plant pathogen Sclerotium rolfsii was found to be the best-performing fungus and it was chosen for further research. Current research activities were focused on the optimization of the hydrolytic process using a factorial design approach and response surface methodology (RSM). The effect of various experimental parameters and their interactions with each other was evaluated using those methods and consequently the optimum conditions were accomplished. The set-up parameters were the enzyme loads (1, 2, 3, 4 μL total enzymes / mg SBP), pH (3, 4, 5, 6, 7) and temperature (20, 30, 40, 50 oC). Using statistical design methodology, it was observed dramatically reduce of the required enzyme loads to only 2 μL total enzymes / g SBP, at the optimal pH and temperature value. Optimal parameters of hydrolytic process would apply in the further investigation of SBP hydrolysis to obtain more economical process for biofuels, biopolymers or biochemicals production.

The production of sustainable liquid fuels such as bioethanol from renewable resources has become a primary worldwide concern. To date, the major problem of biorefinery processes is the high production cost of enzymes used for the degradation of lignocelluloses into fermentable sugars. Here we report the production of lignocellulolytic, hemicellulolytic and pectinolytic enzymes by three fungi using sugar beet pulp as relatively cheap and readily available substrate. The fungi under investigation were the white-rot basidiomycete Trametes multicolor and the plant pathogens Sclerotium rolfsii and Neurospora crassa. In cultivation experiments the activities of extracellular enzymes, namely cellulases, pectinases, xylanases, arabinases, mannanases, manganese peroxidase, laccase and cellobiose dehydrogenase were differently induced and monitored. By optimization of the cultivation conditions highly efficient enzyme preparations were obtained, which have hydrolytic activity towards sugar beet pulp. The highest arabinase (1.3 U mL−1), pectinase (0.78 U mL−1) and carboxymethyl cellulase (0.49 U mL−1) activities were observed in cultures of Sclerotium rolfsii grown on sugar beet pulp. The highest xylanase (0.88 U mL−1), manganese peroxidase (0.3 U mL−1) and laccase activities were detected in Trametes multicolor cultures. Neurospora crassa does not perform equally well as the other two fungi. The clearified culture supernatants were used to degrade sugar beet pulp enzymatically and found to hydrolyze all polymers efficiently and fast.

A recently proposed coenzyme regeneration system employing laccase and a number of various redox mediators for the oxidation of NAD(P)H was studied in detail by kinetic characterization of individual reaction steps. Reaction engineering by modeling was used to optimize the employed enzyme, coenzyme as well as redox mediator concentrations. Glucose dehydrogenase from Bacillus sp. served as a convenient model of synthetic enzymes that depend either on NAD+ or NADP+. The suitability of laccase from Trametes pubescens in combination with acetosyringone or syringaldazine as redox mediator was tested for the regeneration (oxidation) of both coenzymes. In a first step, pH profiles and catalytic constants of laccase for the redox mediators were determined. Then, second-order rate constants for the oxidation of NAD(P)H by the redox mediators were measured. In a third step, the rate equation for the entire enzymatic process was derived and used to build a MATLAB model. After verifying the agreement of predicted vs. experimental data, the model was used to calculate different scenarios employing varying concentrations of regeneration system components. The modeled processes were experimentally tested and the results compared to the predictions. It was found that the regeneration of NADH to its oxidized form was performed very efficiently, but that an excess of laccase activity leads to a high concentration of the oxidized form of the redox mediator – a phenoxy radical – which initiates coupling (dimerization or polymerization) and enzyme deactivation.

Additional file 1: Table S1. Purification schemes of recombinant CtDH/CtCDH. Table S2. Comparison of recombinant DH domain and intact CDHs from literatures. Figure S1. SDS-PAGE of recombinant CtDH expressed in E. coli. Figure S2. Thermostability of CtDH/CtCDHs measured by the tryptophan fluorescence (A-D) and the ThermoFAD method (E-H).

Additional file 1: Table S1. Purification schemes of recombinant CtDH/CtCDH. Table S2. Comparison of recombinant DH domain and intact CDHs from literatures. Figure S1. SDS-PAGE of recombinant CtDH expressed in E. coli. Figure S2. Thermostability of CtDH/CtCDHs measured by the tryptophan fluorescence (A-D) and the ThermoFAD method (E-H).