Acid Thermal Hydrolysis Research Articles

Kinetics of in Vitro Guanine- N7-Alkylation in Calf Thymus DNA by (2 S,3 S)-1,2-Epoxybutane-3,4-diol 4-methanesulfonate and (2 S,3 S)-1,2:3,4-Diepoxybutane: Revision of the Mechanism of DNA Cross-Linking by the Prodrug Treosulfan.

Prodrug treosulfan, originally registered for treatment of ovarian cancer, has gained a use in conditioning prior to hematopoietic stem cell transplantation. Treosulfan converts nonenzymatically to the monoepoxide intermediate (EBDM), and then to (2 S,3 S)-1,2:3,4-diepoxybutane (DEB). The latter alkylates DNA forming mainly (2' S,3' S)- N-7-(2',3',4'-trihydroxybut-1'-yl)guanine (THBG) and (2 S,3 S)-1,4-bis(guan-7'-yl)butane-2,3-diol cross-link (bis-N7G-BD) via the intermediate epoxide adduct (EHBG). It is believed that DNA cross-linking by DEB is a primary mechanism for the anticancer and myeloablative properties of treosulfan, but clear evidence is lacking. Recently, we have proved that EBDM alkylates DNA producing (2' S,3' S)- N-7-(2',3'-dihydroxy-4'-methylsulfonyloxybut-1'-yl)-guanine (HMSBG) and that free HMSBG converts to EHBG. In this paper, we investigated the kinetics of HMSBG, bis-N7G-BD, and THBG in DNA in vitro to elucidate the contribution of EBDM and DEB to treosulfan-dependent DNA-DNA cross-linking. Calf thymus DNA was exposed to ( A) 100 μM treosulfan, ( B) 200 μM treosulfan, and ( C) DEB at a concentration 100 μM, exceeding that produced by 200 μM treosulfan. Following mild acid thermal hydrolysis of DNA, ultrafiltration, and off-line HPLC purification, the guanine adducts were quantified by LC-MS/MS. Both bis-N7G-BD and THBG reached highest concentrations in the DNA in experiment B. Ratios of the maximal concentration of bis-N7G-BD and THBG to DEB (adduct Cmax/DEB Cmax) in experiments A and B were 1.7-3.0-times greater than in experiment C. EHBG converted to the bis-N7G-BD cross-link at a much higher rate constant (0.20 h-1) than EBDM and DEB initially alkylated the DNA (1.8-3.4 × 10-5 h-1), giving rise to HMSBG and EHBG, respectively. HMSBG decayed unexpectedly slowly (0.022 h-1) compared with the previously reported behavior of the free adduct (0.14 h-1), which revealed the inhibitory effect of the DNA environment on the adduct epoxidation to EHBG. A kinetic simulation based on the obtained results and the literature pharmacokinetic parameters of treosulfan, EBDM, and DEB suggested that in patients treated with the prodrug, EBDM could produce the vast majority of EHBG and bis-N7G-BD via HMSBG. In conclusion, EBDM can produce DNA-DNA lesions independently of DEB, and likely plays a greater role in DNA cross-linking after in vivo administration of treosulfan than DEB. These findings compel revision of the previously proposed mechanism of the pharmacological action of treosulfan and contribute to better understanding of the importance of EBDM for biological effects.

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of three N-7-guanine adducts of the active epoxides of prodrug treosulfan in DNA in vitro

Prodrug treosulfan undergoes a pH and temperature-dependent activation to the monoepoxide intermediate (EBDM) and (2S,3S)-1,2:3,4-diepoxybutane (DEB). The latter DNA cross-linker is presently believed to mainly account for the pharmacological action of treosulfan. However, neither respective monoadducts nor cross-links have been isolated from treosulfan-treated DNA, and the exact alkylation mechanism of the treosulfan epoxides is unclear. In this paper, liquid chromatography method with tandem mass spectrometry detection (LC-MS/MS) for simultaneous determination of the N-7-guanine adducts of EBDM and DEB − (2′S,3′S)-N-7-(2′3′-dihydroxy-4′-methylsulfonyloxybut-1′-yl)guanine (HMSBG), N-7-(2′,3′,4′-trihydroxybut-1′-yl)guanine (THBG), and 1,4-bis(N-7-guanyl)butane-2,3-diol cross-link (bis-N7G-BD) − in calf-thymus DNA has been developed and validated for the first time. The mixture of drug-free nucleic acid with the analytes and 15N-isotope labeled internal standards underwent a mild acid thermal hydrolysis and ultrafiltration (cut-off 10 kDa). Following offline LC purification, the analytes and internal standards were determined in the LC-MS/MS system with an electrospray interface. Complete resolution of THBG, HMSBG, and bis-N7G-BD was accomplished on a Zorbax Eclipse C18 column using gradient elution with a mobile phase composed of 0.1% formic acid and acetonitrile. Calibration curves were linear in the ranges: THBG 0.2–200 pmol, HMSBG 0.2–20 pmol, and bis-N7G-BD 0.4–40 pmol. The limits of quantitation allowed to determine the adducts at concentration of 330 or 660 per 109 DNA nucleotides. The LC-MS/MS method was adequately precise (coefficient of variation ≤ 16.7%) and accurate (relative error ≤ 17.7%). Calibration standards were stable for 14 days at –25 °C. The validated method enabled determination of THBG, HMSBG, and bis-N7G-BD in calf thymus DNA treated with treosulfan at pH 7.2 and 37 °C, which constitutes a novel bioanalytical application. To the authors’ best knowledge, the quantification of THBG and bis-N7G-BD in one analytical run is also reported for the first time.

Alkaline and acid thermal hydrolysis of biological excess sludge in sequencing batch reactor

The efficiency of biological wastewater treatment processes is based on pH sensitivity which leads to the reduction in the amount of sludge production. In order to achieve the best result, thermal hydrolysis is recognized and used, although acid thermal hydrolysis has serious drawbacks (corrosion, required post-neutralization, etc.). Alkaline thermal hydrolysis has been less remarkable, as the subject of the detailed pilot-scale research of two sequencing batch reactors (SBR) reported in this study. Long-term (about 8 months) continuous experiments were conducted to identify the effect of pH increasing from 4 to 11 in controlled temperature of 60°C to achieve the optimum pH for the reduction of excess biological sludge. After providing a steady state in the reactors, sampling and testing parameters including chemical oxygen demand (COD), mixed liquor suspended solids (MLSS), dissolved oxygen (DO), specific oxygen uptake rate (SOUR), sludge volume index (SVI), and yield (Y) coefficient were evaluated. Results showed that in pH of 9, the kinetic yield coefficient decreased from 0.63 to 0.33. Therefore, excess sludge declined to 44%. Moreover, the soluble COD increased slightly in the effluent, whereas the removal percentage decreased to 79 in the reactor while the amount of SVI and SOUR in this pH dropped to 65 mg/l and 12 mgO2/l.g VSS, respectively. However, no sludge was seen in the higher pH or lower pH, whereas effluent soluble chemical oxygen demand (SCOD) and turbidity was increased. Additionally, the wastewater disposal standards were not achieved and had a bad odor.

Industrial-scale bioethanol production from brown algae: Effects of pretreatment processes on plant economics

Brown algae, considered as the third generation biomass, offer several advantages over lignocellulosic biomass. However, the current high cost of seaweed cultivation hampers the industrialization of macroalgae-based biofuel production. The aim of this study is to determine the maximum dry seaweed price (MDSP) as an upper limit for purchasing price of brown algae at bioethanol plant gates. In addition, a minimum ethanol-selling price (MESP) was calculated by considering the state-of-the-art bioethanol production technology and current brown algae-cultivation costs. A new simple pretreatment process was economically validated and compared with the traditional acid thermal hydrolysis known as combined pretreatment. The processes were simulated at the scales of 80,000 and 400,000ton/year of dry brown algae using the Aspen Plus v.8.4 software, and techno-economic models were developed based on mass and energy balance. MDSP for the simple and combined processes was calculated as 64.6 and 26$/ton (80,000ton/year) and 91.3 and 71.5$/ton (400,000ton/year), respectively. In addition, MESP for the simple and combined processes was determined as 2.39 and 2.85$/gal (80,000ton/year) and 2.08 and 2.33$/gal (400,000ton/year), respectively. These results indicate that the simple pretreatment is economically superior to the combined pretreatment. A comprehensive sensitivity analysis showed that seaweed price had the highest impact on MESP, thereby confirming that the cost-effective large-scale seaweed cultivation is the key to the success of macroalgae-based biofuel production.

Process simulation of bioethanol production from brown algae

Steady state ethanol production from brown algae (Saccharina japonica) based on 100,000 ton/year dry feed was simulated using Aspen Plus V7.3 software. Different process units including saccharification, fermentation and purification were modeled based on experimental works obtained from literature. Acid thermal hydrolysis using H2SO4 and simultaneous saccharification and fermentation (SSF) were used and modeled in this simulation. Distillation columns along with molecular sieves were used to recover ethanol from the raw fermentation broth to produce 99.5% ethanol. This simulation is the first attempt in literature to evaluate large-scale production of ethanol from macroalgae and allows its economic analysis.

Production of functional oligosaccharides through limited acid hydrolysis of agave fructans

A controlled acid thermal hydrolysis process of fructans from agave (Agave tequilana Weber var. azul) was designed to produce a mixture of functional prebiotic fructooligosaccharides and sweetening power. Despite its highly branched structure, first-order kinetic behaviour with respect to substrate concentration was found with an activation energy of 95kJ/mol, similar to the value found for other linear fructans such as chicory inulin. Fructose equivalent (FE) an analogous parameter to dextrose equivalent (DE) used in the starch industry was introduced to characterise fructan hydrolysis; maximum oligosaccharide production was observed when fructose equivalent (FE) reached 27–48 with a structural profile analysed by high-performance anion-exchange chromatography HPAEC-PAD. After hydrolysis, glucose and fructose may be eliminated through a biological purification step involving the addition of Pichia pastoris cells, which selectively consume these sugars but are unable to metabolise fructooligosaccharides.

Enzymatic Hydrolysis of Fructans in the Tequila Production Process

In contrast to the hydrolysis of reserve carbohydrates in most plant-derived alcoholic beverage processes carried out with enzymes, agave fructans in tequila production have traditionally been transformed to fermentable sugars through acid thermal hydrolysis. Experiments at the bench scale demonstrated that the extraction and hydrolysis of agave fructans can be carried out continuously using commercial inulinases in a countercurrent extraction process with shredded agave fibers. Difficulties in the temperature control of large extraction diffusers did not allow the scaling up of this procedure. Nevertheless, batch enzymatic hydrolysis of agave extracts obtained in diffusers operating at 60 and 90 degrees C was studied at the laboratory and industrial levels. The effects of the enzymatic process on some tequila congeners were studied, demonstrating that although a short thermal treatment is essential for the development of tequila's organoleptic characteristics, the fructan hydrolysis can be performed with enzymes without major modifications in the flavor or aroma, as determined by a plant sensory panel and corroborated by the analysis of tequila congeners.

Reducing the Heavy Metal Content of Sewage Sludge by Advanced Sludge Treatment Methods

Waste-activated sludge (WAS) processes are key technologies to treat wastewater: their effluents can meet stringent discharge standards, thus ensuring a minimum residual impact on the aquatic environment. The presence of heavy metals in the excess sludge poses, however, serious problems, and considerably hampers the final disposal alternatives, especially in the agricultural use (soil improvement/amendment). This article studies the effect of thermal hydrolysis and Fenton's peroxidation on the heavy metal content of the dewatered sludge. Acid thermal hydrolysis reduces the heavy metal content in the filter cake except for Cu, Hg, and Pb. Alkaline thermal hydrolysis releases Cu, Pb, and Cr. Fenton's peroxidation transfers Cd, Cu, and Ni from the filter cake into the filtrate. Land application of the residual cake can hence be reconsidered.

Alkaline thermal sludge hydrolysis

The waste activated sludge (WAS) treatment of wastewater produces excess sludge which needs further treatment prior to disposal or incineration. A reduction in the amount of excess sludge produced, and the increased dewaterability of the sludge are, therefore, subject of renewed attention and research. A lot of research covers the nature of the sludge solids and associated water. An improved dewaterability requires the disruption of the sludge cell structure. Previous investigations are reviewed in the paper. Thermal hydrolysis is recognized as having the best potential to meet the objectives and acid thermal hydrolysis is most frequently used, despite its serious drawbacks (corrosion, required post-neutralization, solubilization of heavy metals and phosphates, etc.). Alkaline thermal hydrolysis has been studied to a lesser extent, and is the subject of the detailed laboratory-scale research reported in this paper. After assessing the effect of monovalent/divalent cations (respectively, K +/Na + and Ca 2+/Mg 2+) on the sludge dewaterability, only the use of Ca 2+ appears to offer the best solution. The lesser effects of K +, Na + and Mg 2+ confirm previous experimental findings. As a result of the experimental investigations, it can be concluded that alkaline thermal hydrolysis using Ca(OH) 2 is efficient in reducing the residual sludge amounts and in improving the dewaterability. The objectives are fully met at a temperature of 100 °C; at a pH≈10 and for a 60-min reaction time, where all pathogens are moreover killed. Under these optimum conditions, the rate of mechanical dewatering increases (the capillary suction time (CST) value is decreased from approximately 34 s for the initial untreated sample to approximately 22 s for the hydrolyzed sludge sample) and the amount of DS to be dewatered is reduced to approximately 60% of the initial untreated amount. The DS-content of the dewatered cake will be increased from 28 (untreated) to 46%. Finally, the mass and energy balances of a wastewater treatment plant with/without advanced sludge treatment (AST) are compared. The data clearly illustrate the benefits of using an alkaline AST-step in the system.