Alkaline Heat Research Articles

Nano-size hydroxyapatite extracted from tilapia scale using alkaline heat treatment method

In biomedical research, hydroxyapatite (HAp) has been commonly used as the coating materials, filler and as a drug delivery system. This is mainly due to the similarity of the chemical composition of HAp to human bone and teeth that make it have excellent bioactivity and biocompatibility. Hence, the recovery of HAp from a natural resource such as from the animal scale and bone is a promising alternative to produce natural HAp, which is much similar to the human bone. Thus, in this research, the nano-size HAp was extracted from tilapia fish scale using alkaline heat treatment. The fish scale was pre-treated with 0.1% HCl to deproteinize the scale prior to the alkaline heat treatment. The alkaline heat treatment was performed in two stages to ensure complete removal of the organic compound from the tilapia scale. Upon completing the procedure, off white HAp powder was obtained. The HAp powder was characterised using X-ray diffraction analysis (XRD), field emission scanning electron microscopy analysis (FESEM), energy dispersive spectroscopy (EDS), and fourier-transform infrared spectroscopy (FTIR). The XRD pattern demonstrated that the obtained powder was a pure crystalline HAp. The FESEM micrograph revealed that the HAp powder exhibited rod shapes with a size ranged between 50 and 60 nm width and 30–200 nm length. The EDS result revealed that the extracted HAp powder had a Ca/P ratio of 1.74, which was higher compared to stoichiometric HAp. Lastly, the corresponding functional groups of PO43- and CO32– were identified using FTIR were corresponded to the HAp functional group. In summary, the natural HAp extracted from the tilapia scales using alkaline heat treatment method has the potential to be used as biomaterials for the biomedical implant.

Efficient H2O2 generation and electro-Fenton degradation of pollutants in microchannels of oxidized monolithic-porous-carbon cathode.

A monolithic-porous carbon (MPC) was fabricated by carbonizing a pine rod, and then the oxidized monolithic-porous carbon (O-MPC) was obtained via alkaline heat treatment of MPC. With well-ordered axial channels (diameters were 6-14 μm and 40-50 μm) and oxygen-containing functional groups, O-MPC as a flow-through cathode exhibited good capability in continuously generating H2O2 (422 μmol/L) via oxygen reduction. O-MPC and additional Fe2+ composed an electro-Fenton system and the performance of the system in degradation of organic pollutants was evaluated. For phenol, bisphenol A or sulfamethoxazole at initial concentration of 10 mg/L the removal efficiency reached 74%-82% in a hydraulic retention time of 100 seconds, nearly close to that of the homogeneous Fenton process (90%). This efficiency may be maintained for at least 10 hours, indicating a good stability of O-MPC. The results demonstrate the potential of O-MPC as a flow-through electrode to realize homogeneous-like degradation of organic pollutants in wastewater.

Extraction of Hydroxyapatite from Fish Scales and Its Integration with Rice Husk for Ammonia Removal in Aquaculture Wastewater

Aquaculture plays an important role in providing food and generate high income in many developing countries but the abundance of ammonia discharged from aquaculture wastewater gives the problem to the environment. This study focused on the extraction of hydroxyapatite (HAp) from fish scales and its modification with rice husk to produce bio-adsorbent for ammonia removal from aquaculture wastewater. The comparison has been made for the HAp preparation via microwave irradiation, alkaline heat treatment, and thermal decomposition method. X-ray Diffraction (XRD) and Fourier Transform Infrared Spectroscopy (FTIR) were used to analyze the surface chemistry and crystallinity of HAp, respectively. While the morphology of the HAp was observed under a Scanning Electron Microscope (SEM). Hydroxyapatite extracted via thermal decomposition method shows the best performance about 79% of ammonia removal at 210 min contact time which selected for coating agent of rice husk (RH/HAp) and was successfully removed 84% at 60 min saturation time. The result shows thermal decomposition is the best technique to extract HAp from fish scales and its integration with rice husk exhibited a better performance of bio-adsorbent. The findings of this study provide useful fundamental knowledge and platform for the development and improvement of aquaculture wastewater treatment system in the future.

Green synthesis of biocompatible nanostructured hydroxyapatite from Cirrhinus mrigala fish scale – A biowaste to biomaterial

In this study, we report a green approach to prepare biocompatible nanostructured hydroxyapatite (NHAp) crystalline powders from Cirrhinus mrigala fish scale wastes by a simple alkaline heat treatment process. The crystallinity and functional groups of the as-prepared material were confirmed using X-ray diffraction and fourier-transform infrared spectroscopy. The surface morphology was characterized using transmission electron microscopy. The formation of NHAp and Calcium/Phosphorus (Ca/P) molar ratio was confirmed using Energy-dispersive X-ray spectroscopy and Ca/P molar ratio was 1.67. The thermal stability of NHAp was examined by thermogravimetric analysis. Further, the biocompatibility of as-synthesized NHAp was examined towards the cell viability of MG-63 cells (human osteosarcoma cells) with various dosages. NHAp boosts up the growth of MG-63 cells which showed superior cell viability and ALP activity when compared with commercial HAp (CHAp). This result illustrates that NHAp synthesized from Cirrhinus mrigala fish scale biowaste showed excellent biocompatibility, and it can be a potential alternative biomaterial for various biomedical applications.

The effect of sintering temperature on characteris-tic and properties of hydroxyapatite extracted from fish scale bio-waste

This present study focused on the extraction of HAp from fish scale waste using alkaline heat treatment sintered at different sintering tem-perature (high range) between 300° C to 1000°C. White powder hydroxyapatite was characterized in term of morphology, surface chemistry and crystallinity structure using Scanning Electron Microscopy, Fourier transform infrared spectroscopy (FTIR) and X-ray Diffraction (XRD), respectively. Analysis from XRD shows that FSHAp-1000 has good sharp peak indicating for high crystallinity of hydroxyapatite. While, the functional analysis performed FTIR determined several functional group attributed to PO42-, CO23- and OH-. As sintering tem-perature increase, the broad peak of PO42- becomes narrower. The intensity of CO32- is observed decreased at higher calcination temperature since they are release as volatile gases. Meanwhile, adsorbed water become narrower under treatment of water. The morphology of FSHAp changed upon treatment of heat especially at high temperature. As sintering temperature increase, the particle size of FSHAp becomes fines and a regular shape of FSHAp was found agglomerated.

Effects of hydrothermal sterilization on properties of biological coating fabricated by alkaline-heat treatment on titanium

Alkaline-heat (AH) treatment is a feasible method to prepare Titanium (Ti) based metals with an active ceramic coating for biological applications. Currently, the AH treated Ti substrates are usually sterilized with ethylene oxide gas (EO) or Gamma-ray irradiation (GI) before biological experiments and clinical implantation. However, the EO process is complex and residual gas is toxic for tissue and cells whereas radioactive source is difficult to access for GI and therefore both methods are not practical for dental offices and laboratories. Previously, hydrothermal sterilization (HS) was proposed for all kinds of Ti based endosseous implants and had been proven promising for clinical applications due to its advantage of restoring the osseointegration of Ti implants stored for long-term. In this study, the effects of HS on properties of AH treated Ti were studied in comparison with conventional autoclaving (AC). Both sterilizations were carried out at 121 °C for 20 min in a common autoclave and distilled water was used as medium for HS only. Results showed that, both AC and HS altered the nano-structure of the ceramic coating formed by AH and caused significant Sodium (Na) loss. AC decreased crystallinity of the coating by forming numerous nano-sheets, whereas HS increased the crystallinity, densified the coating and improved its scratch resistance. AC depressed the formation of apatite, in contrast, a complete apatite layer was formed on the HS specimen after being immersed in simulated body fluid (SBF) for 4 days. Both attachment and proliferation of osteoblast-like MC3T3-E1 cells on the HS specimens were better than that on the AC ones. Decontamination, superhydrophilicity and high crystallinity from HS were believed to be the main contributors. In summary, the current results showed that HS can be used as a sterilization method for AH treated Ti implants in biomedical applications.

Evaluation of antibacterial and Antibiofilm activity of Synthesized Zinc-Hydroxyapatite Biocomposites from Labeo rohita fish scale waste

Materials based on hydroxyapatite (HAp) Synthesized from bio-wastes have been regarded as useful, novel, eco-friendly medical applications that are targeted primarily for their antibacterial nature. In the present study, HAp was Synthesized from the fish scales of Labeo rohita using alkaline heat treatment and subsequently mixed with 1, 2 and 3 wt% of zinc (Zn) at 800 °C using calcination method to yield Zn-HAp composites. A detailed characterization of the generated composites was analysed by XRD, FT-IR, SEM, EDX and DLS methods. Further, antibacterial and biofilm inhibitory activity of the generated composites was determined using strains of Staphylococcus aureus and Escherichia coli. The confirmation of the presence of zinc, confirmed by EDAX spectra, XRD, FT-IR, SEM and DLS observations, established that HAp and Zn-HAp composites were without impurities, irregular in shape and were 848 nm sized particles. Although 1–3 wt% Zn-HAp composites showed antibacterial activity, the 3 wt% Zn-HAp composite was found suitable to kill the surrounding bacterial growth and showed potent inhibitory activity against biofilm formation.

Development and Validation of Stability-Indicating RP-HPLC Method for the Estimation of Cuminaldehyde

Cumin aldehyde is an herbaceous plant (Cuminumcyminum L.) volatile oil that used as a regular spice in kitchen foods. It also has some pharmacological properties such as analgesic, hepato-protective, anti-inflammatory, antibacterial, antimicrobial, antifungal, antiviral, antioxidant and anticancer. The key objective of the present study was to develop and validate the RP-HPLC method for the estimation of cumin aldehyde. According to the ICH guidelines, RP-HPLC stability indicating method was used in which reverse phase enable Cosmosil C18 column (250 × 4.6 mm, 5μm) used in isocratic mode. For the estimation of cumin aldehyde, sodium sulphate: acetonitrile: methanol (20:73:7 v/v) was used as a mobile phase and it was delivered at flow rate of 1.0 mL min- 1. The injection volume was 20 µL and elute was analyzed by a UV detector at 326 nm. Linearity was observed between the concentration range of 20 μg mL-1 -140 μg mL-1 and the correlation coefficient R2 value was found to be 0.997 ± 0.5. The method was accurate and recovery was found to be in the range of 98.06 -100.40 %. The limit of detection of cumin aldehyde was found to be 1.04 μgmL-1and limit of quantitation was found to be 3.16 μgmL-1. Cumin aldehyde was subjected to stress conditions including acidic, alkaline, neutral, oxidation, and dry heat degradation. Cumin aldehyde was more sensitive to acidic, dry heat and oxidative degradation and it is stable at alkaline conditions. The method was validated according to ICH guidelines.

Cytotoxicity Analysis of Ti-7.5Mo Alloy After Biomimetic Surface Treatment to Use as Dental Materials

Titanium (Ti) and its alloys are widely used for medical and dental fields due to their excellent biocompatibility, high corrosion resistance, high specific strength and excellent mechanical properties. Different methods have been developed to improve the surface properties of titanium-based implant materials, and consequently the bone-bonding ability. The Ti-7.5Mo alloy was activated by an alkaline treatment with 5M NaOH, heat treatment and subsequent immersion in SBFx5 to investigate the in vitro response of osteoblastic-like cells (MG-63) on altered biomimetic surfaces. Sample surfaces were characterized by scanning electron microscopy. Cytotoxicity was assessed by the MTT assay, total protein content, alkaline phosphatase activity (ALP) and mineralized bone-like nodule formation. It was shown the que alkali treatment led to the formation of sodium titanate and immersion in SBFx5 formed a film of calcium phosphate. The alkaline treatment and heat treatment of 7.5 Ti-Mo alloys followed by soaking them in SBFx5 for 24 hours is a suitable technique once the final samples were biocompatible, allowed the attachment of the osteoblastic-like cells (MG-G3), and increased the mineralized like-bone nodules formation by these cells.

EXTRACTION, CHARACTERIZATION AND ANTIMICROBIAL ACTIVITY OF HYDROXYAPATITE FROM SEABASS AND SEABREAM SCALE

The present study investigates the characterization of hydroxyapatite (HAp) extracted from seabass and seabream scales as by-product. Fish scales obtained from a seafood processing company were used to extract natural HAp powder. HAp powder was extracted by alkaline heat treatment of fish scales and the synthesized HAp (FS-HAp) was extensively characterized with Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and X-ray diffraction (XRD) analysis. Calcium to phosphate ratio of the HAp was confirmed by inductively coupled plasma (ICP) and elemental analysis of HAp were also carried out using energy dispersive x-ray spectroscopy (EDS). The results of the characterization analysis were compared with commercial hydroxyapatite standard (CHAp) and it was clearly confirmed that the extracted FS-HAp exactly showed CHAp characteristics physicochemically which is used as biomaterial. However, well diffusion assay revealed out that synthesized hydroxyapatite showed no activity against C. albicans , S. aureus and E. coli . It was concluded that, instead of synthetic apatite, extracted FS-HAp presents a potential promising biomaterial as the raw materials are by-product which economically cheap and sustainable substances.

Multilevel structural responses of β-conglycinin and glycinin under acidic or alkaline heat treatment

The structural responses of soy protein isolate under various processing conditions are of practically important for broadening its functionalities. In this work, multilevel structural responses of β-conglycinin and glycinin under acidic or alkaline heat treatment were investigated. Our results suggested that heat treatment under acidic (i.e., pH2.5) or alkaline (i.e., pH8.5) conditions induced multilevel structural responses of β-conglycinin and glycinin: under acidic heat treatment, both β-conglycinin and glycinin underwent hydrolysis and experienced disruption and reorganization in ordered secondary structure. This process was accompanied with changes in tertiary structure where previously buried regions were exposed to the aqueous phase to different extent. Small-angle x-ray scattering (SAXS) results indicated that the protein conformations evolved from globular ones to elongated ellipsoids, with a more remarkable elongation effect in glycinin than β-conglycinin. Upon solvent evaporation, β-conglycinin favored aggregation with a low “lateral to vertical ratio”, glycinin tended to aggregate in a high “lateral to vertical ratio” style. In contrast, alkaline heat treatment did not induce hydrolysis but disturbed the secondary structure instead. The protein monomers maintained the globular conformation and assembled into irregular large aggregates during solvent evaporation. Under either treatment, glycinin responded more sensitively than β-conglycinin at all structure levels. The observed multi-level structural responses can be used to guide the rational modification of soy protein isolate with controlled conformation.

Effect of surface treatments on the surface morphology, corrosion property, and antibacterial property of Ti–10Cu sintered alloy

Ti–10Cu sintered alloy has shown strong antibacterial properties against S. aureus and E. coli and good cell biocompatibility in vitro and in vivo, displaying potential application as an implant material. Surface treatments are always applied to implants to improve the surface biocompatibility. In this paper, several typically used surface treatments, including sandblasting (SB), sandblasted and large-grits acid etching (SLA), and alkaline heat treatment (AH) were chosen to modify the Ti–10Cu. A cp-Ti (commercially pure titanium) sample was used as control sample. The effect of surface treatments on the corrosion properties and antibacterial properties of the Ti–10Cu sintered alloy was investigated. After SB and SLA treatments, a rough surface with a TiO2 layer was formed on the surface, which reduced the corrosion resistance and enhanced the Ti and Cu ion release. After AH treatment, a smooth but microporous surface with a TiO2/titanate layer was formed, which improved slightly the corrosion resistance. However, the Cu ion and Ti ion release from the Ti–10Cu sample was promoted by AH treatment due to the fact that more Ti2Cu phases were exposed on the AH-treated Ti–10Cu sample. It was demonstrated that the Ti–10Cu samples after surface treatments still exhibited good antibacterial properties against S. aureus, which indicated that the surface treatment did not reduce the antibacterial activity. The control mechanism was thought to be related to the high Cu ion release even after surface treatments. It was expected that the surface treatments provided Ti–10Cu sintered alloy with good surface bioactivity without reduction in antibacterial activity.

Molecular Detection Methods for Vibrio parahaemolyticus in Seafood

To detect Vibrio parahaemolyticus in seafood, we evaluated efficient combinations of molecular methods with DNA extraction methods using heat extraction and alkaline heat extraction, and PCR, real-time PCR and loop-mediated isothermal amplification (LAMP) assays were performed targeting V parahaemolyticus species-specific genes (tlh and rpoD) and pathogenic factors genes (tdh and trh). The species-specific genes were detected in all combinations of two strains (a tdh * trh1-positive strain and a trh2-positive strain), two kinds of shellfish (oyster and bloody clams) and molecular methods with tlh-real time PCR or rpoD-LAMP assays with DNA of alkaline heat extraction at 85-145cfu/test level. tdh was detected in both seafoods with real time PCR assay with DNA of heat extraction at 85cfu/test level, and detected with the LAMP and real time PCR assays with DNA of alkaline heat extraction at 85cfu/test level. Detection of both trh1 and trh2 with the PCR assay with DNA of alkaline heat extraction was comparatively high though trh2 was detected with the LAMP assay with DNA of alkaline heat extraction at 145cfu/test level. It, however, is necessary to investigate more sensitive trh-detection methods. In this study, the results indicated that tlh-real time PCR or rpoD-LAMP, tdh-real time PCR and tdh-LAMP assays with DNA of alkaline heat extraction are relatively-sensitive methods to detect V. parahaemolyticus in seafood.

Stability and cytotoxicity of angiotensin-I-converting enzyme inhibitory peptides derived from bovine casein

This study investigated the effect of heat treatment combined with acid and alkali on the angiotensin-I-converting enzyme (ACE) inhibitory activity of peptides derived from bovine casein. The free amino group content, color, and cytotoxicity of the peptides were measured under different conditions. When heated at 100 °C in the pH range from 9.0 to 12.0, ACE inhibitory activity was reduced and the appearance of the peptides was significantly darkened. After thermal treatment in the presence of acid and alkali, the free amino group content of ACE inhibitory peptides decreased markedly. High temperature and prolonged heating also resulted in the loss of ACE inhibitory activity, the loss of free amino groups, and the darker coloration of bovine casein-derived peptides. However, ACE inhibitory peptides, within a concentration range of from 0.01 to 0.2 mg/ml, showed no cytotoxicity to Caco-2 and ECV-304 cell lines after heat treatment. This indicated that high temperature and alkaline heat treatment impaired the stability of bovine casein-derived ACE inhibitory peptides.

A Sensitive and Selective RP-LC Method for the Simultaneous Determination of the Antihypertensive Drugs, Enalapril, Lercanidipine, Nitrendipine and Their Validation

A RP-LC method is presented, which is sensitive and selective for the simultaneous determination of enalapril–lercanidipine and enalapril–nitrendipine binary mixtures in their pharmaceutical dosage forms. The analyte peaks were detected using the LC method with the mobile phase ratio of methanol: water (70:30 v/v, pH 3.0) and a 1.0 mL min−1 flow rate. The detection wavelength was selected at 210 nm using photo diode array detector and column temperature was optimized to 30 °C. Linearity was obtained at different concentration ranges for all working pharmaceutically active compounds between 0.5 and 25 μg mL−1. The proposed methods were extensively validated according to USP 27 requirements and ICH guidelines. The methods were applied to the analysis of pharmaceutical dosage forms containing binary mixtures of enalapril–lercanidipine and enalapril–nitrendipine. Moreover, the proposed methods were applied for the degradation studies of the selected compounds. Degradation studies were conducted using stress conditions such as UV light, acidic and alkaline hydrolysis, oxidation and heat in oven, to evaluate the ability of the separation of the response of standard compounds from their degradation products.

Nanocrystalline hydroxyapatite from fish scale waste: Preparation, characterization and application for selenium adsorption in aqueous solution

Nanocrystalline hydroxyapatite prepared from fish scale waste (FHAp) using the alkaline heat treatment method was used as an adsorbent for the removal of selenite. The FHAp was characterized by BET surface analysis, TG/DTG/DTA, FTIR, XRD and TEM–EDX. Batch adsorption experiments were performed to investigate the effects of various parameters including pH of solution, contact time and initial selenium concentration on adsorption efficiency. The results revealed that FHAp nanocrystals (15–20nm) possessed a good ability to adsorb selenite. First order kinetic, pseudo-second order and intraparticle diffusion model were used to evaluate the kinetic models and the mechanism of adsorption. A comparison of FHAp with other adsorbents was illustrated. Both kinetics and adsorption behavior using Freundlich and Langmuir isotherms were tested. It was found that the kinetics data were fitted well by a pseudo-second order model and the Freundlich isotherm suitably ascribed the adsorption. The adsorption capacities of the FHAp, CHAp (commercial hydroxyapatite), CS (chitosan) and FS (fish scale) itself towards Se (IV) ion were found to be 5.51, 1.42, 1.35 and 2.12 (mgg−1) (Lmg−1)1/n, respectively. Therefore, it is evident that the FHAp nanopowders from the fish scale waste can be used as a natural adsorbent with high adsorption efficiency.

DNA extraction from formalin-fixed laryngeal biopsies: Comparison of techniques

Conclusion: PCR-quality DNA could be extracted from formalin-fixed paraffin-embedded (FFPE) samples with amplicons of at least 390 bp. Paraffin removal was not a necessary step. Proteinase K digestion was as efficient as the commercial kit for DNA extraction with a lower cost. Objectives: To compare different DNA extraction protocols for FFPE samples and to describe the suitability of the extracted DNA for PCR reactions. Methods: For deparaffinization the following techniques were compared: alkaline heat, xylene, and no removal. For DNA extraction, proteinase K digestion and organic extraction were compared. A commercial extraction kit was included as standard. DNA quality was assessed by PCR amplification of the HFE gene, for amplicons of 208 and 390 bp. Results: Extraction with the commercial kit and proteinase K digestion were more efficient than other techniques, with no statistical difference between them for both amplicons. The proteinase K digestion buffer had a cost of U$ 0.2 per sample and the commercial kit of U$7 per sample.

A study on alkaline heat treated Mg–Ca alloy for the control of the biocorrosion rate

To reduce the biocorrosion rate by surface modification, Mg–Ca alloy (1.4 wt.% Ca content) was soaked in three alkaline solutions (Na 2HPO 4, Na 2CO 3 and NaHCO 3) for 24 h, respectively, and subsequently heat treated at 773 K for 12 h. Scanning electron microscopy and energy-dispersive spectroscopy results revealed that magnesium oxide layers with the thickness of about 13, 9 and 26 μm were formed on the surfaces of Mg–Ca alloy after the above different alkaline heat treatments. Atomic force microscopy showed that the surfaces of Mg–Ca alloy samples became rough after three alkaline heat treatments. The in vitro corrosion tests in simulated body fluid indicated that the corrosion rates of Mg–Ca alloy were effectively decreased after alkaline heat treatments, with the following sequence: NaHCO 3 heated < Na 2HPO 4 heated < Na 2CO 3 heated. The cytotoxicity evaluation revealed that none of the alkaline heat treated Mg–Ca alloy samples induced toxicity to L-929 cells during 7 days culture.

Biosynthesis of (&lt;I&gt;R&lt;/I&gt;)-3-Hydroxyalkanoic Acids by Metabolically Engineered &lt;I&gt;Escherichia coli&lt;/I&gt;

An efficient system for the production of (R)-hydroxyalkanoic acids (RHAs) was developed in natural polyhydroxyalkanoate (PHA)-producing bacteria and recombinant Escherichia coli. Acidic alcoholysis of purified PHA and in vivo depolymerization of PHA accumulated in the cells allowed the production of RHAs. In recombinant E. coli, RHA production was achieved by removing CoA from (R)-3-hydroxyacyl-CoA and by in vivo depolymerization of PHA. When the recombinant E. coli harboring the Ralstonia eutropha PHA biosynthesis genes and the depolymerase gene was cultured in a complex or a chemically defined medium containing glucose, (R)-3-hydroxybutyric acid (R3HB) was produced as monomers and dimers. R3HB dimers could be efficiently converted to monomers by mild alkaline heat treatment. A stable recombinant E. coli strain in which the R. eutropha PHA biosynthesis genes were integrated into the chromosome disrupting the pta gene was constructed and examined for the production of R3HB. When the R. eutropha intracellular depolymerase gene was expressed by using a stable plasmid containing the hok/sok locus of plasmid R1, R3HB could be efficiently produced.

Alkaline subcritical-water treatment and alkaline heat treatment for the increase in biodegradability of newsprint waste

This work describes two alkaline semicontinuous processes for the conversion of refractory organic materials into biodegradable substances. Newsprint was used as a lignocellulosic waste. Methane conversion efficiencies and cellulose removals were investigated for the two following processes: alkaline subcritical-water treatment (ASWT) coupled with methane fermentation and alkaline heat treatment (newsprint heated with steam in an autoclave; AHT) coupled with methane fermentation with a neutral subcritical-water treatment (NSWT) recycle. Results showed that for ASWT coupled with methane fermentation higher methane conversion efficiencies and higher cellulose removals were achieved as HRT increased. At HRT = 20 days, average CH4 conversion efficiency and average cellulose removal reached 26% and 44%, respectively. After a final HRT of 40 days, average CH4 conversion efficiency and average cellulose removal reached 50% and 60%, respectively. On the other hand, for AHT coupled with methane fermentation, methane conversion efficiencies did not show a greater improvement using this pretreatment process. Average conversion reached 9% with an average cellulose removal of 20%. In order to improve the yield of the reactor, approximately one-third of the effluent was recycled using NSWT (150 degrees C; neutral pH). Methane conversion efficiency of this process increased as more recycles were performed. For the fifth operation, the total average methane conversion efficiency was 44% with a total average cellulose removal of 55%.