Application of Thermoplastic Microbioreactor to the Single Cell Study of Budding Yeast to Decipher the Effect of 5-Hydroxymethylfurfural on Growth

Yeast cells are generally used as a model system of eukaryotes due to their complex genetic structure, rapid growth ability in optimum conditions, easy replication and well-defined genetic system properties. Thus, yeast cells increased the knowledge of the principal pathways in humans. During fermentation, carbohydrates (hexoses and pentoses) degrade into some toxic by-products such as 5-hydroxymethylfurfural (5-HMF or HMF) and furfural. HMF influences the ethanol yield, and ethanol productivity; it interferes with microbial growth and is considered as a potent inhibitor of bioethanol production. In this study, yeast single cell behavior under HMF application was monitored by using a continuous flow single phase microfluidic platform. Microfluidic device in operation is fabricated by hot embossing and thermo-compression techniques from cyclo-olefin polymer (COP). COP is biocompatible, transparent and rigid material and it is suitable for observing fluorescence of cells considering its low auto-fluorescence characteristic. The response of yeast cells was recorded through Red Fluorescent Protein (RFP) tagged Nop56 gene product, which is an essential evolutionary-conserved nucleolar protein, and also a member of the box C/D snoRNP complexes. With the application of HMF, yeast cell proliferation continued but HMF slowed down the cell growth, and after HMF treatment the cell proliferation stopped. By the addition of fresh nutrient medium, the yeast cells recovered after 6 hours of HMF exposure. Thus, HMF application suppresses normal functioning of cell cycle but it does not cause cells to die. The monitoring of Nop56 expression phases of the individual cells shed light on the protein and ribosome synthesis cycles along with their link to growth. Further computational study revealed that the mechanisms underlying the inhibitory or inductive effects of HMF on growth are enriched in functional categories of protein degradation, protein processing, DNA repair and multidrug resistance. The present microfluidic device can successfully be used for studying the effects of inhibitory agents on growth by single cell tracking, thus capturing cell to cell variations. By metabolic engineering techniques, engineered strains can be developed, and the metabolic network of the microorganism can thus be manipulated such that chemical overproduction of target metabolite is achieved along with the maximum growth/biomass yield.  

Processing and Economic Analysis of Rain Tree (Samanea saman) Pods for Village Level Hydrous Bioethanol Production

Biofuel is one of the renewable energy sources adapted by the Philippine government in order to lessen the dependency on foreign fuel and to reduce carbon dioxide emissions. Rain tree pods were seen to be a promising source of bioethanol since it contains significant amount of fermentable sugars. The study was conducted to establish the complete procedure in processing rain tree pods for village level hydrous bioethanol production. Production processes were done for village level hydrous bioethanol production from collection, drying, storage, shredding, dilution, extraction, fermentation, and distillation. The feedstock was sundried, and moisture content was determined at a range of 20% to 26% prior to storage. Dilution ratio was 1:1.25 (1 kg of pods = 1.25 L of water) and after extraction process yielded a sugar concentration of 22 0Bx to 24 0Bx. The dilution period was three hours. After three hours of diluting the samples, the juice was extracted using extractor with a capacity of 64.10 L/hour. 150 L of rain tree pods juice was extracted and subjected to fermentation process using a village level anaerobic bioreactor. Fermentation with yeast (Saccharomyces cerevisiae) can fasten up the process, thus producing more ethanol at a shorter period of time; however, without yeast fermentation, it also produces ethanol at lower volume with slower fermentation process. Distillation of 150 L of fermented broth was done for six hours at 85 °C to 95 °C temperature (feedstock) and 74 °C to 95 °C temperature of the column head (vapor state of ethanol). The highest volume of ethanol recovered was established at with yeast fermentation at five-day duration with a value of 14.89 L and lowest actual ethanol content was found at without yeast fermentation at three-day duration having a value of 11.63 L. In general, the results suggested that rain tree pods had a very good potential as feedstock for bioethanol production. Fermentation of rain tree pods juice can be done with yeast and without yeast.

Application of Acinetobacter sp. KKU44 for Cellulase Production from Agricultural Waste

Due to a high ethanol demand, the approach for  effective ethanol production is important and has been developed  rapidly worldwide. Several agricultural wastes are highly  abundant in celluloses and the effective cellulase enzymes do exist  widely among microorganisms. Accordingly, the cellulose  degradation using microbial cellulase to produce a low-cost substrate  for ethanol production has attracted more attention. In this  study, the cellulase producing bacterial strain has been isolated  from rich straw and identified by 16S rDNA sequence analysis as Acinetobacter sp. KKU44. This strain is able to grow and exhibit the cellulase activity. The optimal temperature for its growth and  cellulase production is 37°C. The optimal temperature of bacterial  cellulase activity is 60°C. The cellulase enzyme from  Acinetobacter sp. KKU44 is heat-tolerant enzyme. The bacterial culture of 36h. showed highest cellulase activity at 120U/mL when  grown in LB medium containing 2% (w/v). The capability of  Acinetobacter sp. KKU44 to grow in cellulosic agricultural wastes as a sole carbon source and exhibiting the high cellulase activity at high temperature suggested that this strain could be potentially developed further as a cellulose degrading strain for a production of low-cost substrate used in ethanol production.   

Pseudo-Homogeneous Kinetic of Dilute-Acid Hydrolysis of Rice Husk for Ethanol Production: Effect of Sugar Degradation

Rice husk is a lignocellulosic source that can be converted to ethanol. Three hundreds grams of rice husk was mixed with 1 L of 0.18 N sulfuric acid solutions then was heated in an autoclave. The reaction was expected to be at constant temperature (isothermal), but before that temperature was achieved, reaction has occurred. The first liquid sample was taken at temperature of 140 0C and repeated every 5 minute interval. So the data obtained are in the regions of non-isothermal and isothermal. It was observed that the degradation has significant effects on the ethanol production. The kinetic constants can be expressed by Arrhenius equation with the frequency factors for hydrolysis and sugar degradation of 1.58 x 105 1/min and 2.29 x 108 L/mole/min, respectively, while the activation energies are 64,350 J/mole and 76,571 J/mole. The highest ethanol concentration from fermentation is 1.13% v/v, attained at 220 0C.

Conversion of Sugarcane Shoots to Reducing Sugars

Sugarcane Shoots is an abundantly available residual resources consisting of lignocelluloses which take it into the benefit. The present study was focused on utilizing of sugarcane shoot for reducing sugar production as a substrate in ethanol production. Physical and chemical pretreatments of sugarcane shoot were investigated. Results showed that the size of sugarcane shoot influenced the cellulose content. The maximum cellulose yield (60 %) can be obtained from alkaline pretreated sugarcane shoot with 1.0 M NaOH at 30 oC for 90 min. The cellulose yield reached up to 93.9% (w/w). Enzymatically hydrolyzed of cellulosic residual in 0.04 citrate buffer (pH 5) with celluclast 1.5L (0.7 FPU/ml) resulted in the highest amount of reducing sugar at a rate of 32.1 g/l after 4 h incubation at 50°C, and 100 oC for 5 min . Cellulose conversion was 55.5%.

Pseudo-Homogeneous Kinetic of Dilute-Acid Hydrolysis of Rice Huskfor Ethanol Production: Effect of Sugar Degradation

Rice husk is a lignocellulosic source that can be converted to ethanol. Three hundreds grams of rice husk was mixed with 1 L of 0.18 N sulfuric acid solutions then was heated in an autoclave. The reaction was expected to be at constant temperature (isothermal), but before that temperature was achieved, reaction has occurred. The first liquid sample was taken at temperature of 140 0C and repeated every 5 minute interval. So the data obtained are in the regions of non-isothermal and isothermal. It was observed that the degradation has significant effects on the ethanol production. The kinetic constants can be expressed by Arrhenius equation with the frequency factors for hydrolysis and sugar degradation of 1.58 x 105 min-1 and 2.29 x 108 L/mole-min, respectively, while the activation energies are 64,350 J/mole and 76,571 J/mole. The highest ethanol concentration from fermentation is 1.13% v/v, attained at 220 0C.

Water Vapor Plasma Torch: Design, Characteristics and Applications

The atmospheric pressure plasma torch with a direct current arc discharge stabilized by water vapor vortex was experimentally investigated. Overheated up to 450K water vapor was used as plasma forming gas. Plasma torch design is one of the most important factors leading to a stable operation of the device. The electrical and thermal characteristics of the plasma torch were determined during the experimental investigations. The design and the basic characteristics of the water vapor plasma torch are presented in the paper. Plasma torches with the electric arc stabilized by water vapor vortex provide special performance characteristics in some plasma processing applications such as thermal plasma neutralization and destruction of organic wastes enabling to extract high caloric value synthesis gas as by-product of the process. Syngas could be used as a surrogate fuel partly replacing the dependence on the fossil fuels or used as a feedstock for hydrogen, methanol production.

Bioethanol Production from Enzymatically Saccharified Sunflower Stalks Using Steam Explosion as Pretreatment

Sunflower stalks were analysed for chemical compositions: pentosan 15.84%, holocellulose 70.69%, alphacellulose 45.74%, glucose 27.10% and xylose 7.69% based on dry weight of 100-g raw material. The most optimum condition for steam explosion pretreatment was as follows. Sunflower stalks were cut into small pieces and soaked in 0.02 M H2SO4 for overnight. After that, they were steam exploded at 207 C and 21 kg/cm2 for 3 minutes to fractionate cellulose, hemicellulose and lignin. The resulting hydrolysate, containing hemicellulose, and cellulose pulp contained xylose sugar at 2.53% and 7.00%, respectively.The pulp was further subjected to enzymatic saccharification at 50 C, pH 4.8 citrate buffer) with pulp/buffer 6% (w/w)and Celluclast 1.5L/pulp 2.67% (w/w) to obtain single glucose with maximum yield 11.97%. After fixed-bed fermentation under optimum condition using conventional yeast mixtures to produce bioethanol, it indicated maximum ethanol yield of 0.028 g/100 g sunflower stalk.