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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.107, #1-3)
Permeation associated with three-phase-partitioning method on release of green fluorescent protein by Thereza Christina Vessoni Penna; Eb Chiarini; Adalberto Pessoa Junior (pp. 481-491).
Transformed cells of Escherichia coli expressing recombinant green fluorescent protein (GFPuv) were subjected to two methods of extraction: (1) freezing/thawing/sonication (FTS) cycles prior to the three-phase partitioning (TPP) method, or (2) directly to TPP extraction. The amount of GFPuv released by the FTS plus TPP method varied: 374µg/mL (first cycle), 93–442 µg/mL (second cycle), 32–359 µg/mL (third cycle), 18–115 µg/mL (fourth cycle). The GFPuv yields by the second method (TPP only) were, 23–54 µg/mL for the first extract and 33–91 µg/mL for the second. The FTS plus TPP method released similar amounts of GFPuv to that extracted by TPP; and provided a better mixture elution through the hydrophobic interaction column: 13–63 µg/mL for FTS plus TPP methods, and 2.5–13 µg/mL for TPP. The results showed that although selective permeation is a more laborious methodology, it was more efficient for obtaining of GFPuv in relation to the direct extraction of the cells for TPP.
Keywords: Recombinant green fluorescent protein; Escherichia coli ; protein purification; three-phase-partitioning method
Comparison of growth characteristics of Panax ginseng hairy roots in various bioreactors by Gwi-Taek Jeong; Don-Hee Park; Baik Hwang; Je-Chang Woo (pp. 493-503).
This study investigated the effects of flask-to-liquid volume ratio on the growth of Panax ginseng hairy root, transformed by Agrobacterium rhizogenes, in flask cultures and compared the characteristics of various bioreactors for scale-up. The flask-to-liquid volume ratio was optimum at 1.5 mL of air/mL of medium in flask cultures, and hairy root growth was not affected above the optimum ratio. In 500-mL flask culture, hairy root showed two growth phases. After the first exponential growth, specific growth rate decreased. The growth characteristics of P. ginseng hairy root in various bioreactors were investigated. Hairy root growth was about 55-fold of inoculum after 39 d in a 5-L bioreactor and about 38-fold of inoculum after 40 d in a 19-L bioreactor. Carbon yield was higher in a 19-L bioreactor than in others, but it did not show any linear relationship to the growth rate of hairy roots in bioreactors.
Keywords: Panax ginseng ; transformed hairy root; ginseng polysaccharide; bubble bioreactor; flask-to-liquid volume ratio
Heterogeneous aspects of acid hydrolysis of α-cellulose by Qian Xiang; Y. Y. Lee; Pär O. Pettersson; Robert W. Torget (pp. 505-514).
Hydrolysis of α-cellulose by H2SO4 is a heterogeneous reaction. As such the reaction is influenced by physical factors. The hydrolysis reaction is therefore controlled not only by the reaction conditions (acid concentration and temperature) but also by the physical state of the cellulose. As evidence of this, the reaction rates measured at the high-temperature region (above 200°C) exhibited a sudden change in apparent activation energy at a certain temperature, deviating from Arrhenius law. Furthermore, α-cellulose, once it was dissolved into concentrated H2SO4 and reprecipitated, showed a reaction rate two orders of magnitude higher than that of untreated cellulose, about the same magnitude as cornstarch. The α-cellulose when treated with a varying level of H2SO4 underwent an abrupt change in physical structure (fibrous form to gelatinous form) at about 65% H2SO4. The sudden shift of physical structure and reaction pattern in response to acid concentration and temperature indicates that the main factor causing the change in cellulose structure is disruption of hydrogen bonding. Finding effective means of disrupting hydrogen bonding before or during the hydrolysis reaction may lead to a novel biomass saccharification process.
Keywords: Acid hydrolysis; cellulose; hydrogen bonding; kinetics; crystallinity
Characterization of molecular weight distribution of oligomers from autocatalyzed batch hydrolysis of xylan by Xia Li; Alvin O. Converse; Charles E. Wyman (pp. 515-522).
Oat spelt xylan was treated with water in a batch reactor at temperatures of 180 and 200°C. Ion-moderated partition (IMP) chromatography was then applied to separate oligomers in solution according to their molecular size. Calibration of the IMP measurements based on peak height was found to quantify dissolved monomer and oligomer yields well. Oligomer concentrations in the liquid hydrolysate were also determined from the difference in xylose monomer concentrations measured by high-performance liquid chromatography before and after posthydrolysis of dissolved xylooligosaccharides to xylose. Delayed formation and then rapid disappearance of oligomers from DP10 to DP2 was observed by IMP, and total oligomer yields measured by IMP and posthydrolysis were very similar at these times. However, while IMP detected virtually no oligomers initially, posthydrolysis measurements gave significant amounts of soluble oligomers at these times, indicating that oligomers with chain lengths >10 were in solution but not detectable by the IMP system used.
Keywords: Autohydrolysis; hydrolysis; ion-moderated partition chromatography; oligomers; thermochemical; xylan
Conversion of sugarcane bagasse to carboxylic acids using a mixed culture of mesophilic microorganisms by Piyarat Thanakoses; Nagat Abd Alla Mostafa; Mark T. Holtzapple (pp. 523-546).
Using the MixAlco process, biomass can be converted into carboxylic acids, which can be chemically converted into mixed alcohol fuels. This study focused on the use of countercurrent fermentation to anaerobically convert sugarcane bagasse and chicken manure to mixed carboxylic acids using a mixed culture of mesophilic microorganisms from terrestrial and marine sources. Bagasse was pretreated with lime to increase digestibility. The continuum particle distribution model (CPDM) simulated continuous fermentors based on data collected from batch experiments. This model saves considerable time in determining optimum operating conditions. For an 80% bagasse/20% chicken manure fermentation with terrestrial inoculum at a volatile solids loading rate (VSLR) of 7.36 g/(L of liquid·d) and a liquid residence time (LRT) of 8.88 d, total carboxylic acid productivity, total acid selectivity, and yield were 2.49 g/(L of liquid·d), 0.581 g of total acid/g of VS digested, and 0.338 g of total acid/g of VS fed, respectively, at a concentration of 18.7 g of total acid/L. At the same VSLR and LRT, fermentation with marine inoculum gave higher total acid productivity, total acid selectivity, and yield than fermentation with terrestrial inoculum. For an 80% bagasse/20% chicken manure fermentation with marine inoculum at a VSLR of 3.83 g/(L of liquid·d) and an LRT of 12.1 d, total carboxylic acid productivity, total acid selectivity, and yield were 1.38 g/(L of liquid·d), 0.667 g of total acid/g of VS digested, and 0.359 g of total acid/g of VS fed, respectively, at a concentration of 16.2 g of total acid/L.
Keywords: Bagasse; carboxylic acids; lime pretreatment; sugarcane; fermentation
Alternative approach for utilization of pentose stream from sugarcane bagasse by an induced flocculent Pichia stipitis by Heizir F. de Castro; Samuel C. Oliveira; Sandra A. Furlan (pp. 547-555).
A new approach for the utilization of hemicellulosic hydrolysate from sugarcane bagasse is described. This approach consists of using the hydrolysate to dilute the conventional feedstock (sugarcane juice) to the usual sugar concentration (150 g/L) employed for the industrial production of ethanol. The resulting sugar mixture was used as the substrate to evaluate the performance of a continuous reactor incorporating a cell recycle module, operated at several dilution rates. An induced flocculent pentose-fermenting yeast strain was used for this bioconversion. Under the conditions used, the reactor performance was satisfactory at substrate feed rates of 30 g/(L·h) or less, corresponding to an ethanol productivity of about 11.0 g/(L·h) and an overall sugar conversion >95%. These results show real advantages over the existing alternatives for a better exploitation of surplus bagasse to increase industrial alcohol production.
Keywords: Sugarcane bagasse; sugar mixture; Pichia stipitis ; flocculation; ethanol
Hydrogen production from paper sludge hydrolysate by Zsófia Kádár; Truus De Vrije; Miriam A. W. Budde; Zsolt Szengyel; Kati Réczey; Pieternel A. M. Claassen (pp. 557-566).
The main objective of this study was to develop a system for the production of “renewable” hydrogen. Paper sludge is a solid industrial waste yielding mainly cellulose, which can be used, after hydrolysis, as a feedstock in anaerobic fermentation by (hyper)thermophilic organisms, such as Thermotoga elfii and Caldicellulosiruptor saccharolyticus. Tests on different medium compositions showed that both bacteria were able to produce hydrogen from paper sludge hydrolysate, but the amount of produced hydrogen and the requirement for other components differed. Hydrogen production by T. elfii strongly depended on the presence of yeast extract and salts. By contrast, C. saccharolyticus was less dependent on medium components but seemed to be inhibited by a component present in the sludge hydrolysate. Utilization of xylose was preferred over glucose by C. saccharolyticus.
Keywords: Hydrogen production; paper sludge; Thermotoga elfii ; Caldicellulosiruptor saccharolyticus
Single-stage anaerobic codigestion for mixture wastes of simulated Korean food waste and waste activated sludge by Nam Hyo Heo; Soon Chul Park; Jin Suk Lee; Ho Kang; Don Hee Park (pp. 567-579).
Korean food waste was treated with a single-stage anaerobic codigester (SSAD) using waste activated sludge (WAS) generated from a municipal wastewater treatment plant. The stability and performance of the system was analyzed. The C/N ratio was improved with increasing food waste fraction of feed mixture. The pH, alkalinity, and free ammonia nitrogen concentration were the parameters used to evaluate the digester’s stability. The experimentally determined values of the parameters indicated that there were no methane inhibitions in the digester. Digester performance was determined by measuring the total chemical oxygen demand TCOD), volate solids (VS) removal, methane content in biogas, methane production rate (MPR), and specific methane productivity. Methane content in biogas and MPR were significantly dependent on hydraulic retention time (HRT) and ratio of food waste to WAS. The methane content in biogas decreased at shorter HRT or higher organic loading rate (OLR) with increased food waste fraction. Concerning the performance of the codigester, the optimum operating condition of the SSAD was found to be at an HRT of 10 d with a feed mixture ratio of 50% food waste and 50% WAS. A TCOD removal efficiency of 53.6% and a VS removal efficiency of 53.7% were obtained at an OLR of 5.96 kg of TCOD/(m3·d) and 3.14 kg of VS/(m3·d), respectively. A maximum MPR of 1.15 m3 CH4/(m3·d) and an SMP of 0.37 m3 CH4/kg of VSfeed were obtained at an HRT of 10 d with a methane content of 63%.
Keywords: Alkalinity; ammonia nitrogen; anaerobic codigestion; food waste; methane; volatile fatty acids; waste activated sludge
Biosorption and desorption of copper (II) ions by Bacillus sp. by Waihung Lo; Lau Mei Ng; Hong Chua; Peter H. F. Yu; Shirley N. Sin; Po-Keung Wong (pp. 581-591).
Batch biosorption experiments were conducted to investigate the removal of Cu2+ ions from aqueous solutions by a series of bacterial strains isolated from a local activated sludge process. The characteristics of 12 isolates were identified and examined for their ability to bind Cu2+ ions from aqueous solution. Among the isolates, two species exhibited biosorption capacity >40 mg of Cu/g of dry cell. Isotherms for the biosorption of copper on bacterial cells were developed and compared, and the equilibrium data fitted well to the Langmuir and Freundlich isotherm models. The biosorption of copper increased significantly with increasing pH from 2.0 to 6.0 regardless of the species. More than 90% of copper sorbed on the cells of Bacillus sp. could be recovered by washing with 0.1 M HNO3 for 5 min. The performance of two different desorption processes was also tested and compared. The results show that five biosorption and desorption cycles are a better operation process than five successive biosorptions followed by one desorption to remove and recover copper from aqueous solution. The biosorbent could be used for at least five biosorptions and desorption cycles without loss of copper removal capacity. It can be concluded that the activated sludge or sludge-isolated bacteria could be a potential biosorbent for copper removal.
Keywords: Activated sludge; bacteria; bioremediation; copper; desorption; heavy metal; metal removal; wastewater treatment process
Flow field in a shrinking-bed reactor for pretreatment of cellulosic biomass by Yinkun Wan; Thomas R. Hanley (pp. 593-602).
A shrinking-bed reactor was designed by the National Renewable Energy Laboratory to maintain a constant bulk packing density of cellulosic biomass. The high solid-to-liquid ratio in the pretreatment process allows a high sugar yield and avoids the need to flush large volumes of solution through the reactor. The shrinking-bed reactor is a promising pretreatment reactor with the potential for scale-up for commercial applications. To scale up the shrinking-bed reactor, it is necessary to understand the flow pattern in the reactor. In this study, flow field is simulated with computational fluid dynamics using a porous medium model. Different discrete “snapshots” and multiple steady states are utilized. The bulk flow pattern, velocity distribution, and pressure drop are determined from the simulation and can be used to guide reactor design and scale-up.
Keywords: Shrinking-bed reactor; computational fluid dynamics; flow field; cellulosic biomass; hydrolysis
Lactic acid production through cell-recycle repeated-batch bioreactor by Hurok Oh; Young-Jung Wee; Jong-Sun Yun; Hwa-Won Ryu (pp. 603-613).
The effect of various nitrogen sources on cell growth and lactic acid production was investigated. The most effective nitrogen source was yeast extract; more yeast extract gave higher cell growth and lactic acid productivity. Yeast extract dosage and cell growth were proportional up to a yeast extract concentration of 30 g/L, and lactic acid productivity was linearly correlated up to a yeast extract dosage of 25 g/L. However, increasing the yeast extract content raises the total production cost of lactic acid. Therefore, we attempted to find the optimum yeast extract dosage for a repeated-batch operation with cell recycling. The results show that when using Enterococcus faecalis RKY1 only 26% of the yeast extract dosage for a conventional batch fermentation was sufficient to produce the same amount of lactic acid, whereas the lactic acid concentration in the product stream (92–94 g/L) and lactic acid productivity (6.03–6.20 g/[L·h]) were similar to those of a batch operation. Furthermore, long-term stability was established.
Keywords: Lactic acid; repeated-batch; cell-recycle; hollow-fiber module; nitrogen source; yeast extract
Limits for alkaline detoxification of dilute-acid lignocellulose hydrolysates by Nils-Olof Nilvebrant; Per Persson; Anders Reimann; Filipe de Sousa; Lo Gorton; Leif J. Jönsson (pp. 615-628).
In addition to fermentable sugars, dilute-acid hydrolysates of lignocellulose contain compounds that inhibit fermenting microorganisms, such as Saccharomyces cerevisiae. Previous results show that phenolic compounds and furan aldehydes, and to some extent aliphatic acids, act as inhibitors during fermentation of dilute-acid hydrolysates of spruce. Treatment of lignocellulose hydrolysates with alkali, usually in the form of overliming to pH 10.0, has been frequently employed as a detoxification method to improve fermentability. A spruce dilute-acid hydrolysate was treated with NaOH in a factorial design experiment, in which the pH was varied between 9.0 and 12.0, the temperature between 5 and 80°C, and the time between 1 and 7 h. Already at pH 9.0, >25% of the glucose was lost when the hydrolysate was treated at 80°C for 1 h. Among the monosaccharides, xylose was degraded faster under alkaline conditions than the hexoses (glucose, mannose, and galactose), which, in turn, were degraded faster than arabinose. The results suggest that alkali treatment of hydrolysates can be performed at temperatures below 30°C at any pH between 9.0 and 12.0 without problems with sugar degradation or formation of inhibiting aliphatic acids. Treatment with Ca(OH)2 instead of NaOH resulted in more substantial degradation of sugars. Under the harsher conditions of the factorial design experiment, the concentrations of furfural and 5-hydroxymethylfurfural decreased while the total phenolic content increased. The latter phenomenon was tentatively attributed to fragmentation of soluble aromatic oligomers in the hydrolysate. Separate phenolic compounds were affected in different ways by the alkaline conditions with some compounds showing an increase in concentration while others decreased. In conclusion, the conditions used for detoxification with alkali should be carefully controlled to optimize the positive effects and minimize the degradation of fermentable sugars.
Keywords: Bioethanol; lignocellulose hydrolysates; fermentation inhibitors; alkali detoxification; sugar degradation
Genetics and genomics in bioenergy and bioproducts
by James S. McLaren; Thomas W. Jeffries (pp. 631-632).
Production of lactic acid from food wastes by Kwang Il Kim; Woo Kyung Kim; Deok Ki Seo; In Sang Yoo; Eun Ki Kim; Hyon Hee Yoon (pp. 637-647).
Conversion of food wastes into lactic acid by simultaneous saccharification and fermentation (SSF) was investigated. The process involves saccharification of the starch component in food wastes by a commercial amylolytic enzyme preparation (a mixture of amyloglucosidase, α-amylase, and protease) and fermentation by Lactobacillus delbrueckii. The highest observed overall yield of lactic acid in the SSF was 91% of theoretical. Lactic acid concentration as high as 80 g/L was attainable in 48 h of the SSF. The optimum operating conditions for the maximum productivity were found to be 42°C and pH 6.0. Without supplementation of nitrogen-containing nutrients, the lactic acid yield in the SSF decreased to 60%: 27 g/L of lactic acid from 60 g/L of food waste. The overall performance of the SSF, however, was not significantly affected by the elimination of mineral supplements.
Keywords: Food waste; lactic acid; simultaneous saccharification and fermentation; Lactobacillus delbrueckii ; amyloglucosidase
Microbiologic oxidation of isosafrole into piperonal by Alberdan Silva Santos; Nei Pereira Jr.; Iracema I. da Silva; Maria Ines Sarquis; Octavio A. C. Antunes (pp. 649-657).
The biotransformation of isosafrole by Cladosporium sphaerospermum yielded piperonal, which is a compound of great commercial importance in the flavor and fragrance industries. The experiments were performed in 500-mL conical flasks containing 100 mL of Czapek-modified medium in an orbital shaker with controlled agitation and temperature. Spores of C. sphaerospermum were used as inocula, and after 96 h of incubation the substrate was added to the culture. Samples of 2 mL were withdrawn at 24-h intervals and analyzed by gas chromatography, (GC) and/or GC/MS spectroscopy.
Keywords: Biotransformation; Cladosporium sphaerospermum ; phenylpropanoids; isosafrole; piperonal
Breathing air from protein foam by Douglas M. Ackermann Jr.; David N. Jewell; Matthew L. Stedman; Vorakan Burapatana; Prabhani V. Atukorale; Michelle L. Pinson; Alison E. Wardle; Wenyan Zhu; Robert D. Tanner (pp. 659-671).
Protein foams can be used to extinguish fires. If foams are to be used to extinguish fires where people are present, such as in high-rise buildings or ships, then a method for allowing people to breathe in a foam-filled environment is needed. It is proposed that the air, used to create the foam be used for breathing. A canister that will break incoming air-filled foam has been designed for attachment to a standard gas mask, in order to provide breathable air to a trapped person. Preliminary results for the modified mask indicate feasibility of breathing air from air-filled protein foam.
Keywords: Protein foams; egg albumin; canister; polypropylene sheet; airflow; breakthrough
Partial purification and kinetic characterization of acid phosphatase from garlic seedling by Begüm Yenigün; Yüksel Güvenilir (pp. 677-687).
The objective of this study was to obtain purer acid phosphatases than produced by prior art by operating under conditions that improve the final product. The study features are the use of a mild nonionic detergent, 40–80% saturation with (NH4)2SO4, maintained at low temperature to remove impurity, and the use of chromatografic columns to concentrate the acid phosphatase and remove non-acid phosphatase proteins with lower or higher molecular weights. Acid phosphatase was isolated and purified from garlic seedlings by a streamline method without the use of proteolytic and lipolytic enzymes, butanol, or other organic solvents. Grown garlic seedlings of 10–15 cm height were homogenized with 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. After homogenization, the supernatant was filtered with paper filters. Filtrated supernatant was cooled to 4°C, followed by a threestep fractionation of the proteins with ammonium sulfate. The crude enzyme was isolated as a green precipitate that was dissolved in a small amount of 0.1 M acetate buffer containing 0.1 M NaCl and 0.1% Triton X-100. Garlic seedling acid phosphatase was purified with ion-exchange chromatography (DEAE cellulose). The column was equilibrated with 0.1 M acetate buffer. Acid phosphatase was purified 40-fold from the starting material. The specific activity of the pure enzyme was 168 U/mg. A variety of stability and activity profiles were determined for the purified garlic seedling acid phosphatase: optimum pH, optimum temperature, pH stability, temperature stability, thermal inactivation, substrate specificity, effect of enzyme concentration, effect of substrate concentration, activation energy, and effect of inhibitor and activator. The molecular mass of acid phosphatase was estimated to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH was 5.7 and the optimum temperature was 50°C. The enzyme was stable at pH 4.0–10.0 and 40–60°C. Activation energy was between 10 and 20 kcal, and as Michaelis Menten coefficients, V m values were 100 and 20 mM/s and K m values were 21.27 and 8.33 mM for paranitrophenylphosphate and paranitrophenyl, respectively. Studies of the effect of metal ions on enzyme activity showed both an activating and a deactivating effect. While Cu, Mo, and Mn showed strong inhibitory effects, Na, Ca, and K were the significant activators of acid phosphatase.
Keywords: Enzyme; acid phosphatase; enzyme purification; garlic seedlings
Automated filter paper assay for determination of cellulase activity by Stephen R. Decker; William S. Adney; Edward Jennings; Todd B. Vinzant; Michael E. Himmel (pp. 689-703).
Recent developments in molecular breeding and directed evolution have promised great developments in industrial enzymes as demonstrated by exponential improvements in β-lactamase and green fluorescent protein (GFP). Detection of and screening for improved enzymes are relatively easy if the target enzyme is expressible in a suitable high-throughput screening host and a clearly defined and usable screen or selection is available, as with GFP and β-lactamase. Fungal cellulases, however, are difficult to measure and have limited expressibility in heterologous hosts. Furthermore, traditional cellulase assays are tedious and time-consuming. Multiple enzyme components, an insoluble substrate, and generally slow reaction rates have plagued cellulase researchers interested in creating cellulase mixtures with increased activities and/or enhanced biochemical properties. Although the International Union of Pure and Applied Chemists standard measure of cellulase activity, the filter paper assay (FPA), can be reproduced in most laboratories with some effort, this method has long been recognized for its complexity and susceptibility to operator error. Our current automated FPA method is based on a Cyberlabs C400 robotics deck equipped with customized incubation, reagent storage, and plate-reading capabilities that allow rapid evaluation of cellulases acting on cellulose and has a maximum throughput of 84 enzyme samples per day when performing the automated FPA.
Keywords: Filter paper assay; cellulase; cellulose; Trichoderma reesei ; filter paper unit