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Showing papers in "Applied Biochemistry and Biotechnology in 1993"


Journal ArticleDOI
TL;DR: In this article, the mass-transfer kinetics of the immobilized bacteria and the electrode surface were investigated and a computer-controlled carbohydrate feed system enabled the cell to generate a constant output with improved efficiency compared to the performance obtained with single large additions of fuel.
Abstract: Microbial fuel cells containingProteus vulgaris and oxidation-reduction (“redox”) mediators were investigated. The bacteria were chemically immobilized onto the surface of graphite felt electrodes, which supported production of continuous electric current and could be reused after storage A computer-controlled carbohydrate feed system enabled the cell to generate a constant output with improved efficiency compared to the performance obtained with single large additions of fuel. The response to additions of substrate when immobilized bacteria were used was faster than that achieved with freely suspended organisms. This is attributed to the advantageous mass-transfer kinetics resulting from the proximity of the immobilized bacteria and the electrode surface.

590 citations


Journal ArticleDOI
TL;DR: The proteolytic activity produced by aBacillus subtilis isolated from a hot spring was investigated and effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production.
Abstract: The proteolytic activity produced by a Bacillus subtilis isolated from a hot spring was investigated. Maximum protease production was obtained after 38 h of fermentation. Effects of various carbon and nitrogen sources indicate the requirement of starch and bacteriological peptone to be the best inducers for maximum protease production. Requirement for phosphorus was very evident, and the protease was secreted over a wide range of pH 5-11. The partially purified enzyme was stable at 60 degrees C for 30 min. Calcium ions were effective in stabilizing the enzyme, especially at higher temperature. The enzyme was extremely salt tolerant and retained 100% activity in 5M NaCl over 96 h. The molecular weight of the purified enzymes as determined by SDS-PAGE was 28,000. The enzyme was completely inactivated by PMSF, but little affected by urea, sodium dodecyl sulfate, and sodium tripoly phosphate.

329 citations


Journal ArticleDOI
TL;DR: This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to thesilanized carrier.
Abstract: Enzymes were first immobilized on inorganic supports through silane coupling agents over 25 yr ago. Since that initial report, literally hundreds of laboratories have utilized this methodology for the immobilization of enzymes, antigens, antibodies, receptors, and other high and low mol wt compounds. Today silane coupling is one of the commonly used techniques in the arsenal of the biochemist for the binding of material of all sorts to inorganic surfaces. Inorganic materials come in a variety of shapes, sizes, and characteristics. Today silane coupling is one of the most used coupling methods for the preparation of biosensing devices. Sol-gel entrapped enzymes are also produced by the application of silane technology by the polymerization of the silane to form glass-like materials with entrapped protein. This review will discuss the general preparation and characterization of silane coupled proteins with special emphasis on enzymes and describe in detail the actual methods for the silanization and specific chemical coupling of proteins to the silanized carrier.

285 citations


Journal ArticleDOI
TL;DR: In this paper, the results from a medium development study based on the aerobic bacteriumEscherichia coli were presented, and the results of continuous-reactor studies in a continuously stirred tank reactor (CSTR) with and without cell recycle were shown to demonstrate the utility of this improved medium.
Abstract: The anaerobic bacteriaClostridium ljungdahlii produces ethanol and acetate from CO, CO2, and H2 in synthesis gas. Early studies with the bacterium showed that relatively high concentrations of ethanol could be produced by lowering the fermentation pH and eliminating yeast extract from the medium in favor of a defined medium. This article presents the results from a medium development study based on the aerobic bacteriumEscherichia coli. The results of continuous-reactor studies in a continuously stirred tank reactor (CSTR) with and without cell recycle are shown to demonstrate the utility of this improved medium.

180 citations


Journal ArticleDOI
TL;DR: To improve the expression of the XR gene in yeast (Saccharomyces cerevisiae), its 5′-noncoding sequence containing the genetic elements for transcription and translation was systematically replaced by that from the yeast genes.
Abstract: The intactPichia stipitis xylose reductase gene (XR) has been cloned and expressed inSaccharomyces cerevisiae. The possible further improvement of the expression of thePichia gene in the new host was studied. To improve the expression of the XR gene in yeast (Saccharomyces cerevisiae), its 5′-noncoding sequence containing the genetic elements for transcription and translation was systematically replaced by that from the yeast genes. It was found that thePichia genetic signal for transcription of XR is more effective than the yeast TRP5 promoter, but is about half as effective as the yeast strong promoter of the alcohol dehydrogenase gene (ADC1). However, the nucleotide sequence immediately adjacent to the initiation codon of XR, which controls the translation of the gene product, seemed to be five times less effective than the corresponding sequence of the ADC1 gene. By totally replacing its 5′-noncoding sequence with that of the yeast ADC1 gene, the expression of XR in yeast was found to be nearly ten times higher. Furthermore, the clonedPichia XR described in this article contains very little of its 3′-noncoding sequence. In order to study whether the 3′-noncoding sequence is important to its expression inS. cerevisiae, the intact 3′-noncoding sequences of the yeast xylulokinase gene was spliced to the 3′ end of theP ADC1-XR structural gene. This latter modification has resulted in a twofold further increase in the expression of thePichia XR in yeast.

167 citations


Journal ArticleDOI
TL;DR: Using very thin polyesterbacked polyacrylamide gels, a further simplified protocol was compared to other widely used silver staining procedures and was the most sensitive, the fastest to perform, and had relatively few steps and reagents.
Abstract: Nucleic acids can be detected at the picogram level using a quick and simple silver staining method (2). Using very thin polyesterbacked polyacrylamide gels, a further simplified protocol was compared to other widely used silver staining procedures. The improved protocol described here was the most sensitive, the fastest to perform, and had relatively few steps and reagents. This method also produced the least number of staining artifacts and offered images of high contrast.

167 citations


Journal ArticleDOI
TL;DR: In this article, the effects of carbon dioxide discharged from a boiler in a power plant, CO2 fixation by microalgae photosynthesis was studied, and the results of the field test showed that the productivity of both strains using direct flue gas was almost on a par with that of bombed CO2 gas.
Abstract: To mitigate the effects of carbon dioxide discharged from a boiler in a power plant, CO2 fixation by microalgae photosynthesis was studied. For the algae cultivation, actual flue gas from a boiler was used in two sets of small-sized raceway-type cultivators installed at Tohoku Electric Power Company's Shin-Sendai power station. UsingNannochloropsis sp. NANNP-2 andPhaeodactylum sp. PHAEO-2 strains from the SERI collection, the microalgae were cultured semicontinuously and harvested periodically. From the results of the field test, the productivity of both strains using direct flue gas was almost on a par with that of bombed CO2 gas. Therefore, it was clarified that the direct blowing of flue gas into the cultivator did not adversely affect algal growth.

142 citations


Journal ArticleDOI
TL;DR: In this article, very high gravity (VHG) wheat mashes containing more than 300 g of dissolved solids per liter were prepared and fermented with active dry yeast at 20, 25, 30, and 35°C with and without yeast extract as nutrient supplement.
Abstract: Very high gravity (VHG) wheat mashes containing more than 300 g of dissolved solids per liter were prepared and fermented with active dry yeast at 20, 25, 30, and 35°C with and without yeast extract as nutrient supplement. At 20°C, mashes with 38% (w/v) dissolved solids end-fermented without any nutrient supplementation and maximum ethanol yields of 23.8% (v/v) were obtained. With increasing temperatures, the sugar consumption decreased. Addition of yeast extract stimulated the rate of fermentation at all temperatures, but did not increase the total amount of sugar consumed. The stimulatory effect of yeast extract on cell multiplication decreased with increasing sugar concentration, and virtually no difference in cell number was observed between yeast extract-supplemented and unsupplemented mashes at sugar concentrations above 33% (w/v). The fermentative capacity of the yeast (expressed as maximum specific rate of sugar consumption) remained the same at all sugar concentrations in unsupplemented mashes, but decreased in yeast extract-supplemented mashes at sugar concentrations below 33% (w/v). When the sugar concentration was above 33% sugar (w/v), the fermentative capacity in yeast extract-supplemented mashes was greater than that observed in unsupplemented samples.

131 citations


Journal ArticleDOI
TL;DR: Several molecular biological, analytical chemistry, and mathematical and computational tools of relevance to cellular and metabolic engineering are reviewed in this paper, where the importance of host selection and gene selection is emphasized.
Abstract: Metabolic engineering is defined as the purposeful modification of intermediary metabolism using recombinant DNA techniques. Cellular engineering, a more inclusive term, is defined as the purposeful modification of cell properties using the same techniques. Examples of cellular and metabolic engineering are divided into five categories: Over 100 examples of cellular and metabolic engineering are summarized. Several molecular biological, analytical chemistry, and mathematical and computational tools of relevance to cellular and metabolic engineering are reviewed. The importance of host selection and gene selection is emphasized. Finally, some future directions and emerging areas are presented.

120 citations


Journal ArticleDOI
TL;DR: The calculated potential ATP production suggests that, overall, glutamine is the major contributor of cellular energy.
Abstract: The energy metabolism of a mammalian cell line grown in vitro was analyzed by substrate consumption rates and metabolic flux measurements. The data allowed the determination of the relative importance of the pathways of glucose and glutamine metabolism to the energy requirements of the cell. Changes in the substrate concentrations during culture contributed to the changing catalytic activities of key enzymes, which were determined. 1. A murine B-lymphocyte hybridoma (PQXB1/2) was grown in batch culture to a maximum cell density of 1–2×106 cells/mL in 3–4 d. The intracellular protein content showed a maximum value during the exponential growth phase of 0.55 mg/106 cells. Glutamine was completely depleted, but glucose only partially depleted to 50% of its original concentration when the cells reached a stationary phase following exponential growth. 2. The specific rates of glutamine and glucose utilization varied during culture and showed maximal values at the midexponential phase of 2.4-nmol/min/106 cells and 4.3 nmol/min/106 cells, respectively. 3. A high proportion of glucose (96%) was metabolized by glycolysis, but only limited amounts by the pentose phosphate pathway (3.3%) and TCA cycle (0.21%). 4. The maximum catalytic activity of hexolinase approximates to the measured flux of glycolysis and is suggested as a rate-limiting step. In the stationary phase, the hexokinase activity reduced to 11% of its original value and may explain the reduced glucose utilization at this stage. 5. The maximal activities of two TCA cycle enzymes were well above the measured metabolic flux and are unlikely to pose regulatory barriers. However, the activity of pyruvate dehydrogenase was undetectable by spectrophotometric assay and explains the low level of flux of glycolytic metabolites into the TCA cycle. 6. A significant proportion of the glutamine (36%) utilized by the cells was completely oxidized to CO2. 7. The measured rate of glutamine transport into the cells approximated to the metabolic flux and is suggested as a rate-limiting step. 8. Glutamine metabolism is likely to occur via glutaminase and amino transaminase, which have significantly higher activities than glutamate dehydrogenase. 9. The calculated potential ATP production suggests that, overall, glutamine is the major contributor of cellular energy. However, at the midexponential phase, the energy contribution from the catabolism of the two substrates was finely balanced—glutamine (55%) and glucose (45%).

110 citations


Journal ArticleDOI
TL;DR: The simultaneous saccharification and fermentation (SSF) process is employed to convert the cellulose fraction into ethanol at improved rates, higher yields, and higher ethanol concentrations than using sequential processing through careful selection of improved cellulase enzymes and fermentative microorganisms.
Abstract: Biological systems can convert renewable resources, including lignocellulosic biomass, starch crops, and carbon dioxide, into fuels, chemicals, and materials. Ethanol and other products are now derived from starch crops, such as corn. Enzyme-based technology is under development for conversion of lignocellulosic biomass (e.g., wood, grasses, and agricultural and municipal wastes) into fuel ethanol. The simultaneous saccharification and fermentation (SSF) process is employed to convert the cellulose fraction into ethanol at improved rates, higher yields, and higher ethanol concentrations than using sequential processing through careful selection of improved cellulase enzymes and fermentative microorganisms. Medium-BTU gas can be derived from lignocellulosic biomass by anaerobic digestion and cleaned up to a pipeline-quality gas. A high-solids fermenter achieves higher gas generation rates than conventional devices and promises to help make such gas economical. An extensive collection of more than 500 productive strains of microalgae has been established to produce lipid oils for diesel fuel and other compounds from carbon dioxide. Acetyl CoA carboxylase (ACC) has been shown to be a key enzyme in lipid oil synthesis, and genetic engineering approaches are being applied to enhance the rates and yields of product formation. In addition to fuels., a biorefinery could produce a wide range of chemicals and materials through microbial conversion of renewable resources, and technology is being developed for production of chemicals and materials from biomass.

Journal ArticleDOI
TL;DR: The enzymes covalently bounded to chitosan showed the highest operational stability, however, the enzymes immobilized by physical adsorption or by ionic binding showed a low operational stability.
Abstract: Aspergillus niger NRC 107 xylanase and β-xylosidase were immobilized on various carriers by different methods of immobilization, including physical adsorption, covalant binding, ionic binding, and entrapment. The immobilized enzymes were prepared by physical adsorption on tannin-chitosan, ionic binding onto Dowex-50W, covalent binding on chitosan beads through glutaraldehyde, and entrapment in polyacrylamide had the highest activities. In most cases, the optimum pH of the immobilized enzymes were shifted to lower than those of free enzymes. The optimum reaction temperature of immobilized xylanase was shifted from 50°C to 52.5–65°C, whereas that of immobilized β-xylosidase was shifted from 45°C to 50–60°C. TheK m values of immobilized enzymes were higher than those of native enzymes. The operational stability of the immobilized enzymes was evaluated in continuous operation in packed-bead column-type reactors. The enzymes covalently bounded to chitosan showed the highest operational stability. However, the enzymes immobilized by physical adsorption or by ionic binding showed a low operational stability. The enzymes entrapped in polyacrylamide exhibited lower activity, but better operational stability.

Journal ArticleDOI
TL;DR: In this article, a large scale solid state fermenter, designated as Zymotis, is proposed for cellulase production by Trichoderma harzianum, which can be operated at different capacities simply by adding or removing the compartments.
Abstract: A novel design of a large scale solid state fermenter, designated asZymotis—the condensed term based on Greek word “Zymothiras,” which means the fermenter, offers efficient control of various fermentation parameters such as temperature, moisture, and aeration of the fermenting moist solids. A large quantity of metabolic heat can be easily removed by the novel cooling system employed. The unit can be operated at different capacities simply by adding or removing the compartments. Its evaluation at different capacities for cellulase production byTrichoderma harzianum gave similar performance as in the parallel fermentation under optimized parameters in laboratory scale column fermenter of high efficiency. The design is entirely different from all the known fermenter designs and is of potential promise in facilitating scale up of solid state fermentation for leading to industrial exploitation and harvesting numerous socioeconomic advantages of the system.

Journal ArticleDOI
TL;DR: The neutralized hydrolyzate exhibited strong inhibition toward cell growth and ethanol production by yeasts and can be alleviated by treating hydrolyZate either with ion-exchange resins or with acidified activated charcoal.
Abstract: A total of six known xylitol-producing yeast strains were screened for production of xylitol from xylose. Candida sp. 11-2 proved to be the best producer. It was chosen to study its ability to produce xylitol from hemicellulose hydrolysate derived from sugar cane bagasse. The hydrolysate was prepared by dilute sulfuric acid (2-3% [w/v]) hydrolysis, with a high-solid, low-liquid ratio followed by leaching. Owing to the inhibitors present in the hydrolysate, different treatments were studied to overcome its effect. In order to reach higher xylitol productivity, treated hydrolysates were concentrated by vacuum evaporation in rotavapor to provide a higher initial xylose concentration. After treatment, Candida sp. 11-2 was able to ferment xylose in hemicellulose hydrolysate to produce xylitol.

Journal ArticleDOI
TL;DR: This review aims to provide a summary of the impact of this expression system in biochemistry and biotechnology, highlighting important advances that have been made utilizing the system.
Abstract: The use of recombinant baculoviruses as high level expression systems is becoming more and more popular. This review aims to provide a summary of the impact of this expression system in biochemistry and biotechnology, highlighting important advances that have been made utilizing the system. The potential of newly developed multiple baculovirus expression systems to enable the reconstruction of complex biological molecules and processes is also reviewed.

Journal ArticleDOI
TL;DR: In this paper, a nontoxic immiscible solvent, oleyl alcohol, was added to, and removed directly from, the fermenting columnar reactor and extracted the majority of the inhibitory butanol from the aqueous broth.
Abstract: ImmobilizedClostridium acetobutylicum was used to ferment glucose into acetone and butanol in a fluidized-bed bioreactor. A nontoxic immiscible solvent, oleyl alcohol, was added to, and removed directly from, the fermenting columnar reactor and extracted the majority of the inhibitory butanol from the aqueous broth. The extracting solvent had a distribution coefficient of near 3 for butanol. Nonfermenting system tests indicated that equilibrium between the phases could be reached in one pass through the column. Steady-state results are presented for the fermentation with and without the extractive solvent addition. One run, with a continuous aqueous feedstream containing 40 g/L glucose, was operated for 23 d. A steady state was established with just the aqueous feedstream. Approximately half of the glucose was consumed, and the pH fell to 4.5 from 6.5. Then, during multiple intervals, the flow of organic extractive solvent (oleyl alcohol) was begun into the fermenting columnar reactor. A new apparent steady state was reached in about 4 h. The final aqueous butanol concentration was lowered by more than half. The total butanol production rate increased by 50–90% during the solvent extraction, as the organic-to-aqueous ratio increased from 1 to 4, respectively. There was an observed maximum volumetric productivity of 1.8 g butanol h−1L−1 in this nonoptimized system. The butanol yield apparently improved because of the removal of the inhibition. More substrate is going to the desired product, butanol, and less to maintenance or acid production, resulting in 10–20% increases in the ratio of butanol relative to all products.

Journal ArticleDOI
TL;DR: The use of polyacrylamide gel electrophoresis and silver staining can adequately resolve the spectrum of DAF products into detailed and reproducible patterns.
Abstract: DNA amplification fingerprinting (DAF) is a strategy for genetic typing and mapping that uses one or more very short (≥5 nt) arbitrary oligonucleotides to direct the enzymatic amplification of discrete portions of a DNA template resulting in a spectrum of products characteristic of the DNA starting material. Polymorphisms from simple banding patterns are useful as genetic markers while more complex and informative patterns are suitable for DNA fingerprinting. The use of polyacrylamide gel electrophoresis and silver staining can adequately resolve the spectrum of DAF products into detailed and reproducible patterns.

Journal ArticleDOI
TL;DR: Results demonstrated that the chlorinated pyridinyl ring of chlorpyrifos and the phenyl ring of fonofos undergo cleavage during biodegradation by the fungus, demonstrating the usefulness ofP.
Abstract: Organophosphorous insecticides are used extensively in agriculture. As a group, they are easily degraded by bacteria in the environment. However, a number of them have half-lives of several months. Little is known about their biodegradation by fungi. We showed thatPhanerochaete chrysosporium mineralized chlorpyrifos, fonofos, and terbufos (27.5, 12.2, and 26.6%, respectively) during an 18-d incubation in nutrient nitrogen-limited cultures. Results demonstrated that the chlorinated pyridinyl ring of chlorpyrifos and the phenyl ring of fonofos undergo cleavage during biodegradation by the fungus. The usefulness ofP. chrysosporium for bioremediation is discussed.

Journal ArticleDOI
TL;DR: A new strain of Bacillus subtilis C 126 was isolated from sugar cane fermentation and produced an antibiotic that inhibited the growth of Micrococcus flavus.
Abstract: A new strain ofBacillus subtilis C 126 was isolated from sugar cane fermentation and produced an antibiotic that inhibited the growth ofMicrococcus flavus. The production of the antibiotic in culture medium followed to extraction withn-butanol, thin layer chromatography, and microbiological tests indicated that a polypeptide antibiotic was produced. The fraction obtained by Sephadex G-25 column and analyzed by HPLC indicated that bacitracin complex was produced.

Journal ArticleDOI
TL;DR: In this paper, some factors that could alter the biochemical composition of Phaeodactylum tricornutum in favor of lipid production have been examinated in order to improve lipid synthesis.
Abstract: The marine microalgaePhaeodactylum tricornutum, with a high lipid content constituting 20–60% of its dry weight under controlled growth conditions (1), has recently come into focus as a potentially rich source of dietary marine vegetable oil. In particular, this species has a characteristic high content of eicosapentenoic acid (EPA), which has potential benefits in human nutrition, since it cannot be synthesized in vivo in the human body. Some factors that could alter the biochemical composition ofPhaeodactylum tricornutum in favor of lipid production have been examinated in this study.

Journal ArticleDOI
TL;DR: The authors describes habitats and methods for isolation and recovery of thermophilic actinomycetes, such asStreptomyces, Thermomonospora, and Thermoactinomyces.
Abstract: Actinomycetes comprise a large and diverse group of largely mycelial bacteria, many of which are important ecologically and are exploited commercially for the production of natural products such as antibiotics and enzymes. Thermophilic species are also known but these are relatively poorly studied compared with the predominant mesophilic genera. This article describes habitats and methods for isolation and recovery of thermophilic actinomycetes, such asStreptomyces, Thermomonospora, andThermoactinomyces. General properties are discussed with respect to mesophilic species and their potential for the production of enzymes concerned with hydrolysis of complex macromolecules described. Their ability to synthesize antibiotics is reviewed and the advantages of production at high temperatures illustrated with reference to granaticin production byStreptomyces thermoviolaceus. Work with some of the better studied thermophilic species indicates the presence of novel properties. More intensive investigations of their properties, as well as those of actinomycetes that grow at extremes of pH or salinity, may lead to the discovery of novel natural products.

Journal ArticleDOI
TL;DR: Controlling the pH at 7 provided an effective means of circumventing acetic acid toxicity without significant loss in fermentation performance of the recombinant biocatalyst.
Abstract: Efficient utilization of the pentosan fraction of hemicellulose from lignocellulosic feedstocks offers an opportunity to increase the yield and to reduce the cost of producing fuel ethanol. The patented, genetically engineered, ethanologenEscherichia coli B (pLOI297) exhibits high-performance characteristics with respect to both yield and productivity in xylose-rich lab media. In addition to producing monomer sugar residues, thermochemical processing of biomass is known to produce substances that are inhibitory to both yeast and bacteria. During prehydrolysis, acetic acid is formed as a consequence of the deacetylation of the acetylated pentosan. Our investigations have shown that the acetic acid content of hemicellulose hydrolysates from a variety of biomass/waste materials was in the range 2–10 g/L (33–166 mM). Increasing the reducing sugar concentration by evaporation did not alter the acetic acid concentration. Acetic acid toxicity is pH dependent. By virtue of its ability to traverse the cell membrane freely, the undissociated (protonated) form of acetic acid (HAc) acts as a membrane protonophore and causes its inhibitory effect by bringing about the acidification of the cytoplasm. With recombinantE. coli B, the pH range for optimal growth with glucose and xylose was 6.4–6.8. With glucose, the pH optimum for ethanol yield and volumetric productivity was 6.5, and for xylose it was 6.0 and 6.5, respectively. However, the decrease in growth and fermentation efficiency at pH 7 is not significant. At pH 7, only 0.56% of acetic acid is undissociated, and at 10 g/L, neither the ethanol yield nor the maximum volumetric productivity, with glucose or xylose, is significantly decreased. The “uncoupling” effect of HAc is more pronounced with xylose and the potency of HAc is potentiated in a minimal salts medium. Controlling the pH at 7 provided an effective means of circumventing acetic acid toxicity without significant loss in fermentation performance of the recombinant biocatalyst.

Journal ArticleDOI
TL;DR: In this article, water hyacinth was subjected to various thermochemical pretreatments and used as a substrate in anaerobic digestion for biomethanation, and the results indicated that the pretreatment increased the solubility of biomass and improved gas production.
Abstract: Water hyacinth was subjected to various thermochemical pretreatments and used as a substrate in anaerobic digestion for biomethanation. Results indicate that the pretreatment increased the solubility of biomass and improved gas production. Best results were obtained when water hyacinth was treated at pH 11.00 and temperature 121‡C. Severe treatment conditions showed a negative effect, especially on methanogenic bacteria caused by toxic compounds produced during treatment.

Journal ArticleDOI
TL;DR: This study assessed the fermentation performance characteristics of a patented (US Pat. 5,000,000), recombinantEscherichia coli B (ATCC 11303 pLOI297) in anaerobic batch fermentations of both nutrient-supplemented soft and hardwood SSL (30–35 g/L total reducing sugars).
Abstract: Although lignocellulosic biomass and wastes are targeted as an attractive alternative fermentation feedstock for the production of fuel ethanol, cellulosic ethanol is not yet an industrial reality because of problems in bioconversion technologies relating both to depolymerization and fermentation. In the production of wood pulp by the sulfite process, about 50% of the wood (hemicellulose and lignin) is dissolved to produce cellulose pulp, and the pulp mill effluent ("spent sulfite liquor" SSL) represents the only lignocellulosic hydrolysate available today in large quantities (about 90 billion liters annually worldwide). Although softwoods have been the traditional feedstock for pulping operations, hardwood pulping is becoming more popular, and the pentose sugars in hardwood SSL (principally xylose) are not fermented by the yeasts currently being used in the production of ethanol from softwood SSL. This study assessed the fermentation performance characteristics of a patented (US Pat. 5,000,000), recombinant Escherichia coli B (ATCC 11303 pLOI297) in anaerobic batch fermentations of both nutrient-supplemented soft and hardwood SSL (30-35 g/L total reducing sugars). The pH was controlled at 7.0 to maximize tolerance to acetic acid. In contrast to the high-performance characteristics exhibited in synthetic media, formulated to mimic the composition of softwood and hardwood SSL (yield approaching theoretical maximum), performance in SSL media was variable with conversion efficiencies in the range of 67-84% for hardwood SSL and 53-76% for softwood SSL. Overlimiting treatment of HSSL, using Ca(OH)2, improved overall volumetric productivity two- to sevenfold to a max of 0.42 g/L/h at an initial cell loading of 0.5 g dry wt/L. A conversion efficiency of 92% (6.1 g/L ethanol) was achieved using diluted Ca(OH)2-treated hardwood SSL. The variable behavior of this particular genetic construct is viewed as a major detractant regarding its candidacy as a biocatalyst for SSL fermentations.

Journal ArticleDOI
TL;DR: In this paper, a colorimetric method for detecting Galacturonic acid using a color-imputed spectrophotometer is presented. But the method is limited to free sugar or in polymer forms, such as pectin.
Abstract: A simple, colorimetric assay that is specific for galacturonic acid is presented. The assay uses sulfuric acid, carbazole, a controlled heat water bath, and a spectrophotometer. Galacturonic acid can be detected as free sugar or in polymer forms, such as pectin.

Journal ArticleDOI
TL;DR: The results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in therange of 5–60 g/L cellulose in the feed.
Abstract: Experimental results are presented for continuous conversion of pretreated hardwood flour to ethanol. A simultaneous saccharification and fermentation (SSF) system comprised ofTrichoderma reesei cellulase supplemented with additional β-glucosidase and fermentation bySaccharomyces cerevisiae was used for most experiments, with data also presented for a direct microbial conversion (DMC) system comprised ofClostridium thermocellum. Using a batch SSF system, dilute acid pretreatment of mixed hardwood at short residence time(10 s, 220°C, 1% H2SO4) was compared to poplar wood pretreated at longer residence time (20 min, 160°C, 0.45% H2SO4). The short residence time pretreatment resulted in a somewhat (10–20%) more reactive substrate, with the reactivity difference particularly notable at low enzyme loadings and/or low agitation. Based on a preliminary screening, inhibition of SSF by byproducts of short residence time pretreatment was measurable, but minor. Both SSF and DMC were carried out successfully in well-mixed continuous systems, with steady-state data obtained at residence times of 0.58–3 d for SSF as well as 0.5 and 0.75 d for DMC. The SSF system achieved substrate conversions varying from 31% at a 0.58-d residence time to 86% at a 2-d residence time. At comparable substrate concentrations (4–5 g/l) and residence times (0.5–0.58 d), substrate conversion in the DMC system (77%) was significantly higher than that in the SSF system (31%). Our results suggest that the substrate conversion in SSF carried out in CSTR is relatively insensitive to enzyme loading in the range 7–25 U/g cellulose and to substrate concentration in the range of 5–60 g/L cellulose in the feed.

Journal ArticleDOI
TL;DR: A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose and wheat bran extract in glucose medium.
Abstract: A potent indigenous bacillus isolate identified asBacillus cereus (RJ-30) was found to produce Cyclodextrin Glucosyl Transferase (CGTase) extracellularly. Process optimization of various fermentation parameters has been established for optimal growth of bacillus and the maximum enzyme synthesis. The organism had the highest specific growth rate (0.7μ) with a generation time of 1 h in glucose containing medium at the conditions of pH 7.0, 37°C at 300 rpm, 1.5 vvm of agitation, and aeration. At these conditions, it exhibited the maximum activity of 54 U/mL at the synthesis rate of 2.7 U/L/h. CGTase was produced from the early exponential growth and peaked during the midsporulating stage of about 16 h thereafter maintained at the same level of 50 U/mL. Saccharides containing media were better inducers than starch, and the influence of carbohydrate substrates has shown that enzyme synthesis is promoted by xylose (65 U/mL) and, more remarkably, by the supplementation of wheat bran extract in glucose medium (106 U/mL). This organism produced CGTase stably in a chemostat culturing over a period of 400 h with a maximum productivity of 5.4 kU/L/h (threefold higher than obtained in batch culturing [1.75 kU/L/h]). Comparatively, CGTase was produced by immobilized cells in a continuous fluidized bed reactor for over approx 360 h, at a relatively high dilution rate of 0.88 h−1 resulting in the productivity of 23.0 kU/L/h.

Journal ArticleDOI
TL;DR: Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing, which would mean that the gel electrophoresis step in DNA sequencing could be eliminated.
Abstract: Determination of the sequence of DNA is one of the most important aspects of modern molecular biology. New sequencing methods currently being developed enable DNA sequence to be determined increasingly faster and more efficiently. One of the major advances in sequencing technology is the development of automated DNA sequencers. These utilize fluorescent rather than radioactive labels. A laser beam excites the fluorescent dyes, the emitted fluorescence is collected by detectors, and the information analyzed by computer. Robotic work stations are being developed to perform template preparation and purification, and the sequencing reactions themselves. Research is currently in progress to develop the technology of mass spectrometry for DNA sequencing. Success in this endeavor would mean that the gel electrophoresis step in DNA sequencing could be eliminated. A major innovation has been the application of polymerase chain reaction (PCR) technology to DNA sequence determination, which has led to the development of linear amplification sequencing (cycle sequencing). This very powerful yet technically simple method of sequencing has many advantages over conventional techniques, and may be used in manual or automated methods. Other recent innovations proposed recently to increase speed and efficiency include multiplex sequencing. This consists of pooling a number of samples and processing them as pools. After electrophoresis, the DNA is transferred to a membrane, and sequence images of the individual samples are obtained by sequential hybridizations with specific labeled oligonucleotides. Multiplex DNA sequencing has been used in conjunction with direct blotting electrophoresis to facilitate transfer of the DNA to a membrane. Chemiluminescent detection can also be used in conjunction with multiplex DNA sequencing to visualize the image on the membrane.

Journal ArticleDOI
TL;DR: In this paper, a β-d-glucosidase purified from commercial preparations of clarified culture broth of Aspergillus niger (Novo SP188) was shown to elute as two distinct species during analytical anion exchange chromatography (AEC).
Abstract: β-d-glucosidase purified from commercial preparations of clarified culture broth ofAspergillus niger (Novo SP188) was shown to elute as two distinct species during analytical anion-exchange chromatography (AEC). However, the two enzyme forms behaved identically on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE), high-performance size-exclusion chromatography (HPSEC), and isoelectric focusing. Also, the N-terminal amino acid sequence, amino acid composition, fingerprint of tryptic-digest peptides, circular dichroism spectra, and reaction kinetics appear identical for these forms. This feature of theA. niger enzyme is distinctly different from β-d-glucosidase isozymes reported from other sources, where multiple forms tend to differ in molecular weight and/or isoelectric pH. Michaelis-Menten kinetic analysis also gave comparable results for the two forms. The distinct behavior on AEC was explained by considering the differences in N-linked carbohydrates liberated from both species following treatment with endoglycosidase H or F.

Journal ArticleDOI
TL;DR: The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved and the principal cellobiohydrolase, subunit S8, has been demonstrated to be synonymous with cellulase component Ss (CelS) from the cellulosome of ATCC strain 27405.
Abstract: The controversy regarding the identity of a major cellulosomal component type from two different strains of Clostridium thermocellum has been resolved. The principal cellobiohydrolase, subunit S8, from the cellulosome of strain YS has been demonstrated to be synonymous with cellulase component Ss (CelS) from the cellulosome of ATCC strain 27405. This component is not related to any other cellulosomal subunit or cloned endoglucanase in this organism.