Xylitol production from a mutant strain of Candida tropicalis.

AIMS: To characterize the kinetics of growth, sugar uptake and xylitol production in batch and fed-batch cultures for a xylitol assimilation-deficient strain of Candida tropicalis isolated via chemical mutagenesis. METHODS AND RESULTS: Chemical mutagenesis using nitrosoguanidine led to the isolation of the xylitol-assimilation deficient strain C. tropicalis SS2. Shake-flask fermentations with this mutant showed a sixfold higher xylitol yield than the parent strain in medium containing 25 g l⁻¹ glucose and 25 g l⁻¹ xylose. With 20 g l⁻¹ glycerol, replacing glucose for cell growth, and various concentrations of xylose, the studies indicated that the mutant strain resulted in xylitol yields from xylose close to theoretical. Under fully aerobic conditions, fed-batch fermentation with repeated addition of glycerol and xylose resulted in 3.3 g l⁻¹ h⁻¹ xylitol volumetric productivity with the final concentration of 220 g l⁻¹ and overall yield of 0.93 g g⁻¹ xylitol. CONCLUSIONS: The xylitol assimilation-deficient mutant isolated in this study showed the potential for high xylitol yield and volumetric productivity under aerobic conditions. In the evaluation of glycerol as an alternative low-cost nonfermentable carbon source, high biomass and xylitol yields under aerobic conditions were achieved; however, the increase in initial xylose concentrations resulted in a reduction in biomass yield based on glycerol consumption. This may be a consequence of the role of an active transport system in the yeast requiring increasing energy for xylose uptake and possible xylitol secretion, with little or no energy available from xylose metabolism. SIGNIFICANCE AND IMPACT OF THE STUDY: The study confirms the advantage of using a xylitol assimilation-deficient yeast under aerobic conditions for xylitol production with glycerol as a primary carbon source. It illustrates the potential of using the xylose stream in a biomass-based bio-refinery for the production of xylitol with further cost reductions resulting from using glycerol for yeast growth and energy production.

Comparative evaluations of cellulosic raw materials for second generation bioethanol production.

AIMS: To evaluate sugar recoveries and fermentabilities of eight lignocellulosic raw materials following mild acid pretreatment and enzyme hydrolysis using a recombinant strain of Zymomonas mobilis. METHODS AND RESULTS: Dilute acid pretreatment (2% H(2) SO(4) ) with 10% (w/v) substrate loading was performed at 134°C for 60 min followed by enzyme hydrolysis at 60°C. The results demonstrated that hydrolysis of herbaceous raw materials resulted in higher sugar recoveries (up to 60-75%) than the woody sources (<50%). Fermentation studies with recombinant Z. mobilis ZM4 (pZB5) demonstrated that final ethanol concentrations and yields were also higher for the herbaceous hydrolysates. Significant reduction in growth rates and specific rates of sugar uptake and ethanol production occurred for all hydrolysates, with the greatest reductions evident for woody hydrolysates. Further studies on optimization of enzyme hydrolysis established that higher sugar recoveries were achieved at 50°C compared to 60°C following acid pretreatment. CONCLUSIONS: Of the various raw materials evaluated, the highest ethanol yields and productivities were achieved with wheat straw and sugarcane bagasse hydrolysates. Sorghum straw, sugarcane tops and Arundo donax hydrolysates were similar in their characteristics, while fermentation of woody hydrolysates (oil mallee, pine and eucalyptus) resulted in relatively low ethanol concentrations and productivities. The concentrations of a range of inhibitory compounds likely to have influence the fermentation kinetics were determined in the various hydrolysates. SIGNIFICANCE AND IMPACT OF THE STUDY: The study focuses on lignocellulosic materials available for second generation ethanol fermentations designed to use renewable agricultural/forestry biomass rather than food-based resources. From the results, it is evident that relatively good sugar and ethanol yields can be achieved from some herbaceous raw materials (e.g. sugarcane bagasse and sorghum straw), while much lower yields were obtained from woody biomass.

Prediction of prion protein genotype and association of this genotype with lamb performance traits of Suffolk sheep.

The association of the prion protein (PrP) gene with susceptibility to scrapie has formed the basis of selection programs aimed at eradicating the disease from sheep populations. Animals are genotyped for the PrP gene and those with the less susceptible genotypes are selected. The objectives of this study were to determine the effectiveness of predicting PrP genotypes by using information from relatives and to investigate the association of the PrP genotype with lamb performance traits in Suffolk sheep. Data were obtained from a scrapie-affected flock maintained in Scotland. A total of 643 were animals genotyped at codon 171 of the PrP gene with 2 alleles, R and Q. The genotypes of these animals were used to predict the genotypes of 5,173 nongenotyped animals in the same flock using segregation analysis. The genotype of nongenotyped animals was predicted from the probabilities for each possible genotype; further, an overall index for each animal was calculated to reflect the accuracy of prediction. Association analyses of the PrP gene (using animals with both known and inferred genotypes) with BW at birth, at weaning (56 d), and at 150 d, and for backfat and muscle depths at 150 d of age were carried out. A linear mixed model with random direct and maternal additive genetic effects, maternal permanent and temporary environmental effects, and year of birth was tested, and the most appropriate model was used for each trait. The expected number of Q alleles carried (from 0 to 2) by each animal was calculated and used in the model as a linear and quadratic covariate to test for associations with possible additive and dominance PrP gene effects, respectively. Results showed that the genotypes of relatively few animals (235) were inferred with certainty (compared with the 5,173 nongenotyped animals). Approximately 25% of the 5,173 predicted genotypes were inferred with a genotype probability index of 50% and greater. There was no significant association of the PrP gene with any of the performance traits studied (there were no significant additive or dominance effects). Such was the case whether data on animals with known or with both known and predicted genotypes were considered. It can be concluded that selection for PrP-resistant alleles in Suffolk sheep is unlikely to affect performance directly.

Zymomonas mobilis for fuel ethanol and higher value products.

High oil prices, increasing focus on renewable carbohydrate-based feedstocks for fuels and chemicals, and the recent publication of its genome sequence, have provided continuing stimulus for studies on Zymomonas mobilis. However, despite its apparent advantages of higher yields and faster specific rates when compared to yeasts, no commercial scale fermentations currently exist which use Z. mobilis for the manufacture of fuel ethanol. This may change with the recent announcement of a Dupont/Broin partnership to develop a process for conversion of lignocellulosic residues, such as corn stover, to fuel ethanol using recombinant strains of Z. mobilis. The research leading to the construction of these strains, and their fermentation characteristics, are described in the present review. The review also addresses opportunities offered by Z. mobilis for higher value products through its metabolic engineering and use of specific high activity enzymes.

Evaluating longevity of composite beef females using survival analysis techniques.

Objectives were to 1) identify risk factors affecting the longevity of beef females, 2) evaluate the utility of measures collected early in life in predicting longevity, and 3) estimate the heritability of longevity when females were culled primarily for not being pregnant following a 45-d breeding season. Data were from 1,379 Composite Gene Combination (CGC; 1/2 Red Angus, 1/4 Charolais, 1/4 Tarentaise) cows born from 1982 through 1999 at the USDA-ARS, Fort Keogh Livestock and Range Research Laboratory, Miles City, MT, and first calving at approximately 2 yr of age. The length of productive life was modeled using Cox regression to identify factors affecting the longevity of beef females. Age at first calving and calf birth weight did not influence longevity. Cows that experienced dystocia were at greater risk of being culled than those that calved without assistance (P < 0.01). On average, as breeding value for cow weight increased, the risk of being culled decreased (P < 0.01), whereas the risk of being culled increased with increasing maternal breeding values for preweaning gain (P < 0.05). Traits measured before 1 yr of age were not useful in predicting the subsequent longevity of cows. The heritability of functional longevity was estimated to be 0.14. Relatively low heritability and the lack of indicators of longevity expressed early in life suggest that genetic improvement of longevity will be difficult. Matching the genetic potential of cows for size and milk production to the production environment such that rebreeding performance is not compromised by concurrent lactation seems to be a consideration in retaining beef females when open cows are culled.

Screening, identification and kinetic characterization of a bacterium for Mn(II) uptake and oxidation.

Following sample collection and screening at a number of Mn-associated mine sites in Northern Australia, a microbial strain was selected for its enhanced rate of Mn uptake. The strain was identified by phylogenetic analysis as a Rhizobium sp. Kinetic studies of Mn(II) uptake and oxidation by this strain in glucose-based media established that the uptake of Mn(II) was much greater than the conversion of Mn(II) to Mn oxide. Chemical analysis and scanning electron microscopy confirmed the production of significant amounts of polysaccharides by this strain. These polysaccharides may play a role both in enhancing Mn(II) accumulation and in minimizing Mn oxide production.

Anions effects on biosorption of Mn(II) by extracellular polymeric substance (EPS) from Rhizobium etli.

Microbial extracellular polymeric substances (EPS) are potential biosorbents for metal remediation and recovery. The Langmuir and Freundlich kinetics of Mn(II) binding by the EPS from a novel Mn(II) oxidising strain of Rhizobium etli were determined. Maximum manganese specific adsorptions (q(max)) decreased in the sequence: sulphate (62 mg Mn per g EPS) > nitrate (53 mg g(-1)) > chloride (21 mg g(-1)). Consideration of the anion during kinetic studies is usually neglected but is important in providing more practical and comparable data between different biosorbent systems.

Production of asporogenous mutants of Bacillus sphaericus 2362 in continuous culture.

AIMS: To report the production of asporogenous mutants (Spo-) of Bacillus sphaericus 2362 in continuous culture. METHODS AND RESULTS: Microbial culture samples were taken at 0.05 h-1 dilution rate and plated out on nutrient agar plates. Translucent colonies were obtained with vegetative morphology under phase contrast microscope. Heat resistance evaluations at different temperature settings showed that the Spo- mutants had lower heat resistance than the Spo+ wild type. Western blots analyses carried out on both wild type and the mutants indicated the presence of binary protein toxins of 42 and 51 kDa in both. Bioassays carried out on the wild type and the Spo- mutants against mosquitoes showed the mutants to be 100-fold less toxic in comparison to the wild type. CONCLUSION: Existence and production of asporogenous mutants of Bacillus sphaericus 2362 in continuous culture at low dilution rates is demonstrated by this study. The organism's ability to produce toxins appears to be significantly reduced by the mutational process. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of asporogenous mutants had not been reported previously among strains of Bacillus sphaericus. The present report on the toxigenic capability of asporogenous mutants also raises the possibility of using continuous culture to significantly improve the productivity of toxin production in future.

Quantifying learning in medical students during a critical care medicine elective: a comparison of three evaluation instruments.

OBJECTIVE: To compare three different evaluative instruments and determine which is able to measure different aspects of medical student learning. DESIGN: Student learning was evaluated by using written examinations, objective structured clinical examination, and patient simulator that used two clinical scenarios before and after a structured critical care elective, by using a crossover design. PARTICIPATION: Twenty-four 4th-yr students enrolled in the critical care medicine elective. INTERVENTIONS: All students took a multiple-choice written examination; evaluated a live simulated critically ill patient, requested data from a nurse, and intervened as appropriate at different stations (objective structured clinical examination); and evaluated the computer-controlled patient simulator and intervened as appropriate. MEASUREMENTS AND MAIN RESULTS: Students' knowledge was assessed by using a multiple-choice examination containing the same data incorporated into the other examinations. Student performance on the objective structured clinical examination was evaluated at five stations. Both objective structured clinical examination and simulator tests were videotaped for subsequent scores of responses, quality of responses, and response time. The videotapes were reviewed for specific behaviors by faculty masked to time of examination. Students were expected to perform the following: a) assess airway, breathing, and circulation; b) prepare a mannequin for intubation; c) provide appropriate ventilator settings; d) manage hypotension; and e) request, interpret, and provide appropriate intervention for pulmonary artery catheter data. Students were expected to perform identical behaviors during the simulator examination; however, the entire examination was performed on the whole-body computer-controlled mannequin. The primary outcome measure was the difference in examination scores before and after the rotation. The mean preelective scores were 77 +/- 16%, 47 +/- 15%, and 41 +/- 14% for the written examination, objective structured clinical examination, and simulator, respectively, compared with 89 +/- 11%, 76 +/- 12%, and 62 +/- 15% after the elective (p <.0001). Prerotation scores for the written examination were significantly higher than the objective structured clinical examination or the simulator; postrotation scores were highest for the written examination and lowest for the simulator. CONCLUSION: Written examinations measure acquisition of knowledge but fail to predict if students can apply knowledge to problem solving, whereas both the objective structured clinical examination and the computer-controlled patient simulator can be used as effective performance evaluation tools.

Etiology and treatment of achalasia in the pediatric age group.

Achalasia in children bears many similarities to the disorder in adults, both in terms of clinical features and in terms of the approach to therapy. Pharmacologic management is of limited temporary benefit until more definitive therapy is undertaken. Intrasphincteric injections of botulinum toxin provides safe but short-term relief from symptoms. Based on our review of the safety and effectiveness of pneumatic dilation, we advocate this procedure as the primary form of definitive therapy for achalasia in children. In patients who do not achieve satisfactory results from a series of graduated pneumatic dilations, Heller myotomy provides safe and effective surgical treatment.

Compensation for teaching in critical care.

OBJECTIVES: To determine the financial or nonclinical time critical care program directors or teaching faculty members receive as compensation for their educational activities. To compare compensation types and amounts among critical care specialties and between university vs. nonuniversity sponsoring institutions. DATA SOURCES AND EXTRACTION: Survey returns (46%) from critical care fellowship directors listed in the American Medical Association Graduate Medical Education Directory. Information was stratified according to fellowship specialty and type of sponsoring hospital and compared by chi-square analysis and the Kruskal-Wallis test. CONCLUSIONS: Most program directors (77%) and faculty (82%) receive no specified compensation for education-related activities. Multidisciplinary programs are more likely to compensate faculty members than other specialty-specific programs (p = .006). Most programs sponsored by university or military/federal hospitals do not provide specified compensation (79% and 100%, respectively). Overall, community hospital-based programs provide a greater percentage of compensation to directors and faculty than university programs (for directors, p = .02; odds ratio, 3.85; for faculty, p = .001; odds ratio, 8.4). When compensation is specified, it is most often financial and it averages 18% of the salary (range, 5% to 100%) for directors and 19% of the salary for faculty (range, 5% to 50%). When reduced clinical time is provided (5% of program directors, 2% of faculty), it averages 13% (range, 8% to 18%) for directors and 18% (range, 10% to 25%) for faculty. Alternative methods for assigning educational compensation are discussed.

Characterization of a high-productivity recombinant strain of Zymomonas mobilis for ethanol production from glucose/xylose mixtures.

The fermentation characteristics of a recombinant strain of Zymomonas mobilis ZM4(pZB5) capable of converting both glucose and xylose to ethanol have been further investigated. Previous studies have shown that the strain ZM4(pZB5) was capable of converting a mixture of 65 g/L of glucose and 65 g/L of xylose to 62 g/L of ethanol in 48 h with an overall yield of 0.46 g/g. Higher sugar concentrations (e.g., 75/75 g/L) resulted in incomplete xylose utilization (80 h). In the present study, further kinetic evaluations at high sugar levels are reported. Acetate inhibition studies and evaluation of temperature and pH effects indicated increased maximum specific uptake rates of glucose and xylose under stressed conditions with increased metabolic uncoupling. A high-productivity system was developed that involved a membrane bioreactor with cell recycling. At sugar concentrations of approx 50/50 g/L of glucose/xylose, an ethanol concentration of 50 g/L, an ethanol productivity of approx 5 g/(L.h), and a yield (YP/S) of 0.50 g/g were achieved. Decreases in cell viability were found in this system after attainment of an initial steady state (40-60 h); a slow bleed of concentrated cells may be required to overcome this problem.

Nuclear magnetic resonance studies of acetic acid inhibition of rec Zymomonas mobilis ZM4(pZB5).

The fermentation characteristics and effects of lignocellulosic toxic compounds on recombinant Zymomonas mobilis ZM4(pZB5), which is capable of converting both glucose and xylose to ethanol, and its parental strain, ZM4, were characterized using 13C and 31P nuclear magnetic resonance (NMR) in vivo. From the 31P NMR data, the levels of nucleoside triphosphates (NTP) of ZM(pZB5) using xylose were lower than those of glucose. This can be related to the intrinsically slower assimilation and/or metabolism of xylose compared to glucose and is evidence of a less energized state of ZM4(pZB5) cells during xylose fermentation. Acetic acid was shown to be strongly inhibitory to ZM4(pZB5) on xylose medium, with xylose utilization being completely inhibited at pH 5.0 or lower in the presence of 10.9 g/L of sodium acetate. From the 31P NMR results, the addition of sodium acetate caused decreased NTP and sugar phosphates, together with acidification of the cytoplasm. Intracellular deenergization and acidification appear to be the major mechanisms by which acetic acid exerts its toxic effects on this recombinant strain.

Medical students can learn the basic application, analytic, evaluative, and psychomotor skills of critical care medicine.

OBJECTIVE: To determine whether fourth-year medical students can learn the basic analytic, evaluative, and psychomotor skills needed to initially manage a critically ill patient. DESIGN: Student learning was evaluated using a performance examination, the objective structured clinical examination (OSCE). Students were randomly assigned to one of two clinical scenarios before the elective. After the elective, students completed the other scenario, using a crossover design. SETTING: Five surgical intensive care units in a tertiary care university teaching hospital. PARTICIPANTS: Forty fourth-year medical students enrolled in the critical care medicine (CCM) elective. INTERVENTIONS: All students evaluated a live "simulated critically ill" patient, requested physiologic data from a nurse, ordered laboratory tests, received data in real time, and intervened as they deemed appropriate. MEASUREMENTS AND MAIN RESULTS: Student performance of specific behavioral objectives was evaluated at five stations. They were expected to a) assess airway, breathing, and circulation in appropriate sequence; b) prepare a manikin for intubation, obtain an acceptable airway on the manikin, demonstrate bag-mouth ventilation, and perform acceptable laryngoscopy and intubation; c) provide appropriate mechanical ventilator settings; d) manage hypotension; and e) request and interpret pulmonary artery data and initiate appropriate therapy. OSCEs were videotaped and reviewed by two faculty members masked to time of examination. A checklist of key behaviors was used to evaluate performance. The primary outcome measure was the difference in examination score before and after the rotation. Secondary outcomes included the difference in scores at each rotation. The mean preelective score was 57.0%+/-8.3% compared with 85.9%+/-7.4% (p<.0001) after the elective. Significant improvement was demonstrated at each station except station I. CONCLUSION: Fourth-year medical students without a CCM elective do not possess the basic cognitive and psychomotor skills necessary to initially manage critically ill patients. After an appropriate 1-month CCM elective, students' thinking and application skills required to initially manage critically ill patients improved markedly, as demonstrated by an OSCE using a live simulated "patient" and manikin.

Kinetic and nuclear magnetic resonance studies of xylose metabolism by recombinant Zymomonas mobilis ZM4(pZB5).

The specific rates of growth, substrate utilization, and ethanol production as well as yields of biomass and ethanol production on xylose for the recombinant Zymomonas mobilis ZM4(pZB5) were shown to be much less than those on glucose or glucose-xylose mixtures. Typical fermentations with ZM4(pZB5) growing on glucose-xylose mixtures followed two-phase growth kinetics with the initial uptakes of glucose and xylose being followed by slower growth and metabolic uncoupling on xylose after glucose depletion. The reductions in rates and yields from xylose metabolism were considered in the present investigation and may be due to a number of factors, including the following: (i) the increased metabolic burden from maintenance of plasmid-related functions, (ii) the production of by-products identified as xylitol, acetate, lactate, acetoin, and dihydroxyacetone by (13)C-nuclear magnetic resonance (NMR) spectroscopy and high-performance liquid chromatography, (iii) growth inhibition due to xylitol by the putative inhibitory compound xylitol phosphate, and (iv) the less energized state of ZM4(pZB5). In vivo (31)P-NMR studies have established that the levels of NTP and UDP sugars on xylose were less than those on glucose, and this energy limitation is likely to restrict the growth of the recombinant strain on xylose media.

Composition of the cell walls of several yeast species.

Cell walls, representing 26%-32% of the cell dry weight, were prepared from several strains of the yeasts Kloeckera apiculata, Debaryomyces hansenii, Zygosaccharomyces bailii, Kluyveromyces marxianus and Saccharomyces cerevisiae. Extraction of the walls with potassium hydroxide at 4 degrees C, followed by saturation of the alkali-soluble extract with ammonium sulphate gave fractions of mannoprotein, alkali-soluble glucan and alkali-insoluble glucan. Chitin was associated with the alkali-insoluble glucan. The proportions of the different fractions within the walls varied with the species and strain. Mannoprotein comprised between 25% and 34% of the walls, the content of alkali-insoluble glucan ranged from 15% to 48%, and the content of alkali-soluble glucan ranged from 10% to 48%. There was significant variation in the physical appearance of the alkali-soluble glucans and the relative viscosity of suspensions of these glucans. The yeasts could represent novel sources of polysaccharides with industrial and medical applications.

A sandwich enzyme-linked immunosorbent assay for the Bacillus sphaericus binary toxin.

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5alpha harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362.

Isolation of Bacillus megaterium mutants that produce high levels of heterologous protein, and their use to construct a highly mosquitocidal strain.

A xylose-regulated plasmid expression system for producing high levels of recombinant proteins in Bacillus megaterium has recently been described [Appl Microbiol Biotechnol 35:594, 1991]. Using an antibiotic resistance protein as the expressed protein, we have been able to select mutant plasmids that produce increased levels of heterologous protein. The mutant plasmids show increased segregational stability and have lost the ability to be transformed into Escherichia coli. The same selection protocol has been used to isolate a mutant strain producing high levels of the Bacillus sphaericus mosquitocidal binary toxin. This strain shows toxicity to Culex quinquefasciatus larvae that is comparable toB. sphaericus 2362 and higher than a B. megaterium strain with the original expression plasmid. This approach may be generally useful for high-level regulated protein expression in B. megaterium.