Fermentation of lignocellulosic hydrolysate by the alternative industrial ethanol yeast Dekkera bruxellensis.

AIM: Testing the ability of the alternative ethanol production yeast Dekkera bruxellensis to produce ethanol from lignocellulose hydrolysate and comparing it to Saccharomyces cerevisiae. METHODS AND RESULTS: Industrial isolates of D. bruxellensis and S. cerevisiae were cultivated in small-scale batch fermentations of enzymatically hydrolysed steam exploded aspen sawdust. Different dilutions of hydrolysate were tested. None of the yeasts grew in undiluted or 1:2 diluted hydrolysate [final glucose concentration always adjusted to 40 g l⁻¹ (0.22 mol l⁻¹)]. This was most likely due to the presence of inhibitors such as acetate or furfural. In 1:5 hydrolysate, S. cerevisiae grew, but not D. bruxellensis, and in 1:10 hydrolysate, both yeasts grew. An external vitamin source (e.g. yeast extract) was essential for growth of D. bruxellensis in this lignocellulosic hydrolysate and strongly stimulated S. cerevisiae growth and ethanol production. Ethanol yields of 0.42 ± 0.01 g ethanol (g glucose)⁻¹ were observed for both yeasts in 1:10 hydrolysate. In small-scale continuous cultures with cell recirculation, with a gradual increase in the hydrolysate concentration, D. bruxellensis was able to grow in 1:5 hydrolysate. In bioreactor experiments with cell recirculation, hydrolysate contents were increased up to 1:2 hydrolysate, without significant losses in ethanol yields for both yeasts and only slight differences in viable cell counts, indicating an ability of both yeasts to adapt to toxic compounds in the hydrolysate. CONCLUSIONS: Dekkera bruxellensis and S. cerevisiae have a similar potential to ferment lignocellulose hydrolysate to ethanol and to adapt to fermentation inhibitors in the hydrolysate. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study investigating the potential of D. bruxellensis to ferment lignocellulosic hydrolysate. Its high competitiveness in industrial fermentations makes D. bruxellensis an interesting alternative for ethanol production from those substrates.

Increased susceptibility of adult rats to azoxymethane-induced aberrant crypt foci.

The purpose of this study was to compare azoxymethane-induced aberrant crypt foci development in the colons of young and adult rats. Young (4 weeks of age) and adult (50 weeks of age) Sprague-Dawley rats were treated with two weekly injections of azoxymethane or saline. Rats were killed either 6 or 14 weeks following the first injection, and the number, size and location of aberrant crypt foci were determined. At both the 6- and 14-week time points, the number of aberrant crypt foci in older rats was significantly greater than in young rats (P<0.01). A higher percentage of aberrant crypt foci were found in the region from the mid-colon to the cecum in older rats as compared to young rats. Colonic cell proliferation was evaluated using bromodeoxyuridine immunohistochemistry. Colonic cell proliferation indices in the rectal, mid-colon and cecal regions of young and older rats were similar in young compared to adult rats. Ten large ACF from each group were screened for mutations in the beta-catenin gene (Ctnnb1) by PCR single strand conformation polymorphism. No mutations were detected. These results demonstrate that older female rats are more susceptible to the induction of aberrant crypt foci by azoxymethane than young female rats. Differences in colonic cell proliferation or beta-catenin mutations in these two age groups do not appear to be responsible for differences in aberrant crypt foci development.

Capsaicin application to central or peripheral vagal fibers attenuates CCK satiety.

Capsaicin treatment destroys small primary sensory neurons including a subpopulation of vagal afferents. Intraperitoneal, fourth ventricular or perivagal application of capsaicin attenuated or abolished cholecystokinin (CCK)-induced suppression of food intake. Capsaicin applied to the thoracolumbar spinal cord or to the pyloric region of the stomach did not alter CCK-induced reductions of food intake. Intraperitoneal capsaicin treatment reduced substance P-like immunoreactivity (SPLI) in the spinal dorsal horn and parts of the dorsal hindbrain. SPLI depletion, therefore, served as a histochemical indicator of the spread of capsaicin from its site of application. Capsaicin applied directly to the vagal trunks did not reduce SPLI in the spinal cord or hindbrain. Intraventricular capsaicin reduced SPLI in the hindbrain but not in the spinal cord. These data indicate that localized capsaicin application attenuates CCK-induced suppression of food intake by impairing the function of either central or peripheral portions of vagal afferent neurons. The data also support the conclusion that intraperitoneal capsaicin attenuates CCK-induced suppression of feeding by impairing vagal sensory function.

Substance P-containing trigeminal sensory neurons project to the nucleus of the solitary tract.

Intense substance P-like immunoreactivity (SPLI) was identified in fiber bundles coursing between the spinal nucleus of the trigeminal nerve and the ventrolateral nucleus of the solitary tract at the level of the area postrema. These bundles were apparent only when tissue was stained for substance P immunoreactivity and were not visible in preparations treated with antisera to somatostatin or neurotensin. Following unilateral section of the trigeminal nerve, the SPLI-containing fiber bundles were absent ipsilateral to the nerve section. The fibers were absent bilaterally in rats which were previously injected with capsaicin. Unilateral removal of the nodose ganglion did not diminish the intensity or apparent number of SPLI fibers. These data indicate the presence of a trigeminosolitary projection which is composed of primary trigeminal sensory neurons containing substance P. The results provide an anatomical route by which substance P of trigeminal origin may modulate vagal or glossopharyngeal sensory information.

Overconsumption of preferred foods following capsaicin pretreatment of the area postrema and adjacent nucleus of the solitary tract.

Lesions which destroy the area postrema (AP) and damage the adjacent nucleus of the solitary tract (NST) produce a constellation of behavioral signs which include overingestion of highly palatable food, exaggerated drinking in response to angiotensin II, diminished feeding in response to glucoprivation and chronically reduced body weight. The diversity of these signs, as well as the anatomical complexities of the AP and adjacent NST, suggest that more than one behaviorally relevant neural population may be damaged by lesions of these areas. Injections of the neurotoxin, capsaicin, made directly into the region of the AP and adjacent NST, cause rats to overconsume highly palatable foods when these foods are available during short (30 min) presentations or when they are available continuously. The capsaicin-treated animals, unlike rats with thermal lesions of the AP and adjacent NST, do not exhibit chronically reduced body weight or overdrink in response to angiotensin II. In addition, feeding in response to glucoprivation is undiminished in capsaicin-injected rats. These results suggest that thermal damage of the AP and adjacent NST causes overingestion of preferred foods by damaging a population of capsaicin-sensitive neurons. The other manifestations of thermal lesions of the AP and adjacent NST are probably mediated by neurons which are not susceptible to capsaicin-induced damage. Since small unmyelinated sensory neurons are most sensitive to damage by capsaicin, it may be that damage to small sensory neuron projections in the AP and/or adjacent NST produces the overconsumption of palatable foods.

Effects of sphingomyelin on aberrant colonic crypt foci development, colon crypt cell proliferation and immune function in an aging rat tumor model.

Sphingomyelin (SPM) was assessed in older rats for in vivo effects on multiple immune responses and the development of colon preneoplastic lesions. Fifty-four-week-old rats were injected with 10 mg/kg body weight of the carcinogen azoxymethane (AOM), and then treated with 35 mg/kg body weight SPM orally for 6 weeks beginning 6 weeks after AOM treatment. None of the immune functions tested (antibody formation, delayed-type hypersensitivity or natural killer cell cytotoxicity) were significantly affected by SPM treatment. Natural killer (NK) cell activity was, however, decreased in all rats that were treated with AOM. There was a tendency for decreased aberrant crypt foci (ACF) numbers in the SPM-treated rats but this reduction was only significant for the largest lesions (> nine crypts per foci). The decreased ACF numbers were most evident in the proximal end of the colon. Colonic crypt cell proliferation was also decreased in SPM treated rats. This reduction was primarily in the base of the crypt column. Also, low numbers of ACF developed spontaneously in rats not treated with AOM, but no ACF were present in non-AOM rats that also received SPM. It appears that SPM may have effects on the post-initiation development of preneoplastic lesions in the rat colon but not on the immune functions assessed in this study.

Dietary indole-3-carbinol alters immune functions in rats.

To further elucidate the physiological mechanisms that may contribute to the dichotomy of effect of indole-3-carbinol (I3C) on cancer development, we examined immune functions representative of the three major branches of the immune system in rats fed either a high (150 mg/kg) or low (50 mg/kg) dose of I3C. Animals fed the high dose of I3C daily for 7 wk had significantly reduced natural killer cell activity. In contrast, T-cell-mediated delayed-type hypersensitivity was significantly elevated. Antibody production in response to the antigen keyhole limpet hemocyanin was not significantly altered compared to controls. These results indicate that exposure to I3C may have differential effects on major immune responses. The significance of these immune function alterations in tumor development will require additional investigation of the effects of dietary I3C on immune functions in appropriate tumor models.

Effect of dietary chlorogenic acid on multiple immune functions and formation of aberrant crypt foci in rats.

Adult male Sprague-Dawley rats were fed 70 mg/kg body weight chlorogenic acid (CHA) for 7 wk. One CHA-fed group was also given 2 injections of the colon carcinogen azoxymethane (AZO) on d 2 and 9 of CHA treatment. Three major types of immune responses were assessed: antibody production, specific cell-mediated immunity, and nonspecific cell-mediated immunity. The formation of AZO-induced aberrant crypt foci (ACF) in the colon were observed, as was colonic cell proliferation. There were no significant effects of CHA treatment on any of the immune parameters examined or on formation of preneoplastic lesions or cell proliferation in the colon. The overall nonsignificant trends in immune function, colon cell proliferation, and ACF development were, however, more consistent with immunosuppression and enhanced preneoplasia.

Effects of indole-3-carbinol on immune responses, aberrant crypt foci, and colonic crypt cell proliferation in rats.

Male Sprague-Dawley rats were treated orally with indole-3-carbinol (13C) for 7 wk at levels of 150, 100, and 50 mg/kg body weight. The rats were injected with 10 mg/kg body weight of the colon carcinogen, azoxymethane (AOM) on d 2 and 9 of 13C treatment. At termination of the study, all rats were assessed for immune function (humoral immunity, specific cell-mediated immunity, and nonspecific cell-mediated immunity). Colonic tissue was collected and examined for the presence of aberrant crypt foci (ACF) and proliferation of crypt cells. Antibody responses to antigen challenge were significantly suppressed in the animals exposed to the high dose of 13C. Delayed-type hypersensitivity responses, natural killer cell activity, the number and multiplicity of ACF, and cell proliferation parameters were not significantly different from those of the controls. Therefore, there was no clear protective or enhancing effect of 13C on ACF numbers or colonic cell proliferation indices. There was no strong correlation between changes in immune responses and the preneoplastic biomarkers of colon cancer.

Cholecystokinin reduces body temperature in vehicle- but not capsaicin-pretreated rats.

Systemic administration of cholecystokinin C-terminal octapeptide (CCK-8) decreases body temperature. However, it remains unclear whether reduction of body temperature is concomitant with suppression of food intake at CCK-8 doses that approach physiological levels. We examined rectal temperature after intraperitoneal CCK-8, 4 micrograms/kg, both in the presence and absence of a preferred food. We found that rectal temperature was significantly reduced by CCK-8 in both conditions and that the reduction of temperature coincided with the time of maximal suppression of food intake by CCK-8. In rats pretreated systemically with 25 or 175 mg/kg of the sensory neurotoxin capsaicin, both suppression of food intake and reduction of body temperature were significantly attenuated or abolished. The 25 mg/kg capsaicin treatment did not alter corneal chemosensitivity or the ability of rats to maintain normothermia at elevated ambient temperature, suggesting that capsaicin damage to neural substrates mediating CCK-8-induced reduction of body temperature 1) did not generalize to cephalic or peripheral warm-sensitive structures, and 2) was limited to fine sensory fibers accessible to intraperitoneal capsaicin application.

Multiple immune functions in rats fed Echinacea extracts.

This study evaluated acquired-immune functions representing the three major branches of the immune system in male rats fed a commercially available echinacea product. An additional comparison of effects on antibody formation in male and female rats was done using the commercial echinacea product and two echinacea tinctures marketed by local herbalists. In initial testing, we found no evidence of altered natural killer cell activity, T cell-mediated delayed-type hypersensitivity, or specific antibody formation in male rats given either a 225 mg/kg or 50 mg/kg of the commercial echinacea for 6 weeks. Antibody formation was significantly suppressed in female but not male rats given 250 mg/kg for 2 weeks of the commercial echinacea. The local products tested had no effect on antibody formation. We concluded that our study provided no supporting evidence for immunostimulatory activity by the echinacea preparations we examined and, in fact, may be immunosuppressive under some conditions.

Dietary curcumin enhances antibody response in rats.

The effects of dietary curcumin on three major types of immune function were examined in rats. Antibody (IgG) production, delayed-type hypersensitivity and natural killer cell activity were evaluated after 5 weeks of dietary exposure to 1, 20 or 40 mg/kg curcumin. The highest dose of curcumin significantly enhanced IgG levels. Rats receiving lower dietary concentrations (1 or 20 mg/kg) of curcumin were not different in IgG production from rats receiving no curcumin in their diet. Neither delayed-type hypersensitivity nor natural killer cell activity was different from control values at any dietary concentration of curcumin. In vitro incubation of YAC-1 and EL4 tumor cells and normal splenocytes in varying concentrations of curcumin for varying times revealed differences between cell types in curcumin's effects on cell proliferation and viability. No cytotoxic effect was seen in EL4 cells at 125 micrograms/ml curcumin at 4, 24 and 48 hrs incubations, however, cell proliferation was reduced by almost 50% at 24 hrs. YAC-1 cell viability and cell numbers were diminished at longer incubations. A lower curcumin concentration (1.25 micrograms/ml) enhanced cell growth in the YAC-1 cells at 24 and 48 hr. This enhancement was not seen in spleen or EL4 cells.

Dietary quercetin, immune functions and colonic carcinogenesis in rats.

Rats fed 100 mg/kg quercetin (QUE) daily for 7 weeks had significantly enhanced natural killer cell activity compared to their vehicle (VEH)-fed control. In contrast, rats fed 100 mg/kg QUE and treated with the colon carcinogen, azoxymethane had significantly reduced natural killer cell activity compared to their VEH-fed azoxymethane-treated control. There was no significant difference in natural killer cell activity between the two control groups. Antibody production and delayed-type hypersensitivity were not altered by QUE feeding in any treatment group. In vitro exposure of splenic natural killer cells to 1mM QUE significantly decreased natural killer cell cytotoxicity. Lower QUE concentrations produced a non-significant reduction in natural killer cell activity that was restored to control values at 1 x 10(-13)M QUE. The distribution, multiplicity and total number of colonic preneoplastic lesions, aberrant crypt foci, was not significantly different in the QUE-fed azoxymethane-treated rats when compared to azoxymethane-treated vehicle-fed rats at the conclusion of 7 week feeding period. We found no correlation between immune function and development of preneoplastic colon lesions in this study.