Consolidated bioprocessing strategy for ethanol production from Jerusalem artichoke tubers by Kluyveromyces marxianus under high gravity conditions

Aims: Developing an innovative process for ethanol fermentation from Jerusalem artichoke tubers under very high gravity (VHG) conditions. Methods and Results: A consolidated bioprocessing (CBP) strategy that integrated inulinase production, saccharification of inulin contained in Jerusalem artichoke tubers and ethanol production from sugars released from inulin by the enzyme was developed with the inulinase‐producing yeast Kluyveromyces marxianus Y179 and fed‐batch operation. The impact of inoculum age, aeration, the supplementation of pectinase and nutrients on the ethanol fermentation performance of the CBP system was studied. Although inulinase activities increased with the extension of the seed incubation time, its contribution to ethanol production was negligible because vigorously growing yeast cells harvested earlier carried out ethanol fermentation more efficiently. Thus, the overnight incubation that has been practised in ethanol production from starch‐based feedstocks is recommended. Aeration facilitated the fermentation process, but compromised ethanol yield because of the negative Crabtree effect of the species, and increases the risk of contamination under industrial conditions. Therefore, nonaeration conditions are preferred for the CBP system. Pectinase supplementation reduced viscosity of the fermentation broth and improved ethanol production performance, particularly under high gravity conditions, but the enzyme cost should be carefully balanced. Medium optimization was performed, and ethanol concentration as high as 94·2 g l^?1 was achieved when 0·15 g l^?1 K₂HPO₄ was supplemented, which presents a significant progress in ethanol production from Jerusalem artichoke tubers. Conclusions: A CBP system using K. marxianus is suitable for efficient ethanol production from Jerusalem artichoke tubers under VHG conditions. Significance and Impact of the Study: Jerusalem artichoke tubers are an alternative to grain‐based feedstocks for ethanol production. The high ethanol concentration achieved using K. marxianus with the CBP system not only saves energy consumption for ethanol distillation, but also significantly reduces the amount of waste distillage discharged from the distillation system.     

ATPase, peroxidase and lipoxygenase activity during post-harvest deterioration of cassava (Manihot esculenta Crantz) root tubers

Alterations in the activity of ATPases, peroxidases and lipoxygenases were studied during early stages of post-harvest deterioration of cassava (Manihot esculenta Crantz cv. Oyolu) root tubers. The peak activities of Ca²⁺-ATPase, (Ca²⁺+Mg²⁺)-ATPase, Na⁺+K⁺-ATPase, Mg²⁺-ATPase and peroxidases were observed after the first 24 h and thereafter decreased. The activity of lipoxygenase was biphasic, probably depicting two distinct isoforms expressed during deterioration. The results indicate that ATPases and peroxidases have a role in the post-harvest deterioration of cassava tuber, but the participation of lipoxygenases seems unlikely.     

Dibucaine,chlorpromazine, and detergents mediate membrane breakdown in potato tuber homogenates

Various strategies were evaluated for their ability to minimize the rate of breakdown of endogenous membrane lipids during cell fractionation studies with potato tubers. Buffering the homogenates at pH 7.5 to 8.0 resulted in significantly lower rates of phosphatidylcholine (PC) hydrolysis than were observed at lower pH values. Several potential inhibitors were added to homogenates to evaluate their ability to inhibit membrane lipid hydrolysis. The addition of bovine serum albumin (1% w/v) inhibited the rate of PC hydrolysis by 50%. The addition of low concentrations (25–100 μM) dibucaine (nupercaine) inhibited PC hydrolysis, but at higher concentrations (1-2 mM) it caused a 5- to 6-fold stimulation. Because dibucaine is a calmodulin antagonist, two other calmodulin antagonists (trifluoperazine and chlorpromazine) were tested and found to exhibit similar patterns of inhibition and stimulation. Similarly, the addition of low concentrations of deoxycholate also inhibited PC hydrolysis and high concentrations stimulated it. These results indicate that the hydrophobic properties of deoxycholate, dibucaine, and other calmodülin antagonists may explain their unusual effects on the rates of PC hydrolysis in potato tuber homogenates. Although the addition of exogenous calcium increased the rate of PC hydrolysis, the addition of calmodulin (bovine brain) had no effect. Other experiments revealed that the addition of 1% bovine serum albumin improved the yield and stability of mitochondrial and microsomal fractions from potato tubers. In constrast, the addition of 100 μM dibucaine caused deleterious effects.     

Potato water content impact on soil moisture measurement by neutron meter

A field study was conducted to examine the effect of the water content of potato tubers on the neutron count ratios of a neutron probe in an irrigated potato field. At the 20 cm depth, where tubers are concentrated, the neutron count ratios within the potato row (CW) were significantly higher than the neutron count ratios between the rows (CB). In contrast, the measured soil water contents within the rows (SW) at this depth, tended to be less than those measured between the rows (SB). At 45 cm depth, results were similar in a dry but not a wet year. The SW values, in contrast, were less than SB. Neither (CW-CB) or (SW-SB) were significant at 75 cm depth and values of (CW-CB) and (SW-SB) were similar. This study provides qualitative information regarding the influence of a potato tuber water content on neutron measurements, resulting in an overestimations of soil moisture in a potato field. Further studies should be conducted to develop relationships for corrections.Contribution no 3879215Contribution no 3879215     

Enzyme inhibitors from plants: Enterokinase inhibitors in tubers and seeds

Of the 22 tubers and 9 pulses screened for inhibitors of enterokinase activity, the following 12 tubers,Curcuma amada, Kyllinga monocephala, Solanum tuberosum, Canna indica, Helianthus tuberosus, Coleus parviformis, Mirabilis jalapa, Colocasia antiquorum (red variety),Alium cepa, Amorphophalus companulatus, Maranta arundinacea, Daucus carota, and 9 pulses namely,Vigna sinensis, Arachis hypogea, Pisum sativum, Phaseolus vulgaris (white bean),Phaseolus vulgaris (kidney bean),Phaseolus mungo, Cicer arietinum, Dolichos lablab and Cajonus cajan contained inhibitory activity. Three tubers,Amorphophalus companulatus, Maranta arundinacea andDaucus carota and all the nine pulses exhibited endogenous esterase activity towards benzoyl arginine ethyl ester. Among the 8 pulses and 3 tubers processed by affinity chromatography on trypsin-sepharose, to separate trypsin inhibitor from enterokinase inhibitor,Phaseolus vulgaris (kidney bean),Phaseolus vulgaris (white bean) andDolichos lablab contained distinct enterokinase inhibitors. These fractions were devoid of trypsin inhibitor activity. The trypsin inhibitor fromColeus parviformis tubers alone did not bind to trypsinsepharose and was recovered in the unbound fraction along with the enterokinase inhibitor.     

Tuber-specific silencing of asparagine synthetase-1 reduces the acrylamide-forming potential of potatoes grown in the field without affecting tuber shape and yield

Simultaneous silencing of asparagine synthetase (Ast)-1 and -2 limits asparagine (ASN) formation and, consequently, reduces the acrylamide-forming potential of tubers. The phenotype of silenced lines appears normal in the greenhouse, but field-grown tubers are small and cracked. Assessing the effects of silencing StAst1 and StAst2 individually, we found that yield drag was mainly linked to down-regulation of StAst2. Interestingly, tubers from untransformed scions grafted onto intragenic StAst1/2-silenced rootstock contained almost the same low ASN levels as those in the original silenced lines, indicating that ASN is mainly formed in tubers rather than being transported from leaves. This conclusion was further supported by the finding that overexpression of StAst2 caused ASN to accumulate in leaves but not tubers. Thus, ASN does not appear to be the main form of organic nitrogen transported from leaves to tubers. Because reduced ASN levels coincided with increased levels of glutamine, it appears likely that this alternative amide amino acid is mobilized to tubers, where it is converted into ASN by StAst1. Indeed, tuber-specific silencing of StAst1, but not of StAst2, was sufficient to substantially lower ASN formation in tubers. Extensive field studies demonstrated that the reduced acrylamide-forming potential achieved by tuber-specific StAst1 silencing did not affect the yield or quality of field-harvested tubers.     

Immunological relatedness of cytoplasmic and membrane-bound acid phosphatases from Dioscorea cayenensis rotundata

Antibodies were raised against one cytoplasmic and two membrane-bound acid phosphatases purified from yam tubers (Dioscorea cayenensis rotundata). Experiments of immunoinactivation and immunoelectrophoresis revealed cross-immunological reactions between the cytoplasmic enzyme (acid phosphatase A) and one of two membrane-bound counterparts (acid phosphatase B) suggesting that these molecules share common antigenic determinants. The antibodies raised against the other membrane-bound enzyme (acid phosphatase C) only inhibited and precipitated this enzyme.     

Improved production of potato microtubers using a temporary immersion system

A temporary immersion system for potato microtuber production was designed using 4-l vessels. This culture technique showed several advantages compared to solid cultures: i.e., three fold increase in shoot length, more internodes per plant and improved vigor. In the tuber induction stage, microtubers can be induced at all plant nodes, indicating that the tuberization is not restricted to specific regions. For both cultivars tested, Desiree and Atlantic, an average of 3.1 and 2.8 tubers per single node cutting was achieved after 9 weeks in culture. The size and weight of the tubers were higher than on solid media. Scale up was performed with cv. Atlantic in 10-l polycarbonate flasks and 12 units were mounted containing 150 single nodal cuttings each. An average of 2.6 tubers per inoculated cutting was obtained, with 1.3 g fresh weight per microtuber. Temporary immersion is a valuable option for potato microtuber production, as well as for shoot production during the planting season. This revised version was published online in June 2006 with corrections to the Cover Date.