James C. Linden

Methyl Jasmonate Induced Production of Taxol in Suspension Cultures of Taxus cuspidata: Ethylene Interaction and Induction Models

Suspension cultures of Taxus cuspidata were challenged with various concentrations and combinations of methyl jasmonate and ethylene. Taxol productivity increased 19‐fold when T. cuspidata suspension cultures were exposed to 5 ppm ethylene and 10 μM methyl jasmonate. This increase was 15‐fold when either 0 or 10 ppm ethylene was combined with 10 μM methyl jasmonate. The induction of taxol occurred within 51 h after elicitation and would reduce fermentation times and costs. Ethylene concentration at 50 ppm had an inhibitory effect on taxol production but not on phosphate uptake rate, suggesting independent regulation of taxol and physiological functions of the cell. A simple induction model is proposed to explain the action and effects of both ethylene and methyl jasmonate with regard to receptor binding and regulatory systems in plants.

Production of phytase by Mucor racemosus in solid-state fermentation.

Phytase production was studied by three Mucor and eight Rhizopus strains by solid‐state fermentation (SSF) on three commonly used natural feed ingredients (canola meal, coconut oil cake, wheat bran). Mucor racemosusNRRL 1994 (ATCC 46129) gave the highest yield (14.5 IU/g dry matter phytase activity) on coconut oil cake. Optimizing the supplementation of coconut oil cake with glucose, casein and (NH4)2SO4, phytase production in solid‐state fermentation was increased to 26 IU/g dry matter (DM). Optimization was carried out by Plackett‐Burman and central composite experimental designs. Using the optimized medium phytase, α‐amylase and lipase production of Mucor racemosusNRRL 1994 was compared in solid‐state fermentation and in shake flask (SF) fermentation. SSF yielded higher phytase activity than did SF based on mass of initial substrate. Because this particular isolate is a food‐grade fungus that has been used for sufu fermentation in China, the whole SSF material (crude enzyme, in situ enzyme) may be used directly in animal feed rations with enhanced cost efficiency.

Simultaneous Saccharification and Cofermentation of Peracetic Acid-Pretreated Biomass

Previous work in our laboratories has demonstrated the effectiveness of peracetic acid for improving enzymatic digestibility of lignocellulosic materials. The use of dilute alkali solutions as a pre-pretreatment prior to peracetic acid lignin oxidation increased carbohydrate hydrolysis yields in a synergistic as opposed to additive manner. Deacetylation of xylan is easily achieved using dilute alkali solutions under mild conditions. In this article, we evaluate the effectiveness of peracetic acid combined with an alkaline pre-pretreatment through simulataneous saccharification and cofermentation (SSCF) of pretreated hybrid poplar wood and sugar can ebagasse. Respective ethanol yields of 92.8 and 91.9% of theoretical are achieved using 6% NaOH/15% peracetic acid-pretreated substrates and recombinant Zymomonas mobilis CP4/p ZB5. Reduction of acetyl groups of the lignocellulosic materials is demonstrated following alkaline pre-pretreatments. Such processing may be helpful in reducing peracetic acid requirements. The influence of deacetylation is more significant in combined pretreatments using lower peracetic acid loadings.

Alkaline and Peracetic Acid Pretreatments of Biomass for Ethanol Production

Prehydrolysis with dilute acid and steam explosion constitute the most promising methods for improving enzymatic digestibility of biomass for ethanol production. Despite world wide acceptance, these methods of pretreatment are quite expensive considering costs for the reactor, energy, and fractionation. Using peracetic acid is a lignin-oxidation pretreatment with low-energy input by which biomass can be treated in a silo-type system without need for expensive capitalization. Experimentally, ground hybrid poplar and sugar cane bagasse are placed in plastic bags and a peracetic acid solution is added to the biomass in different concentrations based on ovendried biomass. The ratio of solution to biomass is 6∶1 and a 7-d storage period at ambient temperature (20°C) has been used. As an auxiliary method, a series of pre-pretreatments using stoichiometri camounts of sodium hydroxide and ammonium hydroxide based on 4-methyl-glucuronic acid and acetyl content in the biomass are performed before addition of peracetic acid. The basic solutions are added to the biomass in a ratio of 14∶1 solution to biomass, and mixed for 24 h at the same ambient temperature. Biomass is filtered and washed to a neutral pH before peracetic acid addition. The aforementioned procedures give high xylan content substrates as a function of the selectivity of peracetic acid for lignin oxidation and the mild conditions of the process. Consequently, xylanase/β-glucosidase combinations were more effective than cellulase preparations in hydrolyzing these materials. The pretreatment efficiency was evaluated through enzymatic hydrolysis and simultaneous saccharification and cofermentation (SSCF) tests. Peracetic ac treatment improves enzymatic digestibility of hybrid poplar and sugar cane bagasse with no need of high temperatures. Alkaline treatments are helpful in reducing peracetic acid requirements in the pretreatment.

Mixed culture solid substrate fermentation for cellulolytic enzyme production

SummaryCellulolytic and hemicellulolytic enzymes were produced on extracted sweet sorghum silage by mixed culture solid substrate fermentation with Trichoderma reesei LM-1 (a Peruvian mutant) and Aspergillus niger ATCC 10864. Optimal cellulose and xylanase levels of 4 IU/g dry weight (DW) and 180 IU/g DW, respectively, were achieved in 120 h-fermentation when T. reesei, inoculated at 0 h, was followed by the inoculation of A. niger at 48 h.

Optimizing peracetic acid pretreatment conditions for improved simultaneous saccharification and co-fermentation (SSCF) of sugar cane bagasse to ethanol fuel

The use of several lignocellulosic materials for ethanol fuel production has been studied exhaustively in the U.S.A.. Strong environmental legislation has been driving efforts by enterprises, state agencies, and universities to make ethanol from biomass economically viable. Production costs for ethanol from biomass have been decreasing year by year as a consequence of this massive effort. Pretreatment, enzyme recovery, and development of efficient microorganisms are some promising areas of study for reducing process costs.

Isolation and characterization of a novel ribosome-inactivating protein from root cultures of pokeweed and its mechanism of secretion from roots.

Ribosome-inactivating proteins are N-glycosidases that remove a specific adenine from the sarcin/ricin loop of the large rRNA, thus arresting protein synthesis at the translocation step. In the present study, a novel type I ribosome-inactivating protein, termed PAP-H, was purified from Agrobacterium rhizogenes-transformed hairy roots of pokeweed (Phytolacca americana). The protein was purified by anion- and cation-exchange chromatography. PAP-H has a molecular mass of 29.5 kD as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its isoelectric point was determined to be 7.8. Yeast (Saccharomyces cerevisiae) ribosomes incubated with PAP-H released the 360-nucleotide diagnostic fragment from the 26S rRNA upon aniline treatment, an indication of its ribosome-inactivating activity. Using immunofluorescence microscopy, PAP-H was found to be located in the cell walls of hairy roots and root border cells. PAP-H was determined to be constitutively secreted as part of the root exudates, with its secretion enhanced by a mechanism mediated by ethylene induction. Purified PAP-H did not show in vitro antifungal activity against soil-borne fungi. In contrast, root exudates containing PAP-H as well as additional chitinase, β-1,3-glucanase, and protease activities did inhibit the growth of soil-borne fungi. We found that PAP-H depurinates fungal ribosomes in vitro and in vivo, suggesting an additive mechanism that enables PAP-H to penetrate fungal cells.

Oligosaccharides potentiate methyl jasmonate-induced production of paclitaxel in Taxus canadensis.

The interdependence of methyl jasmonate (MJ) with chitin and chitosan derived elicitors in formation of paclitaxel was studied using plant cell suspension cultures of Taxus canadensis. Induction of paclitaxel biosynthesis was enhanced when MJ and elicitors were added 8 days after culture transfer compared to treatments in which only MJ or only elicitors were added. The enhancement of the paclitaxel biosynthesis response to MJ concentration was roughly linear between 0 and 200 µM using colloidal chitin or oligosaccharides of chitin and chitosan as elicitors. MJ concentrations greater than 200 µM were inhibitory. In kinetic studies, culture growth and substrate utilization were inhibited when the cultures were elicited with 100 µM MJ and with 0.63 mg l(-1) N-acetylchitohexaose and with 100 µM MJ alone; paclitaxel yields were 10-fold greater under the latter condition than the former. Ethylene biosynthesis by the cell cultures in response to elicitation is implicated in regulation of the response.

Phytoremediation Potential of Myriophyllum aquaticum and Pistia stratiotes to Modify Antibiotic Growth Promoters, Tetracycline, and Oxytetracycline, in Aqueous Wastewater Systems

Antibiotics are frequently used in the United States as feed efficiency promoters and medicines for livestock that is destined for human consumption. These antibiotics are released into the environment through the runoff and wastewater streams from animal feedlots and land applications of manure. The exposure of microorganisms to these antibiotics has reportedly resulted in the development of resistant species of microorganisms, which in turn can lead to human health hazards. Phytoremediation of these antibiotics can be a useful tool for countering this problem. Aquatic plants, Myriophyllum aquaticum (parrot feather) and Pistia stratiotes (water lettuce), were used for studying phytoremediation of tetracycline (TC) and oxytetracycline (OTC) from aqueous media. TC and OTC are two of the most commonly used tetracyclines in veterinary medicine. M. aquaticum and P. stratiotes gave high antibiotic modification rates of both antibiotics. Kinetic analyses dismiss direct enzyme catalysis; the modification rates decreased with increasing OTC concentrations. Sterile, cell-free root exudates (filtered through 0.2 μ m membranes) from both species also exhibited comparable antibiotic modification rates. The involvement of root-secreted metabolites in antibiotic modification is suggested. The changes in the UV absorbance spectra of OTC during treatment with the root exudates confirmed the modification.

Immobilized α-d-galactosidase in the sugar beet industry

Abstract Mycelia containing α-galactosidase have been used commercially for about ten years to hydrolyse raffinose in beet sugar molasses to aid in the production of sugar. The development of α-galactosidase-producing strains of three genera of fungi, the production of the enzyme and its use in beet sugar manufacture are reviewed. The treatment of mycelial pellets containing the α-galactosidase with glutaraldehyde and the consequent stabilization of the activity are described.