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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.77, #1-3)

Feedstocks by Thomas W. Jeffries; Y. Y. Lee (pp. 3-4).

Natural and somatic embryo development in loblolly pine by John Cairney; Nanfei Xu; Gerald S. Pullman; Vincent T. Ciavatta; Barbara Johns (pp. 5-17).

In production biological systems, monitoring and controlling the growth environment is possible, but assessing the metabolic competency of the organism is more difficult. Somatic embryogenesis (SE), a tissue-culture method for multiplying embryos asexually, has great potential to capture at low cost the genetic gain from breeding and genetic-engineering programs. Loblolly pine, however, has proven recalcitrant in the production of somatic embryos suitable in quality for operational use. Many similarities and differences in gene expression were uncovered. We have modified a recent technique called differential display (DD) to examine gene expression, allowing the comparison of somatic and zygotic embryos. Over 400 cDNA “bands” have been cloned and their sequences determined. These bands can serve as “expression markers,” providing rapid, simple, and sensitive assessment of embryo physiology and development. These techniques are applicable to many areas of research where monitoring specific levels of gene expression is important for evaluating the performance of a system.

Keywords: Differential display; zygotic embryogenesis; somatic embryogenesis; loblolly pine

Alkaline and peracetic acid pretreatments of biomass for ethanol production by Lincoln C. Teixeira; James C. Linden; Herbert A. Schroeder (pp. 19-34).

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.

Keywords: Hybrid poplar; sugar cane bagasse; peracetic acid; enzymatic hydrolysis; SSCF; Zymomonas mobilis ; ethanol fuel

Extrusion processing for ammonia fiber explosion (AFEX) by Bruce E. Dale; Justin Weaver; F. Michael Byers (pp. 35-45).

The ammonia fiber explosion (AFEX) process, previously run only in a batch reactor, has been adapted to run on a twin-screw extruder. The sugar yield of AFEX material after enzymatic hydrolysis has been increased up to 3.5 times over that of completely untreated material. The ruminant digestibility of corn fodder has been increased up to 32% (from 54–71%) over completely untreated material, and 23% (from 63–77%) over material extruded with no ammonia. Extrusion-treated material proved more digestible by the ruminant at 48 h than material treated in the batch reactor.

Keywords: AFEX; extrusion; ammonia; hydrolysis; ruminant digestibility

Optimization of steam explosion to enhance hemicellulose recovery and enzymatic hydrolysis of cellulose in softwoods by Michael M. Wu; Kevin Chang; David J. Gregg; Abdel Boussaid; Rodger P. Beatson; John N. Saddler (pp. 47-54).

A combination of Douglas fir heartwood and sapwood chips were steam pretreated under three conditions as measured by the Severity Factor (log R_o), which incorporated the time, temperature/pressure of pretreatment. By adjusting the steam pretreatment conditions, it was hoped to recover the majority of the hemicellulose component as monomers in the water-soluble stream, while providing a cellulosic-rich, water-insoluble fraction that could be readily hydrolyzed by cellulases. These three conditions were chosen to represent either high hemicellulose sugar recovery (low severity [L], log R_o=3.08), high-enzyme hydrolyzability of the cellulosic component (high severity [H], log R_o=4.21), and a compromise between the two conditions (medium severity [M], log R_o=3.45). The medium-severity pretreatment conditions (195°C, 4.5 min, 4.5% SO₂ logR_o=3.45) gave the best compromise in terms of relatively high hemicellulose recovery after stream pretreatment and the subsequent efficiency of enzymatic hydrolysis of the water-insoluble cellulosic fraction. The percent recovery of the original hemicellulose in the water-soluble fraction dropped significantly when the severity was increased (L-76.8%, M-64.7%, and H-37.5%). However, the ease of enzymatic hydrolysis of the cellulose-rich, water-insoluble fraction increased with increasing severity (L-24%, M-86.6%, and H-97.9%). Although more severe pretreatment conditions provided optimum hydrolysis of the cellulosic component, less severe conditions resulted in better recovery of the combined hemicellulose and cellulosic components.

Keywords: Steam explosion; hemicellulose; enzymatic hydrolysis; softwood

Two high-throughput techniques for determining wood properties as part of a molecular genetics analysis of hybrid poplar and loblolly pine by Gerald Tuskan; Darrell West; Harvey D. Bradshaw; David Neale; Mitch Sewell; Nick Wheeler; Bob Megraw; Keith Jech; Art Wiselogel; Robert Evans; Carolyn Elam; Mark Davis; Ron Dinus (pp. 55-65).

Two new high-through put techniques, computer tomography X-ray densitometry (CT scan) and pyrolysis molecular beam mass spectrometry (pyMBMS), coupled with quantitative trait loci (QTL) analysis, were tested as a means to overcome the time and cost associated with conventional characterization of biomass feedstock components. Applications of these two techniques were evaluated using hybrid poplar for the CT scan and loblolly pine for the pyMBMS. Segregating progeny from hybrid poplar varied in specific gravity, with individual mean estimates ranging from 0.21–0.41. Progeny from loblolly pine varied in lignin, α cellulose, and mannan contents, with individual mean estimates of lignin content ranging from 28.7–33.1%, α cellulose content from 28.8–43.5% and mannan content from 4.2–10.1%. QTL analysis of the loblolly pine data suggested that eleven QTLs were associated with individual feedstock characteristics and that two QTLs for several feedstock components were linked to the same position on the loblolly pine genetic map. Each QTL individually accounted for 7–13% of the total phenotypic variation in associated loblolly pine feedstock components.

Keywords: Pyrolysis molecular beam mass spectrometry; computer tomography; genetic markers; QTL; lignin; cellulose; hemicellulose

Modeling the enzymatic hydrolysis of dilute-acid pretreated Douglas Fir by Daniel J. Schell; Mark F. Ruth; Melvin P. Tucker (pp. 67-81).

Glucose yield from the enzymatic hydrolysis of cellulose was investigated as a function of cellulase enzyme loading (7–36 filter paper units [FPU]/g cellulose) and solids concentration (7–18% total solids) for up to 72 h on dilute sulfuric-acid pretreated Douglas Fir. The saccharification was performed on whole hydrolysate with no separation or washing of the solids. Enzyme loading had a significant effect on glucose yield; solids concentration had a much smaller effect even at higher glucose concentrations. The data were used to generate an empirical model for glucose yield, and to fit parameters of a cellulose hydrolysis kinetic model. Both models could be used for economic evaluation of a separate hydrolysis and fermentation process.

Keywords: Cellulose hydrolysis; softwood; ethanol; enzyme kinetics; cellulase

Environmentally friendly lubricating oil candidate by Aykut Özgülsün; Filiz Karaosmanoĝlu (pp. 83-89).

Synthetic lubricating oils based on renewable sources, excluding petroleum, have a great importance among all of the lubricating oil alternatives that are included in the research field about clean and environmentally friendly lubricating oil technologies. One of the environmentally friendly lubricating oils is a vegetable oil-based product. In this study, the esterification product of oleic acid with a fraction of molasses fusel oil as a lubricating oil candidate was determined according to the American Society for Testing and Materials (ASTM) standard tests. The results indicate that the ester product can be used as an environmental friendly lubricating oil or lubricating oil additive.

Comparison of different methods for the detoxification of lignocellulose hydrolyzates of spruce by Simona Larsson; Anders Reimann; Nils-Olof Nilvebrant; Leif J. Jönsson (pp. 91-103).

This study describes different detoxification methods to improve both cell growth and ethanol production by Baker's yeast, Saccharomyces cerevisiae. A dilute-acid hydrolyzate of spruce was used for the all detoxification methods tested. The changes in the concentrations of fermentable sugars and three groups of inhibitory compounds—aliphatic acids, furan derivatives, and phenolic compounds—were determined and the fermentability of the detoxified hydrolyzate was assayed. The applied detoxification methods included: treatment with alkali (sodium hydroxide or calcium hydroxide); treatment with sulfite (0.1% [w/v] or 1% [w/v] at pH 5.5 or 10); evaporation of 10% or 90% of the initial volume; anion exchange (at pH 5.5 or 10); enzymatic detoxification with the phenoloxidase laccase; and detoxification with the filamentous fungus Trichoderma reesei. An ion exchange at pH 5.5 or 10, treatment with laccase, treatment with calcium hydroxide, and treatment with T. reesei were the most efficient detoxification methods. Evaporation of 10% of the initial volume and treatment with 0.1% sulfite were the least efficient detoxification methods. Treatment with laccase was the only detoxification method that specifically removed only one group of the inhibitors, namely phenolic compounds. Anion exchange at pH 10 was the most efficient method for removing all three major groups of inhibitory compounds; however, it also resulted in loss of fermentable sugars.

Keywords: Detoxification; inhibition; ethanol production; S. cerevisiae ; softwood

Alfalfa fiber as a feedstock for ethanol and organic acids by Richard G. Koegel; Hassan K. Sreenath; Richard J. Straub (pp. 105-115).

Valuable co-products derived from fractionation of alfalfa herbage give the resulting fibrous fraction an economic advantage as a feed stock for ethanol or other organic products. Alfalfa fiber was saccharified and fermented with or without a liquid hot water (LHW) pretreatment. The LHW pretreatment hydrolyzed approximately 60% of the original fiber, yielding a high cellulose residue and aliquid extract. These yielded predominantly hexoses and pentoses, respectively, after enzymatic saccharification. Yields of ethanol and lactic acid resulting from fermentations are given.

Keywords: Medicago sativa; pretreatment; saccharification; ethanol; lactic acid

Steam explosion of straw in batch and continuous systems by Francesco Zimbardi; Donato Viggiano; Francesco Nanna; Mario Demichele; Daniela Cuna; Giovanni Cardinale (pp. 117-125).

The effects of the steam-explosion treatment on aqueous fractionation and bioconversion of wheat straw have been investigated. The treatments have been carried out in batch and continuous reactors with capacity of 0.5 Kg/cycle and 150 Kg/h, respectively. The exploded materials have been sequentially extracted with water at 65°C and sodium hydroxide 1.5%. Analytical determinations of liquid fractions and solid residues haveled to the fractionation pattern of the carbohydrates as, monomers, oligomers, and polymers. Evaluations of total acidity, ash content, and lignin recovery have improved understanding of the process. This part of the work has allowed us to derive: the empiric relationship between the batch and the continuous reactors and the yield and availability of pentoses and hexoses in various phases. Selected samples have been tested in enzymatic-hydrolysis experiments, pointing out the effect of treatment severity and reactor used on the saccharification yield.

Keywords: Enzymatic hydrolysis; steam explosion; straw

Oxidation of organosolv lignins in acetic acid by Adilson R. Gonçalves; Ulf Schuchardt (pp. 127-132).

The oxidation of four lignins obtained by organosolv pulping of eucalyptus wood (Acetosolv-eucalyptus Acetosolv lignin [EAL]), sugarcanebagasse (Acetosolv-bagasse Acetosolv lignin [BAL] and in acetone/water/FeCl₃-bagasse acetone/water lignin [BAWL]), and a softwood mixture (Organocell, Munich, Germany) was performed to obtain vanillin, vanillic acid, and oxidized lignin. Experiments were carried out in a cetic acid under oxygen flow using HBr, cobalt(II), and manganese(II) acetates as catalysts. After 10 h the total vanillin and vanillic acid yields were BAL 0.05 mmol, EAL 0.38 mmol, BAWL 0.45 mmol, and Organ ocell 0.84 mmol. Acetosolv lignins are crosslinked, which explains the lower yields in mononuclear products. The reaction volume (Δ V) of this reaction is −817 cm³/mol, obtained in experiments performed under oxygen pressure, showing the high influence of pressure on the oxidation. The major part of the, lignin stays in solution (oxidized lignin), which was analyzed by infrared spectroscopy, showing an increased in carbonyl and hydroxyl groups in comparison with the original lignin. The oxidized lignin can be used as chelating agent in the treatment of effluents containing heavy metals.

Keywords: Oxidation of lignins; organosolv lignins; acidic oxidation; utilization of lignins

Dilute acid hydrolysis of softwoods by Quang A. Nguyen; Melvin P. Tucker; Fred A. Keller; Delicia A. Beaty; Kevin M. Connors; Fannie P. Eddy (pp. 133-142).

Whole tree chips obtained from softwood forest thinnings were converted to ethanol via a two-stage dilute acid hydrolysis followed by yeast fermentation. The chips were first impregnated with dilute sulfuric acid, then pretreated in a steam explosion reactor to hydrolyze, more than 90% of the hemicellulose and approx 10% of the cellulose. The hydrolysate was filtered and washed with water to recover the sugars. The washed fibers were then subjected to a second acid im pregnation and hydrolysis to hydrolyze as much as 45% of the reamining cellulose. The liquors from both hydrolysates were combined and fermented to ethanol by a Saccharomyces cerevisiae yeast that had been adapted to the inhibitors. Based on available hexose sugars, ethanol yields varied from 74 to 89% of theoretical. Oligosaccharide contents higher than about 10% of the total available sugar appear to have a negative impact on ethanol yield.

Keywords: Biomass; softwood; ethanol; pretreatment; bioconversion; acid hydrolysis

Session 2 by Douglas C. Cameron; Min Zhang (pp. 145-145).

Evaluation of recombinant strains of Zymomonas mobilis for ethanol production from glucose/xylose media by Eva Joachimsthal; Kevin D. Haggett; Peter L. Rogers (pp. 147-157).

The fermentation characteristics of two recombinant strains of Zymomonas mobilis, viz. CP4 (pZB5) and ZM4 (pZB5), capable of converting both glucose and xylose to ethanol, have been characterized in batch and continuous culture studies. The strain ZM4 (pZB5) was found to be capable of converting a mixture of 65 g/L glucose and 65 g/L xylose to 62 g/L ethanol in 48h with a yield of 0.46 g/g. Higher sugar concentrations resulted in incompletexylose utilization (80h) presumably owing to ethanol inhibition of xylose assimilation or metabolism. The fermentation results with ZM4 (pZB5) show a significant improvement over results published previously for recombinant yeasts and other bacteria capable of glucose and xylose utilization.

Keywords: Recombinant Zymomonas mobilis ; xylose fermentation; lignocellulosic hydrolysates; ethanol production

Site-directed mutagenesis study on the thermal stability of a chemiric PQQ glucose dehydrogenase and its structural interpretation by Arief Budi Witarto; Takafumi Ohtera; Koji Sode (pp. 159-168).

We have previously reported that a chimeric pyrroloquinoline quinone (PQQ) glucose dehydrogenase (GDH), E97A3, which was made up of 97% of Escherichia coli PQQGDH sequence and 3% of Acinetobacter calcoaceticus PQQGDH, showed increased thermal stability compared with both parental enzymes. Site-directed mutagenesis studies were carried out in order to investigate the role of amino-acid substitution at the C-terminal region, Ser 771, of a chimeric PQQGDHs on their thermal stability. A series of Ser 771 substitutions of a chimeric PQQGDH, E99A1, confirmed that hydrophobic interaction governs the thermal stability of the chimeric enzymes. Comparison of the thermal denaturation of E. coli PQQGDH and E97A3 followed by far-ultraviolet (UV) circular dichroism (CD) spectroscopy revealed that E97 A3 acquired stability at the first step of denaturation, which is reversible, and where no significant secondary structure change was observed. These results suggested that the interaction between C-terminal and N-terminal regions may play a crucial role in maintaining the overall structure of β-propeller proteins.

Keywords: PQQ glucose dehydrogenase; β-propeller protein; site-directed mutagenesis; CD spectroscopy; denaturation pathway

Isolation and analysis of tetraheme-bound-cytochrome from photosynthetic reaction centers of Rhodopseudomonas viridis by Chikashi Nakamura; Miki Hasegawa; Masayuki Hara; Jun Miyake (pp. 169-179).

A tetraheme cytochrome (BCytc) was isolated from the photosynthetic reaction centers (RC) of Rhodopseudomonas viridis while maintaining the redox activity. BCytc was removed from the H-subunit-detached RC by polyacrylamide electrophoresis using an alkyl ether sulfate mixed with sodium dodecyl sulfate. Redox titration of BCytc showed a simple one-step redox titration curve and a lowered midpoint potential than that of one in RC. Direct electron transfer between BCytc and electrode surfaces, such as indium tin oxide, was successfully performed, indicating a potential for molecular electronic material.

Keywords: Tetraheme cytochrome; surfactant; photosynthetic reaction center; photosynthetic bacterium

Invertase production is related to the nitrogen source in Hansenula anomala by Maria Bernadete de Medeiros; Patricia Maria Barroso de Carvalho (pp. 181-189).

Differences in invertase accumulation of Hansenula anomala cultivated in ammonium and nitrate are reported. Media supplemented with sucrose and ammonium sulfateas the solecarbon and nitrogen source, respectively, show strong media acidification (pH 3.0 or lower), and vigorous cell growth. Invertase activity was not detected under such conditions. A cell-free imitation experiment suggests that, under such low pH, extensive chemical break-down of sucrose (>22%) occurs. Thus, H. anomala is able togrow under strong acidic conditions that permit sucrose metabolism by the uptake of monosaccharides generated from chemical hydrolysis. In addition, invertase activity is not present in cells grown in nitrate as nitrogen source at pH 5.0, but at pH 7.0 activity is detected. If ammonium is supplied instead of nitrate, cells grown at pH 5.0 show invertase activity and at pH 7.0 high levels of activity are detected. These results indicate a specific physiological response of the sucrose metabolism to the presence of alternate nitrogen source.

Keywords: Hansenula anomala ; invertase; nitrogen source; sucrose metabolism

Fermentation performance characteristics of a prehydrolyzate-adapted xylose-fermenting recombinant Zymomonas in batch and continuous fermentations by Hugh G. Lawford; Joyce D. Rousseau; Ali Mohagheghi; James D. McMillan (pp. 191-204).

Long-term (149 d) continuous fermentation was used to adapt a xylose-fermenting recombinant Zymomonas mobilis, strain 39676:pZB 4L, to conditioned (overlimed) dilute-acid yellow poplar hemicellulose hydrolyzate (“prehydrolyzate”). An “adapted” variant was isolated from a chemostat operating at a dilution rate of 0.03/h with a 50% (v/v) prehydrolyzate, corn steep liquor, and sugar-supplemented medium, at pH 5.75. The level of xylose and glucose in the medium was kept constant at 4% (w/v) and 0.8% (w/v), respectively. These sugar concentrations reflect the composition of the undiluted hardwood prehydrolyzate. The level of conditioned hardwood prehydrolyzate added to the medium was increased in 5% increments startingata level of 10%. At the upper level of 50% prehydrolyzate, the acetic-acid concentration was about 0.75% (w/v). The adapted variant exhibited improved xylose-fermentation performance in a pure-sugar, synthetic hardwood prehydrolyzate medium containing 4% xylose (w/v), 0.8% (w/v) glucose, and acetic acid in the range 0.4–1.0% (w/v). The ethanol yield was 0.48–0.50 g/g; equivalent to a sugar-to-ethanol conversion efficiency of 94–96% of theoretical maximum. The maximum growth yield and maintenance energy coefficients were 0.033 g dry cell mass (DCM)/g sugars and 0.41 g sugars/g DCM/h, respectively. The results confirm that long-term continuous adaptation is a useful technique for effecting strain improvement with respect to the fermentation of recalcitrant feedstocks.

Keywords: Recombinant Zymomonas ; continuous cofermentation; xylose; hardwood prehydrolyzate; ethanol yield; adaptation; acetic acid

Kinetics of xylitol fermentation by Candida guilliermondii grown on rice straw hemicellulosic hydrolysate by Inês C. Roberto; Ismael M. de Mancilha; Sunao Sato (pp. 205-210).

The fermentation kinetics for the conversion of rice straw hemicellulosic hydrolysate to xylitol by the yeast Candida guilliermondii was evaluated under batch conditions. The fermentation was accomplished in a 1 L working volume stirred-tank reactor with aeration of 1.3 vvm and agitation of 300 rpm (k_La=15/h). The maximum specific rate of xylitol formation (0.12 g/g) was achieved when the specific growth rate was lowered to 1/5 of its highest value. From analysis of the fermentation kinetics, a linear correlation between specific growth rate (μ_x) and specific rate of xylitol formation (q_p) was evident. Based on the Gaden model, this bioprocess was classified as growth-associated production and the relationship between μ_x and q_p can be described by the equation q_p=6.31μ_x.

Keywords: Rice straw; xylitol; fermentation kinetics; Candida guilliermondii

2-Deoxyglucose as a selective agent for derepressed mutants of Pichia stipitis by Hassan K. Sreenath; Thomas W. Jeffries (pp. 211-222).

The glucose analog 2-deoxyglucose (2-DOG) has been used to obtain mutants depressed for pentose metabolism. Some researchers have used 2-DOG alone whereas others have used it in the presence of a glucose-repressible carbon source. We examined both methods and screened mutant strains for improved use of xylose in the presence of glucose. Pichia stipitis mutants selected for growth on d-xylose in the presence of 2-DOG used xylose from a 1∶1 glucose:xylose mixture more rapidly than did their parents. One of these mutants, FPL-DX26, completely consumed xylose in the presence of glucose and produced 33g/L ethanol in 45h from 80 g/L of this sugar mixture. Mutants selected for growth on 2-DOG alone did not show significant improvement. Selection for growth on d-xylose in the presence of 2-DOG has been useful in developing parental strains for further genetic manipulation.

Keywords: Pichia stipitis ; mutation; selection; 2-deoxyglucose (2-DOG); fermentation; ethanol; glucose repression

Optimization of biosurfactant lipopeptide production from Bacillus subtilis S499 by Plackett-Burman design by Philippe Jacques; Choukai Hbid; Jacqueline Destain; Hary Razafindralambo; Michel Paquot; Edwin De Pauw; Philippe Thonart (pp. 223-233).

Bacillus subtilis S499 is well-known for its ability to produce two families of surfactant lipopeptides: Iturin A and Surfactin S1. Fermentation optimization for this strain was performed to amplify the surfactant production. Ten active variables were analyzed by two successive Plackett-Burman designs, consisting respectively of 12 and 16 experiments to give an optimized medium. The amount of biosurfactant lipopeptides in the supernatant of a culture carried out in this optimized medium was about five times higher than that obtained in nonoptimized rich medium. The analysis of the surfactant molecules produced in such optimized conditions has revealed the presence of a third family of lipopeptides: the fengycins.The time-dependent production of these three families of molecules in bioreactors showed that surfactin S1 is produced during the exponential phase and iturin A and fengycins during the stationary phase.

Keywords: Bacillus subtilis ; biosurfactant; lipopeptide; statistical design; fengycin

The effect of glucose on high-level xylose fermentations by recombinant Zymomonas in batch and fed-batch fermentations by Hugh G. Lawford; Joyce D. Rousseau (pp. 235-249).

6% xylose+6% glucose;8% xylose+4% glucose; and4% xylose+8% glucose. At the level of inoculum used in this study, complete fermentation of the 12% sugar mixtures required 2–3 d (equivalent to a volumetric ethanol productivity of 0.83–1.25 g ethanol/L.h). The sugar-to-ethanol conversion efficiency was 94–96% of theoretical maximum.

Keywords: Recombinant Zymomonas ; xylose; ethanol tolerance; cofermentation; prehydrolyzate; glucose feeding

Improving the catabolic functions of desiccation-tolerant soil bacteria by F. Weekers; Ph. Jacques; D. Springael; M. Mergeay; L. Diels; Ph. Thonart (pp. 251-266).

Bacterial strains were selected from a desiccated polluted soil for their drought tolerance and their ability to grow on diesel oil in view of incorporating them in a bioaugmentation product. These products are useful in case of recal citrant xenobiotic pollution, where there is no intrinsic biodegradation activity in the soil. These strains grow on the easily degradable components of diesel oil. In troduction of new catabolic genes into these desiccation-tolerant bacteria in order to improve their catabolic functions was considered.Plasmid-borne catabolic genes coding for enzymes in volved in the degradation of more recalcitrant compounds (Isopropylbenzene, trichloroethene, 3-chloroben zoate, 4-chlorobiphenyl, biphenyl) were successfully introduced in some of the desiccation-tolerant strains by means of natural conjugation. Strains exhibiting good tolerance to desiccation and able to grow on the new carbon sources were obtained. The frequencies of integration of the plasmids ranged from 2×10⁻⁸ to 9.2 10⁻² transconjugants/acceptor.Drought-tolerance is indeed important for bioaugmentation because of its in trinsic ecological significance and because a bioaugmentation starter has to be conditioned in a desic cated form to ensure good shelf-life. The conservation of the properties during storage was evaluated by accelerated storage tests.

Keywords: Bicaugmentation; drought tolerance; conjugation; plasmids; preservation

A high-copy-number plasmid capable of replication in thermophilic cyanobacteria by Masato Miyake; Hiroshi Nagai; Makoto Shirai; Ryuichiro Kurane; Yasuo Asada (pp. 267-275).

A 2.5 kb high-copy-number plasmid, pM A4 in thermophilic cyanobacterium Synechococcus sp. M A4 was isolated and characterized to develop a genetic engineering system for thermophilic cyanobacteria. The copy number of pM A4 was determined to be by densitometry about 350/cell. The pM A4 may be a type of rolling-circle plasmid, because a possible rep gene encoding 34 k D-protein and a consensus sequence of a double-stranded origin nick site of rolling circle plasmids were found in the pM A4 sequence. The pM A4 was electro-introduced into another thermophile, Synechococcus sp. MA 19, which is the strongest poly-β-hydroxybutyrate (PHB) accumulator in photoau totrophic organisms. The pM A4 was incorporated and retained in MA 19. These results indicate that pM A4 could be developed as a useful vector for thermophilic cyanobacteria.

Keywords: Thermophilic cyanobacteria; poly-β-hydroxybutyrate; carbon dioxide fixation; biodegradable plastic; genetic engineering; rolling circle plasmid

A hybrid neural model of ethanol production by Zymomonas mobilis by Anderson W. Silva da Henriques; Aline C. da Costa; Tito L. M. Alves; Enrique L. Lima (pp. 277-291).

A hybrid neural model was developed for the alcoholic fermentation by Zymomonas mobilis. This model is composed by the mass-balance equations of the process and neural networks, which describe the kinetic rates. Strategies that combines scarce experimental data with approximate models of the process were used to generate new data for the training of the networks, minimizing the number of experiments required. The proposed hybrid neural methodology uses all the information avail able about the process to deal with the difficulties in the development of the model.

Keywords: Alcoholic fermentation; Zymomonas mobilis ; hybrid modeling; functional link networks

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate by Tammy Kay Hayward; Jenny Hamilton; David Templeton; Ed Jennings; Mark Ruth; Arun Tholudur; James D. McMillan; Mel Tucker; Ali Mohagheghi (pp. 293-309).

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.

Keywords: Cellulase; pretreated poplarhydrolysate; Trichoderma reesei ; furfural; acetic acid

l-Asparaginase II of Saccharomyces cerevisiae by Edna M. M. Oliveira; Elvira Carvajal; Elba P. S. Bon (pp. 311-316).

The activity profile of the periplasmic asparaginase of Saccharomyces cerevisiae was determined during cell growth in an ure2 mutant; in an ure2 transformed with a plasmid containing the gene URE2 and, for comparison, in the strain D273-10B. Cells were cultivated in media presenting variable quantitative and qualitative nitrogen availability and the enzyme activity was evaluated in fresh and in nitrogen-starved cells. Nitrogen affected the asparaginase II level in fresh and starved cells of all strains. In the best condition, enzyme was produced by the wild-type cells at the late log-phase in the glucose/ammonium medium with a carbon to nitrogen ratio 4.3:1. Upon starvation, the activity doubled. The overall profile of the transformed strain was similar to that of the wild-type strain. In the ure2 mutant, highenzyme levels were observed during growth, as expected. However the activity level, upon starvation, in proline grown cells, increased sixfold, suggesting that in addition to the Ure2p-Gln3p system, another system regulates asparaginase II biosynthesis.

Keywords: Saccharomyces cerevisiae ; URE2 protein; nitrogen regulation; l-asparaginase II; nitrogen nutrition

Construction and characterization of fermentative lactate dehydrogenase Escherichia coli mutant and its potential for bacterial hydrogen production by Koji Sode; Mika Watanabe; Hiroshi Makimoto; Masamitsu Tomiyama (pp. 317-323).

In Escherichia coli, classified as a mixed-acid producer in fermentation, D-lactate is one of the final metabolites from pyruvate. In order to achieve a high efficiency of bacterial hydrogen production from glucose, we have constructed an E. coli strain deficient in fermentative lactate dehydrogenase (LDH-A) by P1 transduction. The mutant, designated as MC13-4, entirely lost LDH-A activity while retaining whole formate hydrogenlyase activity. This mutation resulted in an increase in hydrogen production based on glucose consumed. The effect of uptake hydrogenases on the hydrogen production was also discussed.

Keywords: Metabolic engineering; lactate dehydrogenase; hydrogen production; Escherichia coli

Subunit analyses of a novel thermostable glucose dehydrogenase showing different temperature properties according to its quaternary structure by Tomohiko Yamazaki; Wakako Tsugawa; Koji Sode (pp. 325-335).

We previously reported a novel glucose dehydrogenase (GDH) showing two peaks in the optimum temperature for the reaction at around 45°C and at around 75°C. Each peak derived from hetero-oligomeric enzyme, constructed from two distinct peptides with an α-subunit (MWs 67,000) and β-subunit (MWs 43,000), and a single peptide enzyme containing an α-subunit alone. The function of the two subunits in the thermostable co-factor binding GDH was investigated. The results of spectroscopic analyses indicated that the α-subunit contained an unknown co-factor showing specific fluorescence spectra like pyrroloquinoline quinone (PQQ), and the β-subunit was cytochrome c. Moreover, the results of a urea denaturation and reconstitution experiment suggested that the dissociation of the hetero-oligomeric complex to a single peptide was reversible. The kinetic parameter analyses for glucose and the electron mediator also suggested that the β-subunit was responsible for electron transfer from the catalytic center of the α-subunit to the electron mediator.

Keywords: Glucose dehydrogenase; thermal stability; catalytic subunit; electron transfer subunit; quaternary structures

Expression of luciferase gene under control of the puf promoter from Rhodobacter sphaeroides by Lyudmila Vasilyeva; Masato Miyake; Chikashi Nakamura; Eiji Nakada; Anatoly Tsygankov; Yasuo Asada; Jun Miyake (pp. 337-345).

An expression vector for purple bacteria based on the puf promoter from Rhodobacter sphaeroides was constructed. The 1.2 kb promoter fragment included the pufQ gene, ORFK, as wellas the SD sequence and thestartcodon from pufB. Translational pufduc fusion was constructed toanalyze transcription from the puf promoter. The luciferase expression was negligible under aerobic light conditions and was stimulated more than 700-fold under anaerobic light conditions. When Rb. sphaeroides, (p Pluc-2) strain was grown photoheterotrophically in batch culture, the luciferase expression was observed during 7h. The expression time could be greatly extended using continuous cultivation in a photobioreactor.

Keywords: Rhodobacter sphacroides ; expression vector; puf promoter; luciferase; continuous culture

Aspects of xylitol formation in sugarcane bagasse hydrolysate by Candida guillie rmondii in the presence of tetracycline by Ernesto Acosta Martínez; Silvio S. Silva; Maria G. A. Felipe (pp. 347-354).

The biocon version of xylose intoxylitol using pH values of 4.0, 5.5 and 7.0 and tetracycline concentrations of 20 and 40 mg/L was carried out to verify the influence of these parameters on Candida guilliermondii metabolism for xylitol production. Experiments were performed with sugarcane bagasse hemicellulosi chydrolysate (48.5 g/L of xylose) in 125-mL Erlenmeyer flasks, at 30°C, 200 rpm, during 88 h. The results demostrated that the bioconversion of xylose into xylitol was significantly influenced by the pH. On the other hand, in media containing 20 or 40 mg/L of tetracycline, this bioconversion was not significantly affected. The best results of xylitol production were obtained in hemicellulosic hydrolysate without tetracycline, at pH 7.0 In these conditions, the maxim um specific growth rate was 0.014/h and the yield factor of xylitol and volumetric productivity were 0.85g/g and 0.70g/L/h respectively. Xylitol and cell growth occureed simultaneously.

Keywords: Hemicellulosic hydrolysate; sugarcane bagasse; xylose; xylitol; Candida guilliermondii ; Klebsiella pueumoniae

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.77, #1-3)

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Applied Biochemistry and Biotechnology: Part A: Enzyme Engineering and Biotechnology (v.77, #1-3)