The profile of enzymes relevant to solvent production during direct fermentation of sago starch by <Emphasis Type=Italic>Clostridium saccharobutylicum</Emphasis> P262 utilizing different pH control strategies

The profile of enzymes relevant to solvent production during direct fermentation of sago starch by Clostridium saccharobutylicum P262 in a 2 L stirred tank fermenter was determined utilizing different pH control strategies. During fermentation without pH control (initial pH of 6), the specific activity of crotonase, thiolase, and β-hydroxybutyryl-CoA dehydrogenase increased proportionally with solvent production. The highest crotonase (3,450.7 kat) and phosphotransbutyrylase activity (1,475.6 kat) was observed in fermentation where pH was maintained at 5 during the acidogenic phase and corresponded to a fairly high acid accumulation but low solvent production. During fermentation with a controlled pH of 5.25 during the sol-ventogenic phase, the highest thiolase specific activity (255.7 kat) was obtained and corresponded to the highest production of acetone. On the other hand, the highest specific activities of crotonase, β-hydroxybutyryl-CoA dehydrogenase, and phosphotransbutyrylase were observed at pH 5.5 and corresponded to the highest production of ethanol and butanol. Butyryl-CoA dehydrogenase had no significance role in solvent fermentation. These results suggested that pH control strategies were important for improvement of solvent production during direct fermentation of sago starch by C. saccharobutylicum.     

Effect of carbohydrate source on alpha-amylase and glucoamylase formation byClostridium acetobutylicum SA-1

Summary An examination into the effect of different carbohydrate sources indicated that the production of extracellular alpha-amylase and glucoamylase was under similar biosynthetic control inClostridium acetobutylicum SA-1. Cell-associated starch-hydrolytic enzymes may be more important than extracellular enzymes in the processing of the starch molecule.     

Buffering as a means for increasing growth and butanol production byClostridium acetobutylicum

Summary The objective of this work was to optimize butanol formation in the acetone-butanol-ethanol (ABE) fermentation by examining the level of buffering as it affects the dissociation of butyric acid to the less toxic butyrate anion. Experiments were carried out in batch culture using chemically defined (P2) or complex media containing various buffering agents. These included salts of acetate, citrate, phosphate, nitrate, or bicarbonate, representing a range of pK _a values and buffering capacities. Growth in highly buffered medium was found to increase the stationary phase cell density, carbohydrate utilization, and the final butanol concentration. At higher levels of buffering, increased growth and elevated concentrations of butyric acid were required to initiate solventogenesis, suggesting the involvement of a critical threshold level of undissociated butyric acid.     

Fed-batch production of baker's yeast using millet (Pennisetum typhoides) flour hydrolysate as the carbon source

A fermentation medium based on millet (Pennisetum typhoides) flour hydrolysate and a four-phase feeding strategy for fed-batch production of baker's yeast,Saccharomyces cerevisiae, are presented. Millet flour was prepared by dry-milling and sieving of whole grain. A 25% (w/v) flour mash was liquefied with a thermostable 1,4--d-glucanohydrolase (EC 3.2.1.1) in the presence of 100 ppm Ca²⁺, at 80°C, pH 6.1–6.3, for 1 h. The liquefied mash was saccharified with 1,4--d-glucan glucohydrolase (EC 3.2.1.3) at 55°C, pH 5.5, for 2 h. An average of 75% of the flour was hydrolysed and about 82% of the hydrolysate was glucose. The feeding profile, which was based on a model with desired specific growth rate range of 0.18–0.23 h^–1, biomass yield coefficient of 0.5 g g^–1 and feed substrate concentration of 200 g L^–1, was implemented manually using the millet flour hydrolysate in test experiments and glucose feed in control experiments. The fermentation off-gas was analyzed on-line by mass spectrometry for the calculation of carbon dioxide production rate, oxygen up-take rate and the respiratory quotient. Off-line determination of biomass, ethanol and glucose were done, respectively, by dry weight, gas chromatography and spectrophotometry. Cell mass concentrations of 49.9–51.9 g L^–1 were achieved in all experiments within 27 h of which the last 15 h were in the fedbatch mode. The average biomass yields for the millet flour and glucose media were 0.48 and 0.49 g g^–1, respectively. No significant differences were observed between the dough-leavening activities of the products of the test and the control media and a commercial preparation of instant active dry yeast. Millet flour hydrolysate was established to be a satisfactory low cost replacement for glucose in the production of baking quality yeast.Nomenclature C _ox Dissolved oxygen concentration (mg L^–1) - CPR Carbon dioxide production rate (mmol h^–1) - C _s0 Glucose concentration in the feed (g L^–1) - C _s Substrate concentration in the fermenter (g L^–1) - C _s.crit Critical substrate concentration (g L^–1) - E Ethanol concentration (g L^–1) - F _s Substrate flow rate (g h^–1) - i Sample number (–) - K _e Constant in Equation 6 (g L^–1) - K _o Constant in Equation 7 (mg L^–1) - K _s Constant in Equation 5 (g L^–1) - m Specific maintenance term (h^–1) - OUR Oxygen up-take rate (mmol h^–1) - q _ox Specific oxygen up-take rate (h^–1) - q _ox.max Maximum specific oxygen up-take rate (h^–1) - q _p Specific product formation rate (h^–1) - q _s Specific substrate up-take rate (g g^–1 h^–1) - q _s.max Maximum specific substrate up-take rate (g g^–1 h^–1) - RQ Respiratory quotient (–) - S Total substrate in the fermenter at timet (g) - S ₀ Substrate mass fraction in the feed (g g^–1) - t Fermentation time (h) - V Instantaneous volume of the broth in the fermenter (L) - V ₀ Starting volume in the fermenter (L) - V _si Volume of samplei (L) - x Biomass concentration in the fermenter (g L^–1) - X ₀ Total amount of initial biomass (g) - X _t Total amount of biomass at timet (g) - Y _p/s Product yield coefficient on substrate (–) - Y _x/e Biomass yield coefficient on ethanol (–) - Y _x/s Biomass yield coefficient on substrate (–) Greek letters Moles of carbon per mole of yeast (–) - Moles of hydrogen atom per mole of yeast (–) - Moles of oxygen atom per mole of yeast (–) - Moles of nitrogen atom per mole of yeast (–) - Specific growth rate (h^–1) - _crit Critical specific growth rate (h^–1) - _E Specific ethanol up-take rate (h^–1) - _max.E Maximum specific ethanol up-take rate (h^–1)     

Continuous acetone-butanol-ethanol (ABE) fermentation using immobilized cells of Clostridium acetobutylicum in a packed bed reactor and integration with product removal by pervaporation

An integrated solvent (ABE) fermentation and product removal process was investigated. A stable solvent productivity of 3.5 g/L h was achieved by using cells of Clostridium acetobutylicum immobilized onto a packed bed of bonechar, coupled with continuous product removal by pervaporation. Using a concentrated feed solution containing lactose at 130g/L, a lactose value of 97.9% was observed. The integrated fermentation and product removal system, with recycling of the treated fermentor effluent containing only low amount of solvents (/but lactose and acids), leads to only low acid losses. Therefore, most of the acids are converted to solvents, and this results in a high solvent yield of 0.39 g solvents/g lactose utilized. The pervaporation system provided a high product removal rate even at low solvent concentrations. A solvent membrane flux of 7.1 g/m(2) h with a selectivity of 5 was achieved during these investigations. The system proved to be very reliable.     

Growth kinetics and astaxanthin production of Phaffia rhodozyma on glycerol as a carbon source during batch fermentation

Glycerol was studied as a substrate for astaxanthin by Phaffia rhodozyma PR 190. With co-utilisation of yeast extract and peptone, the maximum specific growth rate was 0.24 ± 0.02 h–1. Astaxanthin percentage in total pigment is constant (0.78 mg/g) and its yield from glycerol is always 0.97 mg/g. The yield of biomass from glycerol alone is 0.50 ± 0.02 g/g. The specific rate of astaxanthin production versus the cell growth rate reached a maximum for an optimal specific growth rate of 0.075 h–1. For this optimal value, the maximum specific astaxanthin production rate is 0.09 ± 0.01 mg/g.h. The best astaxanthin results were : 33.7 mg/l, 0.2 mg/l.h and 1.8 mg/g yeast after a fermentation term of 168 hours. Our results suggest a strategy of astaxanthin production in fed batch culture or chemostat at a growth rate of 0.075 h–1. © Rapid Science Ltd. 1998     

Effect of pH on organic acid production byClostridium propionicum in test tube and fermentor cultures

Summary Clostridum propionicum is a chemical autotroph that metabolizes alanine to propionic acid (reduction product) and acetic acid (oxidation product). The ratio of propionate/acetate predicted by the electron balance is 2:1. This study reports the effect of pH on growth and organic acid production by this organism when grown in both test tube cultures initially buffered from pH 7.0 to 5.0, and in fermentors maintained at pH 7.0 and 6.5. Highest growth and organic acid production was found at pH 7.0 in both cases. HPLC analysis showed that at pH 7.0, the ratios of propionate to acetate were 0.45:1 (stationary tube, 24 h). The highest ratio observed was 1.8:1 (stationary tube, pH 6.0, 24h). This tube produced 8.5% of the acids produced in the pH 7.0 culture tube. The identify of the major portion of the reduction products of the organism remains unknown.     

Arrowroot (Marantha arundinacea) starch as a new low-cost substrate for alkaline protease production

The feasibility of arrowroot (Marantha arundinacea) starch for alkaline protease production using an alkalophilic Bacillus lentus isolate was evaluated in batch fermentations in shake flasks and in a bioreactor under a range of conditions. A new arrowroot starch-casein medium (pH 10.2) was formulated having a composition (%, w/v): arrowroot starch 1, casein 1, sodium succinate 0.25, NH₄Cl 0.05, NaCl 0.05, KH₂PO₄ 0.04, K₂HPO₄ 0.03, MgCl₂ 0.01, yeast extract 0.01 and Na₂CO₃ 1.05. The isolate produced a maximum protease yield (6754.7 U ml^–1) in this medium when grown for 72 h at 250 rev/min and 37 °C. Scaling-up studies in a bioreactor showed a 5-fold increase in alkaline protease yields (31899 U ml^–1) at a lower production time of 45 h, aeration of 1 v/v/m and agitation of 400 rev/min at 37 °C.     

Determination of root biomasses of three species grown in a mixture using stable isotopes of carbon and nitrogen

A method is evaluated that employs variation in stable C and N isotopes from fractionations in C and N acquisition and growth to predict root biomasses of three plant species in mixtures. Celtis laevigata Willd. (C₃), Prosopis glandulosa Torr. (C₃, legume) and Schizachyrium scoparium (Michx.) Nash (C₄), or Gossypium hirsutum L. (C₃), Glycine max (L.) Merr. (C₃ legume), and Sorghum bicolor (L.) Moench (C₄) were grown together in separate, three-species combinations. Surface roots (0–10 cm depth) of each species from each of the two combinations were mixed in various proportions, and the relative abundances of ¹⁵N and ¹⁴N and ¹³C and ¹²C in prepared mixtures, surface roots of single species, and roots extracted from the 80-cm soil profile in which each species combination was grown were analyzed by mass spectrometry. An algebraic determination which employed the δ ¹³C, % ¹⁵N, and C and N concentrations of root subsamples of individual species accounted for more than 95% of the variance in biomass of each species in prepared mixtures with G. max, G. hirsutum, and S. bicolor. A similar analysis demonstrated species-specific differences in rooting patterns. Root biomasses of the C₄ monocots in each combination, S. scoparium and S. bicolor, were concentrated in the upper 20 cm of soil, while those of G. hirsutum and the woody P. glandulosa were largest in lower soil strata. Analyses of stable C and N isotopes can effectively be used to distinguish roots of species which differ in ratios of ¹⁵N to ¹⁴N and ¹³C to ¹²C and thus to study belowground competition between or rooting patterns of associated species with different C and N isotope signatures. The method evaluated can be extended to quantify aboveground and belowground biomasses of component species in mixtures with isotopes of other elements or element concentrations that differ consistently among plants of interest.     

Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation

Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures. Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products. Low-grade glycerol [65% (w/v) purity] could replace commercial glycerol [87% (w/v) purity], leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l^–1 h^–1 and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h^–1; butanol accounted for 94% (w/w) of total solvents. These values are among the highest reported in literature for C. acetobutylicum simple chemostats.     

丙酮丁醇梭菌硫氧还蛋白系统在溶剂生产过程中的功能

[目的]探究丙酮丁醇梭菌硫氧还蛋白系统在生长和代谢过程中的功能.[方法]使用ClosTron系统对硫氧还蛋白系统中的硫氧还蛋白还原酶基因(trxB)进行插入失活,得到突变株,通过Southern杂交方法验证插入内含子的拷贝数;在基本培养基中进行分批发酵,比较并分析突变株的生长特点;通过pH控制,利用限磷的连续发酵方法使...     

Effect of carbon source on enzymes involved in glycerol metabolism inNeurospora crassa

Specific activities of eight enzymes involved in glycerol metabolism were determined in crude extracts of three strains ofNeurospora crassa after growth on six different carbon sources. One of the strains was wild type, which grew poorly on glycerol as sole carbon source; the other two were mutant strains which were efficient glycerol utilizers. A possible basis for this greater effeciency of glycerol utilization was catabolite repression of glyceraldehyde kinase by glycerol in wild type, and two-fold higher glycerate kinase activity in the mutant strains after growth on glycerol, thus apparently allowing two routes for glyceraldehyde to enter the glycolytic pathway in the mutant strains but only one in wild type. The preferential entry of glyceraldehyde to the glycolytic pathway through glycerate was suggested by the lack of glyceraldehyde kinase in all three strains after growth on one or more of the carbon sources and the generally higher levels of aldehyde dehydrogenase and of glycerate kinase than of glyceraldehyde kinase.     

Nannochloropsis (Eustigmatophyceae) as source of commercially valuable pigments

Pigment composition and its variation with culture agewere analyzed in six strains of Nannochloropsis(Eustigmatophyceae). The capacity for accumulationof the ketocarotenoids astaxanthin and canthaxanthinwas higher in N. salina and N. gaditanathan in the other strains studied here. Theinfluence of salinity (15 to 100 practical units) onpigment production was studied in N. gaditana,where a defined pattern of variation could not befound apart from a notable increase in zeaxanthin at100. In cultures grown in a photobioreactor and athigh cell densities of about 10⁹ cells mL^-1,pigment production reached: 350 mg L^-1 forchlorophyll a, 50 mg L^-1 for violaxanthin,5 mg L^-1 for canthaxanthin, 3 mg L^-1 forastaxanthin. The highest contents of canthaxanthin andastaxanthin obtained in experiments with N.gaditana were 19.4 and 14.6 ng pigment (10⁶cells)^-1, respectively, which accounts for 0.7%dry weight. By means of xanthophyll cycle inductionthrough exposure of cells to high irradiance and at40 ^°C, conversion of violaxanthin intozeaxanthin may attain up to 70% of the violaxanthincontent, which corresponds to 0.6% dry weight. Theresults indicate that interest in Nannochloropsis as a source of valuable pigments isnot related to its capacity for single pigmentaccumulation, but the availability of a range ofpigments such as chlorophyll a, zeaxanthin,canthaxanthin and astaxanthin, each with highproduction levels.     

The internal pH of Clostridium acetobutylicum and its effect on the shift from acid to solvent formation

Clostridium acetobutylicum was unable to keep a constant pH inside the cells when grown on a phosphatelimited synthetic medium which allowed production of organic acids in a first phase and of solvents in a second phase. At external pH values between 5.9 and 4.3, the cells kept a constant pH of 0.9 to 1.3. A similar pH was measured in continuous culture under solventproducing conditions. The pH was abolished by protonovorous uncouplers, such as tetrachlorosalicylanilide (TCS) or carbonyl-p-trifluormethoxyphenylhydrazone (FCCP). n-Butanol at concentration of 150 mM and above led also to a complete abolition of the pH gradient.The internal pH stayed above 5.5 in cultures that shifted from acid to solvent formation. It is concluded that this is a prerequisite for the shift. The possible function of high internal concentrations of butyrate, butyryl phosphate and butyryl coenzyme A in the triggering mechanisms of the shift is discussed.Abbreviations TCS Tetrachlorosalicylanilide - FCCP carbonyl-p-trifluormethyoxyphenylhydrazone     

High production of alkaline protease byBacillus licheniformis in a fed-batch fermentation using a synthetic medium

Summary High production (9016 U/ml) of alkaline protease byBacillus licheniformis has been achieved. A 49% increase in production was achieved by the method used as compared with a batch process. By using a synthetic medium and a fed-batch operation controlled by the Advanced Fermentation Software (AFS) package, it was found that the keys to high production of protease are: (i) to maintain a low concentration of glucose (<0.43 g/l) in the medium; (ii) to control pH at a certain level (pH 6.50) in the culture; and (iii) to use rough type colonies as the starting culture. Our fed-batch fermentation process successfully simulates and surpasses ordinary batch fermentation processes. By using ammonium sulfate instead of soy bean flour as the only nitrogen source, an expected benefit was the elimination of unpleasant odors caused by natural organic nitrogenous components in the media. This would improve the industrial production environment.     

Dopamine in tissues of the hydrozoan jellyfishPolyorchis penicillatus as revealed by HPLC and GC/MS

Summary High performance liquid chromatography of alumina extracts of several tissues inPolyorchis penicillatus show the presence of dopamine and a catecholamine resembling norepinephrine. Dopamine is found in the highest concentrations in nerve-rich tissue (120 fmol·mg wet wt^–1), at intermediate concentrations in endoderm-rich tissue (30 fmol·mg^–1), and at the lowest concentrations in the mesoglea (10 fmol·mg^–1). The presence of dopamine was confirmed using gas chromatography/mass spectrometry with negative ion chemical ionization, but norepinephrine and epinephrine could not be detected in nerve-rich tissue.     

Production of 1,3-propanediol by <Emphasis Type="Italic">Clostridium butyricum</Emphasis> VPI 3266 using a synthetic medium and raw glycerol

Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated. C. butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e. above 87% (w/v). A 39% increase of growth inhibition was observed in the presence of 100 g l^–1 of a lower grade raw glycerol (65% w/v). Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium. No significant differences were found in C. butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source. In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.     

Utilization of homoserine lactone as a sole source of carbon and energy by soil Arthrobacter and Burkholderia species

Homoserine lactone (HSL) is a ubiquitous product of metabolism. It is generated by all known biota during the editing of certain mischarged aminoacyl-tRNA reactions, and is also released as a product of quorum signal degradation by bacterial species expressing acyl-HSL acylases. Little is known about its environmental fate over long or short periods of time. The mammalian enzyme paraoxonase, which has no known homologs in bacteria, has been reported to degrade HSL via a lactonase mechanism. Certain strains of Variovorax and Arthrobacter utilize HSL as a sole source of nitrogen, but not as a sole source of carbon or energy. In this study, the enrichment and isolation of four strains of soil bacteria capable of utilizing HSL as a carbon and energy source are described. Phylogenetic analysis of these isolates indicates that three are distinct members of the genus Arthrobacter, whereas the fourth clusters within the non-clinical Burkholderia. The optimal pH for growth of the isolates ranged from 6.0 to 6.5, at which their HSL-dependent doubling times ranged from 1.4 to 4 h. The biodegradation of HSL by these 4 isolates far outpaced its chemical decay. HSL degradation by soil bacteria has implications for the consortial mineralization of acyl-homoserine lactones by bacteria associated with quorum sensing populations.