The metabolism of lactose byClostridium acetobutylicum

Summary Acetone and butanol biosynthesis byClostridium acetobutylicum ATCC 824 was affected by lactose concentration and by agitation during the fermentation. At 1% and 3% lactose concentrations acid production predominated, while butanol production predominated at 5% lactose concentration. Higher solvent production was observed in fermentors without agitation than in fermentors with agitation.     

Silver toxicity to ferrous iron and pyrite oxidation and its alleviation by yeast extract in cultures ofThiobacillus ferrooxidans

Summary Ferrous-ion oxidation byThiobacillus ferrooxidans was inhibited by 10^–6 M Ag⁺ while a slight inhibition of growth was apparent with 10^–7 M Ag⁺. The threshold toxic concentration was the seme for four different test strains. While prolonged lag phases resulted from culture exposure to Ag⁺, Fe²⁺ oxidation rates after the onset of growth showed little variation under these conditions. Yeast extract (0.02%) partially alleviated the toxicity of silver-ion by reducing the lag periods. Pyrite oxidation byT. ferrooxidans and mixed cultures of acidophiles was tested at 8.3×10^–7 to 8.3×10^–5 M Ag⁺. Strong inhibition was apparent at 8.3×10^–5 M Ag⁺ and little to no inhibition was observed at 8.3×10^–7 M Ag⁺.     

Acetone-butanol production by Clostridium acetobutylicum in an ammonium-limited chemostat at low pH values

Clostridium acetobutylicum was grown in continuous culture under ammonium limitation (15.15 mM NH₄ ⁺). At a pH of 6.0 and at various dilution rates only acetate, butyrate and ethanol were formed as non-gaseous products. A decrease of the pH to values between 5.2 and 4.3 resulted in a shift of the fermentation towards acetone-butanol formation.     

Could the improvements in the alcoholic fermentation of high glucose concentrations by yeast immobilization be explained by media supplementation?

Summary The maximal concentration of ethanol produced during the fermentation of 320 g/l glucose bySaccharomyces bayanus was higher when the yeast cells were immobilized either by adsorption on celite or by entrapment in k-carrageenan beads (from 10.5% with free cells up to 14.5% and 13.1% (v/v) respectively). This increase was due to medium supplementation with the compounds present in the immobilization supports.     

Effect of temperature upon growth rate and solvent production in batch cultures ofClostridium acetobutylicum

Summary The effect of temperature on the solvent production byClostridium acetobutylicum has been studied in the range 25 to 40°C. It was found that the solvent yield decreased with increasing temperature; seemingly because of a reduction in acetone production. It appeared that the yield of the other major solvent, butanol, was not affected by the temperature. Considering total solvent yield and productivity only, the optimum fermentation temperature is 35°C.     

Decomposition and humification of plant residues by some soil fungi

Summary Decomposition and humification of powdered plant material ofLeptochloa fusca L. Kunth andSesbania aculeata Pers. by eight soil fungi was studied in pure culture. Maximum decomposition was caused bySporotrichum pruinosum, and maximum humification byStachybotrys atra. Significant differences were observed in some chemical and optical properties of humic compounds produced by these fungi.     

Directed metabolic flow with high butanol yield and selectivity in continuous cultures ofClostridium acetobutylicum

Summary This work addresses the problem of stable butanol formation byClostridium acetobutylicum in continuous culture. Sustained altered electron flow was observed in the presence of benzyl viologen which serves to redirect carbon flow towards primarily butanol formation. A yield of butanol of over 0.28 g.g^–1 glucose was obtained and butanol comprised over 90% of the total solvents formed. Additionally, acid formation decreased significantly with butyric acid as the dominant acid end product.     

By-products formed during direct conversion of sugar beets to ethanol byZymomonas mobilis in conventional submerged and solid-state fermentations

Summary The nature and amounts of by-products formed during conversion of sugar beets to ethanol byZ. mobilis in Conventional Submerged Fermentation (CSF) and Solid-State Fermentation (SSF) were investigated. It was found that the bacterium produced fewer by-products in SSF than CSF, and that by-products profile was different. The influence of fermentation temperature on synthesis of by-products in SSF was also studied. High fermentation temperature favoured sorbitol synthesis and low fermentation temperature the synthesis of levan. The best results were obtained at 35°C. An ethanol yield of up to 95% of the theoretical value with final ethanol concentration of 142 g/L were obtained.     

Corrinoid production byMethanosarcina barkeri in a repeated fed-batch reactor with membrane module

Summary In an attempt to improve corrinoid production byM. barkeri strain Fusaro, a repeated fed-batch culture coupled with a membrane module on methanol-acetate medium was used. Productivity of 22 mg-corrinoid/1. day was obtained during 626 h cultivation with corrinoid and cell mass concentration of 95 mg/l and 25.9 g-dry cell/l, respectively. The minimum value for membrane flux permeation was 18 liter/m². h for cell mass concentration between 25.9 to 31.0 g-dry cell/l. with rejection of 100 %.     

Ethanol production from glucose byClostridium thermosaccharolyticum strains: Effect of pH and temperature

Summary The fermentation of glucose byClostridium thermosaccharolyticum strains IMG 2811^T, 6544 and 6564 was studied in batch culture in a complex medium at different temperatures in defined and free-floating pH conditions. All the strains ferment 5 g glucose.l^–1 completely. The yield of the fermentation products turned out to be independent of the incubation temperature for strain IMG 2811^T. Strain IMG 6544 produced at 60°C significantly more ethanol and less acetic acid, butyric acid, hydrogen gas and biomass than at lower temperatures. With strain IMG 6564, the opposite effect occurred: ethanol appeared to be the main fermentation product at 45°C; at 60°C less ethanol and more acetic acid, butyric acid and hydrogen gas was formed.Experiments, carried out with strain IMG 6564, at defined pH conditions (between 5.5 and 7) and different temperatures (45, 55 and 60°C) revealed no effect of the incubation temperature, but an important effect of the pH on the product formation. At pH 7, ethanol was the main fermentation product while minor amounts of hydrogen gas, acetic and butyric acid were produced. Lowering the pH gradually to 5.5 resulted in a decrease of ethanol and an increase of biomass, hydrogen gas, acetic, butyric and lactic acids. At pH higher than 7 no growth occurred. Similar conclusions could be drawn for strains IMG 2811^T and 6544.     

Overproduction of human insulin-like growth factor-II inEscherichia coli

Summary Human insulin-like growth factor II (hIGF-II) was produced inEscherichia coli as a protein fused to human growth hormone. High level expression of the fusion protein was attained with pIBL-1 plasmid. The hIGF-II obtained byin vitro cleavage of the fusion protein with cyanogen bromide was highly purified and its biological activity was assessed.     

Production of a constitutive,extracellular levansucrase fromErwinia herbicola NRRL B-1678

Summary Levansucrase (E.C. 2.4.1.10), which has long been known to be produced intracellularly byErwinia herbicola, was found in relatively high concentrations in the extracellular culture fluid of bacteria grown in various media. Sucrose was not required for enzyme secretion. A medium consisting of corn steep liquor and sorbitol or mannitol gave the highest yields of enzyme.The mention of firm or trade names does not imply endorsement by the USDA or recommendation over other firms or similar products not mentioned.     

Construction of aClostridium acetobutylicum-Escherichia coli shuttle vector

Summary A 4.1-kb cryptic plasmid, designated pCA134, has been isolated fromClostridium species. In order to develop a vector suitable for transforming saccharolytic clostridia three hybrid plasmids were constructed by inserting pCA134 into pHV32 withEcoRI, orBglII andBamHI. The newly constructed plasmids were propagated inEscherichia coli and were used to transformBacillus subtilis andClostridium acetobutylicum. One of them, pCAB32 (10.1 kb), which contains chloramphenicol acetyltransferase gene and an origin of replication derived from pCA134 was introduced intoB.subtilis andC.acetobutylicum as well asE.coli.     

Ethanol production byZymomonas mobilis in continuous culture at high glucose concentrations

Summary Ethanol production byZ.mobilis has been studied in continuous culture with 10, 15 and 20% glucose media. At 10% glucose, steady state conditions were achieved under glucose-limited conditions. At 15 and 20% glucose, the glucose was not fully metabolized even at low dilution rates and oscillatory behavior was evident. It is proposed that ethanol inhibition of growth is responsible for these phenomena. Comparison of kinetic parameters with those from previously published batch data revealed similar values. The maintenance energy coefficient (m) forZ.mobilis was relatively high and was calculated as 1.6 g/g/h for 10% glucose and 3.1 g/g/h for 15% glucose.     

Somatic embryogenesis and organogenesis in callus cultures of a tree legume — Albizia richardiana King

Hypocotyl explants of 1 and 10 mm lengths were excised from 12-day-old in vitro-grown seedlings of Albizia richardiana. The larger pieces, after 40 days of culture, developed shoots along with green calli on B₅ + BAP (10^–7–10^–5M), while the smaller segments produced only green calli on B₅+BAP (10^–7–10^–4M) medium. Some of the green calli turned morphogenic and started producing somatic embryos with the 2nd sub-culture and shoots from 7th sub-culture onwards. Calli retained the morphogenic potential even after repeated sub-culturing for over two years. The number of embryos in an embryogenic culture varied from 2 to 20 per callus mass of 5–6.5 cm³. Sucrose at the 2% level in MS medium was optimal for embryogenesis while 4% was optimal for shoot bud differentiation. Higher levels of sucrose (6–10%) caused browning of green calli and also inhibited differentiation into embryos and shoot buds. By selective sub-culturing of 0.1 cm³ pieces of embryogenic calli on MS+10^–5M BAP, 46% of the cultures produced somatic embryos. The latter germinated into plantlets on Knop's medium.Abbreviations BAP 6-benzylaminopurine - B₅ Gamborg et al., 1968 medium - IAA Indole-3-acetic acid - MS Murashige and Skoog's (1962) medium     

Increased productivity of <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> fermentation of acetone,butanol, and ethanol by pervaporation through supported ionic liquid membrane

Pervaporation proved to be one of the best methods to remove solvents out of a solvent producing Clostridium acetobutylicum culture. By using an ionic liquid (IL)-polydimethylsiloxane (PDMS) ultrafiltration membrane (pore size 60 nm), we could guarantee high stability and selectivity during all measurements carried out at 37°C. Overall solvent productivity of fermentation connected with continuous product removal by pervaporation was 2.34 g l⁻¹ h⁻¹. The supported ionic liquid membrane (SILM) was impregnated with 15 wt% of a novel ionic liquid (tetrapropylammonium tetracyano-borate) and 85 wt% of polydimethylsiloxane. Pervaporation, accomplished with the optimized SILM, led to stable and efficient removal of the solvents butan-1-ol and acetone out of a C. acetobutylicum culture. By pervaporation through SILM, we removed more butan-1-ol than C. acetobutylicum was able to produce. Therefore, we added an extra dose of butan-1-ol to run fermentation on limiting values where the bacteria would still be able to survive its lethal concentration (15.82 g/l). After pervaporation was switched off, the bacteria died from high concentration of butan-1-ol, which they produced.     

Breeding by protoplast fusion for glucoamylase production

Summary Protoplast fusion was carried out between two strains ofAspergillus niger 8-2, a fast growing culture and poor producer of glucoamylase enzyme andA.niger 8-7, a slow growing culture and good producer of the enzyme. The nonconidiating fused mass in presence of benomyl, produced fast growing segregants showing various combinations of the two parental gene markers. Some of the segregants produced up to 68% more glucoamylase than the better yielding parent 8-7.     

Use of an electrochemical technique to study plasmamembrane redox reactions in cultured cells of Daucus carota L.

By exploiting the electron transfer reactions of ferricyanide-ferrocyanide at a gold electrode, an electrochemical technique was devised and successfully employed for the measurement of extracellular ferricyanide reduction or ferrocyanide oxidation by carrot (Daucus carota L.) cells grown in suspension culture. This technique eliminated problems of cell damage and insensitivity encountered when these activities are measured spectrophotometrically in magnetically stirred cell suspensions. Cells harvested from the mid-exponential phase of culture growth catalysed a rapid reduction of ferricyanide which was accompanied by H⁺ extrusion and was stimulated by ethanol. These cells also oxidised ferrocyanide, which was not associated with H⁺ extrusion. These observations can be explained on the basis of a plasmamembrane located, H⁺-translocating redox system. The electrochemical technique is a useful method of studying this system.     

Batch and membrane-assisted cell recycling in ethanol production byCandida shehatae

Summary During xylose fermentation byCandida shehatae ATCC 22984 with batch cell recycling, the volumetric ethanol fermentation rate increased two-fold, and the xylitol production rate increased three-fold as the cell density increased to ten-fold. In continuous fermentation with membrane-assisted cell recycle, the fermentation rates increased almost linearly with increasing agitation rates up to 300 rpm. The maximum continuous ethanol production rates obtained with 90 and 200 g L^–1 xylose were respectively 2.4 and 4.4 g L^–1h^–1. The cell density was 65–70 g (dry wt) L^–1. Ethanol yields ranged from 0.26 to 0.41 g g^–1.     

Control of Carbon and Electron Flow in Clostridium acetobutylicum Fermentations: Utilization of Carbon Monoxide to Inhibit Hydrogen Production and to Enhance Butanol Yields

Extracts prepared from non-solvent-producing cells of Clostridium acetobutylicum contained methyl viologen-linked hydrogenase activity (20 U/mg of protein at 37°C) but did not display carbon monoxide dehydrogenase activity. CO addition readily inhibited the hydrogenase activity of cell extracts or of viable metabolizing cells. Increasing the partial pressure of CO (2 to 10%) in unshaken anaerobic culture tube headspaces significantly inhibited (90% inhibition at 10% CO) both growth and hydrogen production by C. acetobutylicum. Growth was not sensitive to low partial pressures of CO (i.e., up to 15%) in pH-controlled fermentors (pH 4.5) that were continuously gassed and mixed. CO addition dramatically altered the glucose fermentation balance of C. acetobutylicum by diverting carbon and electrons away from H₂, CO₂, acetate, and butyrate production and towards production of ethanol and butanol. The butanol concentration was increased from 65 to 106 mM and the butanol productivity (i.e., the ratio of butanol produced/total acids and solvents produced) was increased by 31% when glucose fermentations maintained at pH 4.5 were continuously gassed with 85% N₂-15% CO versus N₂ alone. The results are discussed in terms of metabolic regulation of C. acetobutylicum saccharide fermentations to achieve maximal butanol or solvent yield.