Vertical Rotating Immobilized Cell Reactor of the bacteriumZymomonas mobilis for stable long-term continuous ethanol production

Summary Vertical Rotating Immobilized Cell Reactor was designed and built for glucose conversion into ethanol. Immobilized biomass units withZ. mobilis cells attached into polyurethane foam discs were fixed along a rotating shaft inside the bioreactor. The effect of rotation speed on the concentration of immobilized biomass was studied. Stability of the bioreactor over long-term operation was dependent on the concentration of the immobilized biomass. With fermentation carried out at 6 rpm a constant active immobilized cell concentration of only 34.5 g/l was maintained and used to convert up to 140 g glucose/l into more than 70 g ethanol/l with a volumetric ethanol productivity of 63 g/l/h.     

Ethanol production from Jerusalem artichoke juice using flocculent cells of Kluyveromyces marxianus

Summary Flocculent cells ofKluyveromyces marxianus SM 16-10 were used for batch production of ethanol from the inulin sugars derived from Jerusalem artichoke tubers. Using 20% initial sugar concentration, a maximum ethanol concentration of 92 g/l was achieved in 7 h, when the flocculent cell concentration was 30 g dry wt./l bioreactor volume. The same flocculent cells were used repeatedly for 7 batch runs starting with fresh medium at the beginning of each run. The ethanol yield was found to be almost constant at about 94% of the theoretical for all the 7 batch cycles, while the maximum ethanol production rate increased from 17.21 g ethanol/1/h during the first batch run to 21 g ethanol/1/h during the last batch run.     

Accumulation of silver by Chromatium vinosum from solutions containing silver thiosulfate

The photosynthetic sulfur bacterium, Chromatium vinosum, was cultured in inorganic photographic processing solutions containing silver thiosulfate complex salt (AgNa₃(S₂O₃)₂) under light. It was found that Chromatium was resistant to Ag and accumulated granular silver in the membrane during growth. The amount of Ag accumulated in the cells depended on the initial concentrations of the Ag salt in the culture solution. When the concentration of Ag was 300 mg/l, the bacteria accumulated Ag as high as 30% of the dry cell weight. The size of the granules was 0.1 to 0.3 m. Results from X-ray microanalysis indicated that these granules consisted mostly of Ag^o with small fractions of Ag₂S and AgCl.     

Comparative studies of the phytochrome system in cell cultures from different plants

Phytochrome contents have been assayed in vivo in cell suspension cultures of Petroselinum hortense, Daucus carota and Glycine max. After transferring the cells to fresh medium phytochrome increased in parallel with the increase in cell number, whereas the amount of phytochrome per cell remained constant. The rate of phytochrome reaccumulation after pretreatment with 15 h red light was very similar in all three systems (2.8–3.6 (e) 10^–5/h). Dark reversion and a fast and slow P_fr destruction were observed in all systems. The rate constants of these reactions varied strongly between the systems. The phytochrome systems of the cell cultures were compared with those of etiolated and light-grown seedlings and it was concluded that the cell suspension cultures of Petroselinum hortense and Daucus carota behaved similarly to light-grown seedlings. In contrast, those of Glycine max behaved similarly to a dark grown seedling.Abbreviations P_r'fr red, far-red absorbing forms of phytochrome - P_tot P_r+P_fr total amount of phytochrome - fwt fresh weight     

High productivity continuous ethanol fermentation with a flocculating mutant strain of Zymomonas mobilis

High fermenter (volumetric) ethanol productivities (80 g/lh^–1) were attained in a simple single-stage continuous-stirred-tank-reactor (CSTR) employing a flocculent mutant of Zymomonas mobilis with a feed containing 100g/l glucose. Under these conditions a final ethanol concentration of 47.6 g/l was obtained, representing a maximum conversion efficiency of 97% of theoretical.Nomenclature SR = Medium glucose concentration (g/l)X Biomass concentration (g/l) - P Ethanol concentration (g/l) - VP Volumetric productivity (g ethanol/l/h) - Yp/s Product yield coefficient (g ethanol/g glucose consumed) - Qp Specific rate of ethanol formation (g ethanol/g cells/h) - D Dilution rate (h^–1) - Dmax Maximum dilution rate: ie., highest dilution rate at which the effluent glucose concentration 4g/l (h^–1)     

Kinetic studies on alac-containing strain ofZymomonas mobilis

Summary Batch and continuous culture studies have been carried out on a strain ofZ.mobilis (ZM6306) which can convert lactose directly to ethanol. Previous strain development has established that thelac operon encoded on the transposon Tn951 can be expressed inZ.mobilis. Using a medium containing 80 g/l glucose and 40 g/l lactose, it was found that strain ZM6306 could convert about 13 g/l lactose to 4 g/l ethanol and 6 g/l galactose in continuous culture. Further lactose conversion is likely with increased cell concentration using a cell recycle system.     

Potential of Datura innoxia cell suspension cultures for glucosylating hydroquinone

The efficiency of glucosylation of hydroquinone by Datura innoxia cell suspension cultures was investigated. The yield of arbutin was 2.4 g/l medium when 10 mM of hydroquinone was added to a suspension culture that was then incubated for 24 h, but the yield decreased at a higher concentration. This decrease, which could not be overcome by changing the growth phase or increasing the cell density used, could be avoided by the repeated addition of a low concentration of hydroquinone over 3 days. This increased the yield of arbutin to 4.2 g/l at the usual cell density and to 7.1 g/l at a high density. The kinetics of this reaction were explained by the Michaelis-Menten formula. The theoretical maximum velocity of the arbutin-forming reaction was estimated as 0.77 mg/h/g. The velocity increased linearly up to a cell density of 300 g/l under standard aeration, the theoretical maximum yield of arbutin being calculated to be 5.5 g/l/day.     

High ethanol productivities using small Ca-alginate beads of immobilized cells of Zymomonas mobilis

Summary Zymomonas mobilis cells were immobilized into small 1 mm diameter beads of Ca-alginate in order to minimize mass transfer limitations and maximize immobilized cell activity. A combination of small bead size with a high cell concentration of 58 g dry wt. cell per lit. bead volume resulted in high ethanol productivities using a newly designed packed bed bioreactor system. Steady-state dilution rates ranging from 0.4 h^-1 to 3.9 h^-1 were run resulting in a maximum productivity of 102 g ethanol/l/h for an inlet substrate concentration of 100 g glu/l and 87% conversion. The bioreactor was run continuously at a fixed dilution rate for 384 h and short intermittent treatment of the beads with CaCl₂ temporarily increased ethanol productivity to a maximum of 116 g ethanol/l/h.     

Continuous production of acrylamide byBrevibacterium sp. immobilized in a dual hollow fiber bioreactor

Summary Acrylamide was continuously produced from acrylonitrile usingBrevibacterium sp. CHl grown and immobilized in a dual hollow fiber bioreactor of 8.0 cm³. The biomass reached as high as 200 gm/L of the space available for the cell growth. The volumetric productivity of the reactor was 88 gm/L. h and the conversion of acrylonitrile varied with acrylonitrile concentration, pH and feed rate.     

Glycerol production by immobilised cells of Pichia farinosa

Summary Cells ofPichia farinosa were immobilised in calcium alginate and K-Carrageenan and their ability to produce glycerol from glucose under aerobic conditions with acidic as well as alkaline pH was investigated. An average glycerol production rate of 0.07 g/l.h was obtained with immobilised cells (IMC) in shake flasks. Continuous glycerol production in a fluidised bed reactor (FBR) under steady state operation gave a glycerol concentration of 13.5 g/l in the product stream.     

Growth of baculovirus-infected insect cells in microcapsules to a high cell and virus density

Summary Insect cells (Spodoptera Frugiperda), infected with a temperature-sensitive mutant (TS10) of theAutographa Californica nuclear polyhedrosis virus (AcNPV), were cultured in multiple membrane alginate-polylysine (PLL) microcapsules. It was possible to obtain intracapsular cell densities of 8× 10⁷ cells/mL of capsules and virus concentrations of up to 10⁹ IFU/mL of capsules. This was higher by a factor of 10 than that which could be achieved by conventional cell suspension culture.     

Enzyme production in a cell recycle fermentation system evaluated by computer simulations

Enzyme production in a cell recycle fermentation system was studied by computer simulations, using a mathematical model of -amylase production by Bacillus amyloliquefaciens. The model was modified so as to enable simulation of enzyme production by hypothetical organisms having different production kinetics at different fermentation conditions important for growth and production. The simulations were designed as a two-level factorial assay, the factor studied being fermentation with or without cell recycling, repression of product synthesis by glucose, kinetic production constants, product degradation by a protease, mode of fermentation, and starch versus glucose as the substrate carbon source.The main factor of importance for ensuring high enzyme production was cell recycling. Product formation kinetics related to the stationary growth phase combined with continuous fermentation with cell recycling also had a positive impact. The effect was greatest when two or more of these three factors were present in combinations, none of them alone guaranteeing a good result. Product degradation by a protease decreased the amount of product obtained; however, when combined with cell recycling, the protease effect was overshadowed by the increased production. Simulation of this type should prove a useful tool for analyzing troublesome fermentations and for identifying production organisms for further study in integrated fermentation systems.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - c starch concentration (g/l) - c ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - e intrinsic intracellular amylase concentration (g product/g cell mass) - E extracellular amylase concentration (g/l) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA/cell - K ₁ intracellular repression constant - K ₂ intracellular repression constant - K _s Monod saturation constant (g/l) - k ₃ product excretion rate constant (h^–1) - k _I translation constant (g product/g mRNA/h) - k _d first order decay constant (h^–1) - k _dw first order decay constant (h^–1) - k _gl rate constant for glucose production (g/l/h) - k _{m, dgr} saturation constant for product degradation (g/l) - k _st rate constant for starch hydrolysis (g/l/h) - k _t1 proportionality constant for amylase production (g mRNA/g substrate) - k _t2 proportionality constant for amylase production (g mRNA *h/g substrate) - k _w protease excretion rate constant (h^–1) - k _wt1 proportionality constant for protease production (g mRNA/g substrate) - k _wt2 proportionality constant for protease production (g mRNA *h/g substrate) - k _wI translation constant (g protease/g mRNA/h) - m maintenance coefficient (g substrate/g cell mass/h) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function, K₁/K₂ less than or equal to 1.0 - Q _w repression function, K₁/K₂ less than or equal to 1.0 - r intrinsic amylase mRNA concentration (g mRNA/g cell mass) - r _m intrinsic protease mRNA concentration (g mRNA/g cell mass) - R _ex retention by the filter of the compounds x=: C starch, E amylase, or S glucose - R _t amylase transport rate (g product/g cell mass/h) - R _wt protease transport rate (g protease/g cell mass/h) - R _s rate of glucose production (g/l/h) - R _c rate of starch hydrolysis (g/l/h) - S ₀ feed concentration of free reducing sugar (g/l) - s extracellular concentration of reducing sugar (g/l) - t time (h) - V volume (1) - w intracellular protease concentration (g/l) - W extracellular protease concentration (g/l) - X cell mass concentration (dry weight) (g/l) - Y yield coefficient (g cell mass/g substrate) - substrate uptake (g substrate/g cell mass/h) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) - _m,dgr maximum specific rate of amylase degradation (h^–1) This study was supported by the Nordic Industrial Foundation Bioprocess Engineering Programme and the Center for Process Biotechnology, The Technical University of Denmark.     

Production of hirudin by recombinant Saccharomyces cerevisiae in a membrane-recycle fermentor

Summary Recombinant Saccharomyces cerevisiae was employed to continuously produce hirudin in a membrane cell recycle fermentor. The gene cooing for the anticoagulant protein was combined with the GAL10 promoter for controlled expression and the MF 1 signal sequence for secretion to the fermentation broth. A dilution rate of 0.1h^–1 yielded a maximum hirudin concentration of 59mg / l with a specific hirudin concentration of 2.4 mg /g cell mass among dilution rates studied ranging from 0.05h^–1 to 0.3h^–1. Cell bleeding gave the same fermentation results as cell recycle fermentation without cell bleeding. The productivity of the cell recycle fermentation process was 6.0mg hirudin/l · hr, corresponding to a 1.7-fold increase compared with a conventional continuous culture.     

Continuous ethanol production by immobilized cells of Zymomonas mobilis

Summary Studies have been carried out with a highly productive strain of Zymomonas mobilis in an immobilized cell reactor using both Ca alginate and -carrageenan as supporting matrices. Productivities above 50 g/l/h have been found at ethanol concentrations in excess of 60 g/l. With immobilized cells of Z. mobilis, there was a decline of approximately 30s% in activity after 800 h operation.     

Production of L-DOPA from pyrocatechol and DL-serine by bioconversion using immobilized Erwinia herbicola cells

Summary Immobilized cells of Erwinia herbicola were used for L-DOPA production from pyrocatechol and DL-serine. Optimal conditions have been defined and utilized in batch and continuous reactors. A maximal volumetric productivity of 0.46 g/l.h in L-DOPA was obtained with a conversion yield of 18% (L-DOPA concentration 2.3 g/l).     

Kinetic studies on a flocculent strain of Zymomonas mobilis

Summary A flocculent mutant of Zymomonas mobilis has been isolated and kinetic studies carried out in batch and continuous culture. By comparison with the parent strain the specific rates of glucose uptake and ethanol production were decreased by 20%. Cell recycle and semibatch cultures with the flocculent strain resulted in relatively high productivities (viz. 50 g/l/h). However semibatch culture had the additional advantages of an increased ethanol concentration (viz. 82 g/l) and a more stable and controlled environment for cell separation.     

Hydrolysis of cellulose in fed-batch culture by Cl. thermocellum: A way to increase speed of cellulose hydrolysis

Summary Fed batch fermentation ofClostridium thermocellum gives improved conversion rates and high ethanol concentration in the broth; 8.5 g/l of ethanol were obtained in 60 hours with a maximum ethanol productivity of 1.0 g/l h.     

Use of wild type and nickel resistantNeurospora crassa for removal of Ni2+ from aqueous medium

Summary A novel nickel resistant, hyperaccumulatingN.crassa nir-2 mutant, isolated by us, sequestered 90% of Ni²⁺ from medium with 120 mg/l Ni²⁺. The parent wild strain showed comparable efficiency only at much lower concentrations (<10mg/l). The initial rapid rate and efficiency of Ni²⁺ removal could be maintained beyond 2 h by fresh addition of mycelial biomass. The results have been discussed from the stand point of the utility of metal resistant fungi in the control of environmental pollution.     

Growth rate and methane affinity of a turbidostatic and oxystatic continuous culture ofMethylococcus capsulatus (Bath)

Summary A turbidostatic and oxystatic fermentation system was used to study the growth kinetic ofMethylococcus capsulatus (Bath). Dissolved oxygen and methane concentrations were measured continuously with membrane inlet mass spectrometry. The specific growth rate was found to increase from 0.25 h^–1 to 0.37 h^–1 and the saturation constant for methane was found to decrease from 71 M to 1.3 M as the copper content of the medium was varied from a very low to a high value.     

Clonal propagation of Leptospermum spp. by tissue culture

Propagation by axillary and multiple axillary bud development was achieved in three native Leptospermum spp. when axillary buds derived from nodal tissues ex mature plants were placed in benzylaminopurine media (0.04–1.0 M) containing macro- and micro-nutrients, sucrose (0.06 M) and a vitamin/amino acid supplement. Reduction of agar concentration from 0.8 to 0.2% greatly stimulated axillary bud development and growth in L. flavescens and L. brachyandrum. Rooting of axillary shoots was stimulated by 2,4-dichlorophenoxyacetic acid and p-chlorophenoxy acetic acid in L. flavescens at concentrations of 5 and 1 M respectively. In L. petersonii ssp. root initiation and development was favoured by -naphthoxyacetic acid (1 M) and in L. brachyandrum indole butyric acid and -naphthalene acetic acid (1 M) were almost equally effective.