Continuous acetone-butanol fermentation: a global approach for the improvement in the solvent productivity in synthetic medium

Summary The present chemostat culture studies were performed in a partial gas recycle system using Clostridium acetobutylicum ATCC 824. Initiation of chemostat cultures at two different levels of vitamins has demonstrated a more than 3 fold improvement in the solvent productivity. The production of extracellular autobacteriocin was increased markedly, when strict anaerobic conditions of the feed vessel were not maintained. When pH was regulated at 4.4, very little variation was seen in the concentration of acids. The duration of solvent production was reduced significantly when NH₄OH was replaced by NaOH for pH regulation, whereas a marked increase in the production of extracellular autobacteriocin was observed. In the optimised medium conditions, a solvent productivity of 2.09 g l^-1 h^-1 (highest ever) could be obtained in the synthetic medium.     

Establishment of Orthosiphon stamineus Cell Suspension Culture for Cell Growth

Callus of Orthosiphon stamineus could be induced successfully from petiole, leaf and stem tissues but not roots when cultured on MS medium containing different concentration of NAA (0–4.0 mg l^–1) and 2,4-D (0–2.0 mg l^–1). Highest fresh weight callus production was obtained from leaf explants and those with best friability were obtained on MS medium plus 1.0 mg l^–1 2,4-D plus 1.0 mg l^–1 NAA. Cell suspension cultures were established from these cultures. The appropriate cell inoculum size for the best cell growth was 0.75 g of cells in 20 ml culture medium. Cell suspension culture using MS medium supplemented with 1.0 mg l^–1 2,4-D promoted the best cell growth with maximum biomass of 8.609 g fresh weight and 0.309 g dry weight 24 days after inoculation. Cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D reached the stationary growth phase in 15 days as compared to the cells that grew in MS medium supplemented with 1.0 mg l^–1 2,4-D + 1.0 mg l^–1 NAA reached the stationary phase in 24 days. MS medium supplemented with 1.0 mg l^–1 2,4-D was considered as the maintenance medium for maintaining the optimum cell growth of O. stamineus in the cell suspension cultures with 2-week interval subculture.     

Evaluation of solvents for extractive butanol fermentation with Clostridium acetobutylicum and the use of poly(propylene glycol) 1200

Summary In an effort to improve the viability of acetone-butanol-ethanol fermentation by extractive fermentation, 63 organic solvents, including alkanes, alcohols, aldehydes, acids, and esters, were experimentally evaluated for biocompatibility with Clostridium acetobutylicum by observing gas evolution from cultures in contact with candidate solvents. Thirty-one of these solvents were further tested to determine their partition coefficient for butanol in fermentation medium. The biocompatible solvent with the highest partition coefficient for butanol (4.8), was poly(propylene glycol) 1200, which was selected for fermentation experiments. This is the highest partition coefficient reported to date for a biocompatible solvent. Extractive fermentations using concentrated feeds were observed to produce up to 58.6 g·l^–1 acetone and butanol in 202 h, the equivalent of three control fermentations in a single run. Product yields (based on total solvent products and glucose consumed) of 0.234 g·g^–1 to 0.311 g·g^–1 and within run solvent productivities of 0.174 g·l^–1·h^–1 to 0.290 g·l^–1·h^–1 were consistentwith conventional fermentations reported in the literature. The extended run-time of the fermentation resulted in an overall improvement in productivity by reducing the fraction of between-run down-time for fermentor cleaning and sterilization.     

Degradation of 3,4-dichloroaniline in synthetic and industrially produced wastewaters by mixed cultures freely suspended and immobilized in a packed-bed reactor

Summary Degradation of 3,4-dichloroaniline (34DCA) in aqueous by undefined cultures of free and immobilized cells was examined. Batch cultures of freely suspended cells and continuous degradation in a packed-bed reactor were studied using both synthetically concocted and industrially produced waste-waters. 34DCA was found to be degraded with a concomitant evolution of chloride ions into the bulk medium. The [acked bed reactor with biomass immobilized on celite diatomaceous earth was found to be capable of degrading over 98% of the 34DCA present in a synthetically concocted inlet stream at a concentration of 250 mg l^–1. Residence times of less than 4 h were employed, giving an overall volumetric degradation rate for the packed bed of 90 mg l^–1 h^–1. The industrially produced wastewater contained, in addition to 34DCA, aniline, 4-chloroaniline, 2,3-dichloroaniline (23DCA) and 3,4-dichloronitrobenzene. The biomass enriched on the synthetic 34DCA waste-water was found to be capable of degrading these compounds in addition to 34DCA with the exception of 23DCA. 34DCA degradation efficiencies of over 95% were obtained for the industrial waste-water at reactor residence times of 4.6 h, giving volumetric degradation rates of 24 mg l^–1 h^–1. Offprint requests to: A. G. Livingston     

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.     

Improvement of cephalomanine production in Taxus chinensis cells by a combination of sucrose and methyl jasmonate

Cell suspension cultures of Taxus chinensis, supplemented with 25 g sucrose l^–1, produced 11 mg cephalomanine l^–1, 21 g biomass l^–1 and 19 nkat geranylgeranyl diphosphate (GGPP) synthase activity g protein^–1. Supplementation of the cultures with 100 M methyl jasmonate (MJA) produced 17 mg cephalomanine l^–1, 6 g biomass l^–1 and 78 nkat GGPP synthase activity g protein^–1. Addition of sucrose and MJA together produced 24 mg cephalomanine l^–1, 18 g biomass l^–1 and 55 nkat GGPP synthase activity g protein^–1.     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Production of vitamin B12 in an upflow anaerobic filter continuous reactor using Acetobacterium sp.

The accumulation of biofilm by Acetobacterium sp. during continuous culture in an upflow anaerobic filter (UAF) growing on methanol-formate was the result of space velocity and inlet concentrations of substrate and Co⁺². To achieve good development of biofilm, a space velocity of 0.38 h^–1, inlet substrate concentrations of 125 mM of both methanol and formate, and Co⁺² at 0.16 mM were required. Cell productivities in the effluent of the UAF-reactor were about 6-fold higher than in chemostat cultures (0.20 g l^–1 h^–1 for UAF and 0.035 g l^–1 h^–1 for chemostat) (previous studies), and the maximum vitamin B₁₂ specific concentration was 5.1 mg g cell^–1.     

Aerobic nitrate and nitrite reduction in continuous cultures of Escherichia coli E4

Nitrate and nitrite was reduced by Escherichia coli E4 in a l-lactate (5 mM) limited culture in a chemostat operated at dissolved oxygen concentrations corresponding to 90–100% air saturation. Nitrate reductase and nitrite reductase activity was regulated by the growth rate, and oxygen and nitrate concentrations. At a low growth rate (0.11 h^–1) nitrate and nitrite reductase activities of 200 nmol · mg^–1 protein · min^–1 and 250 nmol · mg^–1 protein · min^–1 were measured, respectively. At a high growth rate (0.55 h^–1) both enzyme activities were considerably lower (25 and 12 nmol mg^–1 · protein · min^–1). The steady state nitrite concentration in the chemostat was controlled by the combined action of the nitrate and nitrite reductase. Both nitrate and nitrite reductase activity were inversely proportional to the growth rate. The nitrite reductase activity decreased faster with growth rate than the nitrate reductase. The chemostat biomass concentration of E. coli E4, with ammonium either solely or combined with nitrate as a source of nitrogen, remained constant throughout all growth rates and was not affected by nitrite concentrations. Contrary to batch, E. coli E4 was able to grow in continuous cultures on nitrate as the sole source of nitrogen. When cultivated with nitrate as the sole source of nitrogen the chemostat biomass concentration is related to the activity of nitrate and nitrite reductase and hence, inversely proportional to growth rate.     

Stevioside biosynthesis by callus,root, shoot and rooted-shoot cultures in vitro

Leaf explants of Stevia rebaudiana Bertoni (Compositae), an herb which produces the sweet ent-kaurene glycoside stevioside, were cultured in Murashige and Skoog medium with vitamins, sucrose (30 g l^–1), agar (0.9% w/v) and supplemented with naphthaleneacetic acid (NAA, 0.5 mg l^–1) and benzylaminopurine (BAP, 0.5 mg l^–1). These conditions yielded friable callus cultures. Differentiation of the callus tissue was then achieved by eliminating the agar and modulating the medium's hormone concentrations. Thus, medium containing increased auxin concentration (1.0 mg l^–1) and no cytokinin or increased cytokinin (1.0 mg l^–1) and no auxin yielded root or shoot cultures respectively. Supplementation of the shoot medium with NAA (1.0 mg ml^–1) induced shoot cultures to grow roots thereby differentiating into rooted-shoot cultures. Only the rooted-shoot cultures tasted sweet. Feedings of [2-¹⁴C]acetic acid to callus, shoot or rooted-shoot cultures demonstrated that only the rooted-shoot cultures are capable of de novo biosynthesis of the aglycone moiety of stevioside (steviol). In addition, [methyl-³H(N)steviol feedings to shoot or rooted-shoot cultures illustrated that both types of cultures are capable of the glycosylation reaction. The ability of these tissues to glycosylate steviol to stevioside was also demonstrated employing crude enzyme preparations derived from shoot or rooted-shoot cultures. These results suggest that stevioside biosynthesis is a function of tissue differentiation since both roots and leaves are required for cultured S. rebaudiana to biosynthesize stevioside from acetate, while the final biosynthetic steps can be performed at all levels of differentiation.     

Sequential kinetic parameter estimation of anaerobic fluidized bed bioreactor using an optimization technique

Mathematical model parameters for the methanogenic degradation of propylene glycol were estimated in a sequential manner by means of an optimization technique. Model parameters determined from an initial experimental data set using one bioreactor were then verified with the results from a second bioreactor. The proposed methodology is a useful tool to obtain model parameters for continuous flow reactors with completely mixed regime. Abbrevations: S – substrate concentration (mg COD l^–1); S _in – influent substrate concentration (mg COD l^–1); D _L – dilution rate (day^–1); – stoichiometric coefficients (ND); nx – number of microbial species (ND); X _S – fixed biomass concentration (mg biomass l^–1); X _L – suspended biomass concentration of (mg biomass l^–1); k _d – decay rate of biomass (day^–1); b _S – specific detachment rate of biofilm (day^–1); – specific growth rate of biomass (day^–1); _m – maximum specific growth rate of biomass (day^–1); K _S – half saturation constant (mg COD l^–1); K _I – inhibition constant (mg COD l^–1).     

Comparative investigations of growth and solvent formation in ‘Clostridium saccharoperbutylacetonicum’ DSM 2152 and Clostridium acetobutylicum DSM 792

The butanol and acetone-producing strain DSM 2152, invalidly described as ‘Clostridium saccharoperbutylacetonicum’ is compared with the type strain C. acetobutylicum, DSM 792, with respect to solvent and acid formation at varying pH values and growth rates. Batch cultures, product-limited chemostat and pH-auxostat cultures were used for characterization. Under all conditions strain DSM 2152 produced much lower amounts of butyric and acetic acids than the type strain. The pH optimum for solvent formation was higher, ie 5.5 instead of 4.5. Solvent formation occurred at higher dilution rates, but below 0.1 h⁻¹ a lower solvent concentration was obtained, indicating that acid production was too low to provide a sufficient amount for acetone formation. The results are discussed in the light of recent publications on the taxonomy of butanol-acetone producing clostridia using 16S rRNA sequence analysis and other nucleic acid data. The presently suggested ‘phylogenetic’ classification of the collective species, C. acetobutylicum, is also reflected in the fermentation characteristics. Received 21 December 1998/ Accepted in revised form 22 January 1999     

Denitrification kinetics of simulated fish processing wastewater at different ratios of nitrate to biomass

Denitrification was studied in anoxic batch cultures of a simulated fish processing wastewater at 37 r C and pH 7.5, using a denitrifying enrichment culture from fishery wastewater. Different initial nitrate to biomass ratios (So/Xo) were used: nitrate and biomass varied from 7.5 to 94.7 mg NO₃-N l^–1, and from 20 to 4300 mg volatile suspended solids l^–1, respectively. The specific maximum denitrification rate (r _m) and the cell yield (Y _{X / S}) depended on the So/Xo ratio under anoxic conditions: r _m increased from 1.2 to 1584 mg NO₃-N g^–1 VSS h^–1 and Y _{X / S} decreased from 42 to 0.03 mg VSS mg^–1 NO₃-N when So/Xo varied from 5.5 10^{– 3} to 9.3 mg NO₃-N/mg VSS. Nomenclature C_{NO3 – N} nitrate concentration, mg NO₃-N l^–1 K _S saturation constant, mg NO₃-N l^–1 r _m specific maximum denitrification rate, mg NO₃-N g^–1 VSS h^–1 So initial substrate concentration, mg l^–1 t time, h TOC total organic carbon VSS volatile suspended solids x biomass concentration, g VSS l^–1 Xo initial biomass concentration, g VSS l^–1 Y _X/S substrate to biomass cell yield, mg VSS/mg N Greek symbols: _m maximum specific growth rate of the anoxic microbial population, 1 h^–1     

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l^–1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l^–1.     

Production of acetone,butanol and ethanol (ABE) from potato wastes: fermentation with integrated membrane extraction

The technical possibilities of the microbial production of acetone, butanol and ethanol (ABE) from potato waste using in-line solvent recovery, are evaluated. Clostridium acetobutylicum DSM 1731 produces up to 20 g·l^–1 of solvents when grown on a medium containing 14% (w/v) potato powder. Using a polypropylene perstraction system and a oleyl alcohol/decane mixture as the extractant, the product yield (based on total solvents and potato dry weight) increased from 0.13 g·g^–1 to 0.23 g·g^–1. The recovery system worked well for 50 h, after which membrane fouling frustrated proper operation. In the second system a microfiltration step was incorporated whereas the solvents were extracted through a hydrophilic membrane using fatty acid methyl esters from sunflower oil as an extractant. This process configuration resulted in a comparable increase of ABE production. Correspondence to: G. Eggink     

Improvement of indole alkaloid production in Catharanthus roseus cell cultures by osmotic shock

Catharanthine production in Catharanthus roseussuspension cell cultures was increased by about 4-fold to 28 mg l^–1, 23 mg l^–1and 24 mg l^–1by adding sodium alginate, mannitol or polyvinyl pyrrolidone, respectively. Sodium alginate and polyvinyl pyrrolidone also enhanced ajmalicine production to 28 mg l^–1and 31 mg l^–1, respectively. Up to 55–70% of the total alkaloids were released into the medium. These treatments could stimulate higher alkaloid production in C. roseuscell cultures than NaCl and KCl stresses. The possible mechanisms for these treatment effects are discussed.     

Plant regeneration from suspension cultures of Hosta sieboldiana

Systems for establishing suspension cultures and for inducing plant regeneration from these cultures for the Liliaceous ornamental plant, Hosta sieboldiana (Lodd.) Engl. have been developed. Pale-yellow and nodular calluses were induced from more than 20% of scape segments on MS medium containing 1 mg l^–1 picloram (PIC), 30 g l^–1 sucrose, and 2 g l^–1 gellan gum. Upon transfer of calluses to the same medium lacking gellan gum, stably-growing suspension cultures were established after 1 month. Suspension cell clusters regenerated a large number of adventitious shoots following transfer to MS media containing 0.1 mg l^–1 NAA in combination with either BA or TDZ. Over 20 shoots per 0.3 g FW of cell clusters were obtained on media containing 0.1 mg l^–1 NAA and either 1 or 5 mg l^–1 TDZ. Shoots rooted easily on plant growth regulator (PGR)-free MS medium, and plantlets were successfully transferred to soil. Plants showed no visible morphological alterations and maintained the diploid level as indicated by flow cytometric analysis.     

Fungal biotransformation of p-coumaric acid into caffeic acid by Pycnoporus cinnabarinus: an alternative for producing a strong natural antioxidant

Fungal biotransformation of p-coumaric acid into caffeic acid, potentially a strong antioxidant, was evidenced in Pycnoporus cinnabarinus cultures grown with high feeding of p-coumaric acid. Preliminary experiments showed no toxicity of both p-coumaric and caffeic acids at concentrations ranging from 0 to 500 mg l^–1. Feeding 450 mg p-coumaric acid l^–1 into P. cinnabarinus cultures grown on 20 g l^–1 glucose medium resulted in the production of 257 mg caffeic acid l^–1with a molar yield of 21%.     

Calli and suspension cultures for biomass production ofEuphorbia characias L. subsp.characias

Summary Friable calli and cell suspension cultures were obtained from leaf segments ofEuphorbia characias L. subsp.characias, in Murashige and Skoog (MS) basal medium supplemented with 1g.l^–1 casein hydrolyzate (CH), 5mg.l^–1 ascorbic acid, 1.0mg.l^–1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.75mg.l^–1 benzyl adenine (BA). The highest callus specific growth rate (=0.085.day^–1), calculated for 1 year old calli cultures, was obtained with 0.25 mg.l^–1 2,4-D and 0.50mg.l^–1 BA. Suspension cultures started with an inoculum of 8.0×10⁴ cells.ml^–1 in supplemented liquid MS medium, gave a specific growth rate =0.256.day^–1.     

Improvement of the culture stability of non-anchorage-dependent animal cells grown in serum-free media through immobilization

A murine hybridoma cell line producing a monoclonal antibody against penicillin-G-amidase and a murine transfectoma cell line secreting a monovalent chimeric human/mouse Fab-antibody fragment were cultivated in three different media (serum-containing, low protein serum-free, and iron-rich protein-free) in flask cultures, stirred reactors and a fixed bed reactor. In static batch cultures in flasks both cell lines showed similar good growth in all three media.In suspension in a stirred reactor, the hybridoma cell line could be cultivated satisfactory only in serum-containing medium. In low protein serum-free medium, Pluronic F68 had to be added to protect the hybridoma cells against shear stress. But even with this supplement only batch, not chemostat mode was possible. In iron-rich protein-free medium the hybridoma cells grew also in continuous chemostat mode, but the stability of the culture was low. The transfectoma cell line did not grow in stirred reactors in any of the three media.Good results with both cell lines were obtained in fixed bed experiments, where the cells were immobilized in macroporous Siran^®-carriers. The media, which were optimized in flask cultures, could be used without any further adaptation in the fixed bed reactor. Immobilization improved the stability and reliability of cultures of non-adherent animal cells in serum-free media tremendously compared to suspension cultures in stirred reactors. The volume-specific glucose uptake rate, an, indicator of the activity of the immobilized cells, was similar in all three media. Deviations in the metabolism of immobilized and suspended cells seem to be mainly due to low oxygen concentrations within the macroporous carriers, where the cells are supplied with oxygen only by diffusion.List of symbols c substrate or product concentration mmol l^–1 - c₀ substrate or product concentration in the feed mmol l^–1 - c_Glc glucose concentration mmol l^–1 - c_Gln glutamine concentration mmol l^–1 - c_Amm ammonia concentration mmol l^–1 - c_Lac lactate concentration mmol l^–1 - c_FAB concentration of Fab# 10 antibody fragment g l^–1 - c_MAb monoclonal antibody concentration mg l^–1 - D dilution rate d^–1 - q cell-specific substrate uptake or metabolite production rate mmol cell^–1 h^–1 - q_Glc cell-specific glucose uptake rate mmol cell^–1 h^–1 - q_Gln cell-specific glutamine uptake rate mmol cell^–1 h^–1 - q_MAb cell-specific MAb production rate mg cell^–1 h^–1 - q^* volume-specific substrate uptake or metabolite production rate mmol l^–1 h^–1 - q^*FB volume-specific substrate uptake or metabolite production rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Glc volume-specific glucose uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - q^*FB,Gln volume-specific glutamine uptake rate related to the fixed volume mmol l_FB ^–1 h^–1 - q^*FB,MAb volume-specific MAb production rate related to the fixed volume mg l_FB ^–1 h^–1 - q^*FB,02 volume-specific oxygen uptake rate related to the fixed bed volume mmol l_FB ^–1 h^–1 - t time h - U superficial flow velocity mm s^–1 - V medium volume in the conditioning vessel of the fixed bed reactor l - V_FB volume of the fixed bed l - x_v viable cell concentration cells ml^–1 - y_Amm,Gln yield of Ammonia from glutamine - y_Lac,Glc yield of lactate from glucose - specific growth rate h^–1 - _d specific death rate h^–1