Fed-batch cultivation of Acetobacter suboxydans for the microbial oxidation of D-sorbitol to L-sorbose

Microbial oxidation of D-sorbitol to L-sorbose is commercially important since it is the only biochemical process in Vitamin-C manufacture. The main bottleneck in the batch oxidation of D-sorbitol is that the process is severely inhibited by sorbitol. By conducting fed-batch fermentation, the inherent substrate inhibition present in batch fermentation can be eliminated. Batch fermentation with an initial sorbitol concentration of 200 g l^у featured a productivity of 14.2 g l^у h^у and a final sorbose concentration of 200 g l^у. Fed-batch fermentation conducted by feeding nutrients containing 600 g l^у of sorbitol at a constant feed rate of 0.36 l h^у yielded a productivity of 17.7 g l^у h^у and a final sorbose concentration of 320 g l^у.     

Optimized fed-batch fermentation of L-β-hydroxy isobutyric acid by Yarrowia lipolytica

Yarrowia lipolytica KCCM50506, which transforms isobutyric acid to L-#-hydroxy isobutyric acid (L-#-HIBA), was screened. Chemostat cultures were carried out in jar fermentors at dilution rates of 0.02 h^у to 0.12 h^у. L-#-HIBA fermentation-regulating factors were determined to be specific growth rate, and concentrations of glucose and isobutyric acid in fermentor from analysis of steady-state data. The specific productivity of L-#-HIBA increased as the specific growth rate increased, apparently as a growth-associated type of product formation. A fed-batch culture was carried out under optimum conditions where the concentrations of glucose and isobutyric acid in the fermentor were maintained at 23 g l^у and 9 g l^у, respectively. The concentrations of cells and L-#-HIBA obtained at the end of fermentation were 20 g l^у and 49 g l^у, respectively, corresponding to 2.0 and 2.7 times more than concentrations in batch culture.     

Effect of shear on morphology and erythromycin production in Saccharopolyspora erythraea fermentations

A complex medium was used to investigate the effects of shear on the S. erythraea fermentation at 7-l scale. Maximum biomass was 11.1 - 0.5 g l^у at 1250 rpm (tip speed = 4.45 ms^у), whereas it was 12.7 - 0.2 g l^у at 350 rpm (tip speed = 1.07 ms^у). Specific erythromycin production was not stirrer speed dependent in the range of 350 to 1000 rpm and decreased by 10% at stirrer speed of 1250 rpm. Morphological measurements using image analysis showed that the major axis of the mycelia (both freely dispersed and clumps) decreased after the end of the rapid growth phase to a relatively constant value (equilibrium size) dependent on the stirrer speed. The mechanical properties of the cell wall were examined by disruption of fermentation broth in homogeniser and it was shown that mechanical strength of the cell wall increased in a large extent during deceleration phase.     

Production of manganese peroxidase and laccase in laboratory-scale bioreactors by Phanerochaete chrysosporium

The production of ligninolytic enzymes by the fungus Phanerochaete chrysosporium BKM-F-1767 (ATCC 24725) in laboratory-scale bioreactors was studied. One bioreactor was filled with cubes of polyurethane foam and the other with cubes of nylon sponge, in order to determine the more suitable carrier to produce high ligninolytic enzyme activities by this fungus. Both cultivations were carried out in batch. Manganese-dependent peroxidase activities about 600 U l^у were achieved in the bioreactor filled with cubes of nylon sponge, while up to 500 U l^у were detected in that filled with cubes of polyurethane foam. Furthermore, quite high levels of laccase appeared in both cultures: maximum activities of 114 U l^у and 62 U l^у were obtained on nylon and polyurethane supports, respectively.     

The processing of a plasmid-based gene from E. Coli. Primary recovery by filtration

We describe the primary recovery of plasmid DNA from alkaline lysis mixtures using a nutsche filter operated under pressure. Six different filter cloths constructed of polypropylene, polyester and stainless steel were tested, with pore sizes ranging from 5–160?μm. Both pore size and the material of the filter membranes employed in filtration experiments exerted considerable impact on the purity and yield of the plasmid DNA. The greatest degree of solids extrusion, shearing of chromosomal DNA and subsequent contamination of the filtrate was observed with the 160?μm polyester filter. The best compromise was obtained with a 5?μm polypropylene cloth. For an alkaline lysis mixture containing 101?g wet weight solids per litre, filtration through this cloth proceeded at an average rate of 22.5?cm?h^?1. Virtually complete removal of solids (99.4%) and protein (96.8%) was achieved, with a 8.2-fold purification of plasmid DNA at the expense of a 33% loss in yield. The filtration performance of this membrane was further modified by precoating with diatomaceous earths of different permeabilities (0.07–1.2?darcies). The finest filter aid resulted in very pure plasmid DNA (65%), complete suspended solids removal and ?1), and some losses of plasmid DNA, due to adsorption on to the diatomaceous earth, were also observed (5.7%).     

Biodegradation of ethanol vapour in a biofilter

Biofiltration is a cost effective air pollution control technology for volatile organic compounds. Biofiltration of ethanol vapour from air stream was evaluated in this study. Experimental investigations were conducted on a laboratory scale biofilter, containing mixture of compost and polystyrene inert particles as the filter materials. Mixed consortium of activated sludge was used as a inoculum. The continuous performance of biofilter for ethanol removal was monitored for different concentrations and flow rates. The removal efficiencies decreased at higher concentrations and higher gas flow rates. A maximum elimination capacity of 195 g m^х h^у was achieved. The response of biofilter to shut down and restart-up operation showed that the biofilm has got a good stability.     

Influence of k L a on bioconversion of rice straw hemicellulose hydrolysate to xylitol

The effect of overall oxygen mass transfer coefficient (k_La) on the conversion of xylose to xylitol by Candida guilliermondii FTI 20037 was investigated in batch experiments. Rice straw hemicellulose hydrolysate obtained by acid hydrolysis was employed as a xylose-rich medium. The results showed that this bioconversion strongly depended on the aeration rate. The maximum volumetric productivity (0.52 g/l h^у) and the highest xylitol yield (0.73 g/g) were achieved at an overall oxygen mass transfer coefficient of 15 h^у. Under these conditions 80% efficiency in relation to theoretical yield was attained.     

A novel fiber optic probe for on-line monitoring of biomass concentrations

A fiber optic biomass probe for on-line measurement of biomass concentration was designed. Results of biomass concentration monitoring experiments with suspended cells of baker's yeast as well as an experimental cultivation of S. cerevisiae are presented. The device was able to observe biomass concentrations of 14 g l^у S. cerevisiae. By means of correlations the capability of estimating the biomass concentration from the probe signal is demonstrated.     

Physiological energetics of the protobranch bivalve <Emphasis Type="Italic">Yoldia hyperborea</Emphasis> in a cold ocean environment

Yoldia hyperborea is a deposit-feeding circumpolar protobranch that also inhabits muddy sediments of the cold water boreal system of Conception Bay, Newfoundland, Canada. Little is known about this species, despite its wide distribution and frequent high density in the benthos. The present work deals with oxygen consumption and ammonia excretion under cold ambient conditions. Y. hyperborea showed low basal metabolism [0.051 ml O₂ h^у·(g dry weight)^у, T=у°C] and low ammonia excretion rates [4.212 µg·NH₄-N·h^у·(g dry weight)^у, T=у°C]. Low metabolic activity could prove a useful strategy during periods of low food availability. In addition, Y. hyperborea was able to regulate its O₂ consumption rate at very low pO₂ levels, which may be advantageous for a species that may experience periods of hypoxia.     

Clearance rates in the Arctic bivalves <Emphasis Type="Italic">Hiatella arctica</Emphasis> and <Emphasis Type="Italic">Mya </Emphasis>sp.

Filtration was studied in two Arctic clams, Hiatella arctica and Mya sp., collected in Young Sound, Northeast Greenland. Clearance rates were determined as a function of ambient temperature and algal cell concentration, using the clearance method and feeding with a unicellular flagellate. For both species, clearance rates increased with increasing temperature from <у up to 4-8°C. At higher temperatures, filtration ceased and the clams closed their valves. Clearance rates were also determined in temperate specimens of H. arctica collected on the west coast of Sweden. For these specimens, clearance rates increased with increasing temperature from 0 to 18-20°C. When weight-specific clearance rates were compared between the two populations and between species, there were no differences at 1°C. Clearance rates in Arctic H. arctica were maximal at algal cell concentrations corresponding to 2.5-8 µg chlorophyll a l^-1. Temperature compensation in Arctic bivalves is discussed and it is concluded that adaptations to constant low temperatures consist of a lower minimum temperature, for active filtration. Low clearance rates due to low temperatures did not seem to limit growth, under the prevailing conditions in Young Sound.     

Hypoxia affects root sodium and chloride concentrations and alters water conductance in salt-treated jack pine (<Emphasis Type="Italic">Pinus banksiana</Emphasis>) seedlings

The effects of NaCl were studied in 6-month-old jack pine (Pinus banksiana Lamb.) seedlings growing in solution culture under hypoxic (approximately 2 mg l^у O₂) and well-aerated (approximately 8 mg l^у O₂) conditions. The results showed that hypoxia led to further reduction of stomatal conductance (g_s) in plants treated with 45 mM NaCl. This effect was likely due to a reduction in root hydraulic conductance by both stresses. When applied individually or together, neither 45 mM NaCl nor hypoxia affected cell membrane integrity of needles as measured by tissue electrolyte leakage. Hypoxia did not alter shoot Na⁺ and Cl^m concentrations in NaCl-treated plants. However, root Na⁺ concentrations were lower in NaCl-treated hypoxic plants, suggesting that hypoxia affected the ability of roots to store Na⁺. Hypoxia also induced root electrolyte leakage from NaCl-treated and control plants. The higher root Cl^m concentrations compared with Na⁺ and the positive correlation between root Cl^m concentrations and electrolyte leakage suggest that Cl^m played a major role in salt injury observed in jack pine seedlings. Roots of well-aerated plants treated for 1 week with NaCl contained almost two-fold higher concentration of total non-structural carbohydrates compared with plants from other experimental treatments and these concentrations decreased in subsequent weeks. We suggest that under prolonged hypoxic conditions, roots lose the ability to prevent Cl^m uptake resulting in the increase in root Cl^m concentration, which has damaging effects on root cell membranes.     

Purification of plasmid DNA by an integrated operation comprising tangential flow filtration and nitrocellulose adsorption

There is an increasing interest in the development of scaleable and reproducible plasmid DNA purification protocols for vaccine and gene therapy. The use of an integrated unit operation, comprising tangential flow microfiltration coupled with the adsorption of contaminants onto nitrocellulose membranes as a single processing step was examined in this work. Experiments were performed using a custom-built tangential flow microfiltration rig (membrane area=12.5 cm(2)). Tangential flow filtration-adsorption of E. coli lysates containing a plasmid product removed most solids (>75%) and decreased chromosomal DNA contamination by 75% w/w. Total plasmid DNA concentration and supercoiled content of the permeate were virtually identical to those of the feed, indicating a recovery yield of 100% (transmission equal to 1). Results were similar for E. coli lysates containing either a 6.9 kb or a 20 kb plasmid. Significant reductions in RNA, endotoxin, and protein levels were also observed. The reproducibility and potential for scale up of this integrated filtration-adsorption operation makes it at attractive option for intermediate- to large-scale pharmaceutical-grade plasmid processing.     

Production of inulo-oligosaccharides from inulin by recombinant E. coli containing endoinulinase activity

To utilize intracellular endoinulinase for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was successfully cloned into the plasmid pBR322 by using EcoRI restriction endoinulinase and E. coli HB101 as a host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, showing similar reaction modes as compared to those of the original strain. However, some critical differences existed in optimal reaction conditions and oligosaccharide compositions between the two products catalyzed by the native enzyme of original strain and those by intact cells from recombinant cells. The IOS compositions produced by recombinant E. coli were quite different due to the diffusional restriction of the substrate and products within the cell wall. Optimal reaction conditions for batchwise production of IOS were as follow : optimum temperature, 55v°C; pH, 7.5; substrate concentration, 100 g/l inulin; enzyme dosage, 20 units/g substrate. Continuous production of IOS from inulin was also carried out at 50v°C using a bioreactor packed with the recombinant cells immobilized on calcium alginate gel. The optimal feed concentration and the feed flow rate were 100 g/l inulin and 0.6 h^у as a superficial space velocity, respectively. Under the optimum operation conditions, continuous production of IOS was successfully performed with productivity of 166.7 g/l·h for 15 days at 50v°C without significant loss of initial activity.     

Callus Culture of Sainfoin (Onobrychis viciifolia) and Plant Regeneration through Somatic Embryogenesis

Callus of sainfoin (Onobrychis viciifolia Scop.) was initiatedfrom stem and root explants which were obtained from seedlingsgrowing in vitro, on Linsmaier Skoog (LS) medium supplementedwith 1 mg l^–1 2, 4-D and 1 mg l^–1 BA or only 1 mgl^–1 BA, and the Vacin and Went medium without hormones.Somatic embryos were formed on LS medium containing 1 m l^–1BA. Embryos developed into complete plants on filter paper saturatedwith hormone-free LS medium. Onobrychis viciifolia, somatic embryogenesis, callus culture, plant regeneration     

Continuous production of inulo-oligosaccharides from chicory juice by immobilized endoinulinase

Several carrier materials were examined for endoinulinase immobilization. A polystyrene carrier material (UF93^®) gave the best immobilization capacity (217 units/g carrier) and operational stability. Carbohydrate compositions in the reaction product were quite similar irrespective of the support materials even though each carrier material has different pore structure associated with diffusional restriction. After immobilization the optimal pH for enzyme activity was shifted from 5.0 to 4.5, whereas optimal temperature (55v°C) was unaltered. Continuous production of inulo-oligosaccharides from chicory juice was carried out using the polystyrene-bound endoinulinase. The recommended operating conditions of the enzyme reactor for maximizing productivity were as follows: feed concentration, 100 g/l chicory juice; flow rate, as superficial space velocity 2.0 h^у; temperature, 55v°C. The enzyme reactor was run for 28 days at 55v°C achieving an oligosaccharide yield of 82% without any significant loss of initial enzyme activity, where the volumetric productivity was 200 g/l · h. Furthermore, there was no marked difference in operational stability between the two reactors fed with pure inulin solution and with chicory juice as a substrate even though chicory juice contains a lot of impurities.     

Molecular breeding of a Brevibacterium lactofermentum l-phenylalanine producer using a cloned prephenate dehydratase gene

Summary The prephenate dehydratase gene was cloned from a mutant of Brevibacterium lactofermentum, AJ11957 that produced enzyme free from feedback inhibition. The recombinant plasmids pPH11 and pPH14 complemented a phenylalanine auxotroph of B. lactofermentum, A-15, provided the transformant with the desensitized enzyme and caused an increased level of the enzyme compared to that of a wild strain. Plasmid pPH14 was introduced into l-phenylalanine producers genetically induced from B. lactofermentum; MF358 and FP-1 excreting l-tyrosine and anthranilate, respectively, as by-products. Both transformants predominantly accumulated l-phenylalanine at the expense of by-product formation. Co-existence of pPH14 and pTAR16, a recombinant plasmid expressing desensitized 3-deoxy-d-arabino-hepturosonate-7-phosphate synthase had a marked effect on further improvement in l-phenylalanine productivity, accompanied by an increase in the corresponding enzyme activity. The parent, MF358, accumulating 5.5 g/l l-phenylalanine, 6.8 g/l l-tyrosine and 0.3 g/l anthranilate turned into a potent l-phenylalanine producer producing 18.2 g/l l-phenylalanine and 1.0 g/l l-tyrosine by-product. Offprint requests to: Hisao Ito     

Coagulant selection and sludge conditioning in a slaughterhouse wastewater treatment plant

Attempts were made in this study to examine the effectiveness of polymer addition to the aeration tank effluent prior to sludge flotation as practiced in a slaughterhouse wastewater treatment plant. The plant currently uses 10 mg/l of polymer prior to sludge flotation, but alternative, less-expensive, chemicals such as alum could be equally effective. Therefore, experiments were conducted using the Standard Jar test to determine the performance of both alum (Al2SO4.6H2O) and organic polymer. The dosages used for alum ranged between 0 and 1000 mg/l, whereas polymer dosages varied between 0 and 90 mg/l. The (optimal) removal efficiency for suspended solids in the mixed liquor was obtained at 400 mg/l for alum and 30 mg/l for polymer. It is evident that addition of alum or polymer results in significant removal of suspended solids reaching up to 99% for alum and 96% for polymer but alum produced a more compacted sludge. Removal of filterable COD was much lower in both cases since the chemicals used target the colloidal and suspended portion of the COD rather than the soluble (filterable) part of the COD.     

Statistical optimization of a culture medium for biomass and poly(3-hydroxybutyrate) production by a recombinant Escherichia coli strain using agroindustrial byproducts.

A statistically based Plackett-Burman screening design identified milk whey and corn steep liquor concentrations as well as ionic strength (based on phosphate buffer concentration) as the three main independent components of the culture medium that significantly (p < 0.05) influenced biomass and poly(3-hydroxybutyrate) (PHB) production in recombinant cells of Escherichia coli. This strain carries a plasmid encoding phb genes from a natural isolate of Azotobacter sp. Response surface methodology, using a central composite rotatable design, demonstrated that the optimal concentrations of the three components, defined as those yielding maximal biomass and PHB production in shaken flasks, were 37.96 g deproteinated milk whey powder/l, 29.39 g corn steep liquor/l, and 23.76 g phosphates/l (r2 = 0.957). The model was validated by culturing the recombinant cells in medium containing these optimal concentrations, which yielded 9.41 g biomass/l and 6.12 g PHB/l in the culture broth. Similar amounts of PHB were obtained following batch fermentations in a bioreactor. These results show that PHB can be produced efficiently by culturing the recombinant strain in medium containing cheap carbon and nitrogen sources.     

The annual patterns of photosynthesis in two large, freshwater, ultra-oligotrophic Antarctic lakes

The phytoplankton productivity and biomass of two large, freshwater Antarctic lakes (Vestfold Hills, eastern Antarctica) were investigated over a 12-month period. Crooked Lake was sampled at one site, while Lake Druzhby, a complex lake with two shallow and one deep basin, was subject to a more detailed investigation. Concentrations of chlorophyll a were usually below 1 µg l^-1, indicating ultra-oligotrophic conditions. Despite periodic low nutrient levels, low temperatures (range 0.4-2.8°C) and periodic poor light climate, some degree of photosynthesis was measurable throughout the year, including the dark winter phase. Snow cover had a pronounced impact on the light climate of the water column and inhibited photosynthesis. Mean rates of carbon fixation in the 0- to 15-m water column varied between 0 and 38.47 µg C l^-1 day^-1 in Crooked Lake and 0.24 and 37.68 µg C l^-1 day^-1 in Lake Druzhby. There were significant differences in the seasonal patterns of primary production between the basins of Lake Druzhby. The shallow basins had highest productivity in August, whereas the deep basin had highest rates in summer. Chlorophyll specific rates of photosynthesis or assimilation numbers [µg C (chl. a)^-1 h^-1] varied between 0.05 and 44.9, and photosynthetic efficiency [µg C (chl. a)^-1 h^-1 µmol m^-2 s^-1] between 0.02 and 5.19. The data suggest that the phytoplankton of these lakes is adapted to low irradiance levels, low temperatures and nutrient limitation.     

Fed-batch culture of insect cells with exponential feeding of amino acid and yeastolate solution

Amino acids rather than sugars are the primary limiting substrates for the culture of insect cells in a Grace's medium. When cultures are supplemented with amino acids, the yeastolate components other than the amino acids become the secondary limiting substrates. For the fed-batch culture of insect cells, a solution containing concentrated amino acids and yeastolate was supplied using an exponential feed flow rate calculated from mass balance equations. During the batch period the specific growth rate was 0.02 h^у, whereas during the fed-batch period it was measured as 0.007 and 0.012 h^у on the basis of the cell numbers and the dry cell weight, respectively. This difference in the specific growth rates in the fed-batch period is caused by an increase in the cell size during this period. Furthermore, in fed-batch cultures, dissolved oxygen was found to be a limiting factor for high cell-density cultures.