Improvement of the yield of physiologically active oligosaccharides in continuous hydrolysis of chitosan using immobilized chitosanases

The continuous production of chitosan oligosaccharides using a packed-bed enzyme reactor was investigated as to the effects of the operation conditions on the yield of pentamers and hexamers of chitosan oligosaccharides. A column reactor packed with immobilized chitosanases prepared by the multipoint attachment method was used for continuous hydrolysis of chitosan. In this reactor, the decrease of the yield of the target intermediate oligosaccharides due to axial mixing was negligible. The surface enzyme density of the support and flow rate of the substrate solution significantly affected the maximum yield of pentamers and hexamers. These effects were summarized as a correlation with the Damk?hler number (Da), defined as the ratio of the maximum reaction rate to the maximum mass transfer rate. The optimum condition was determined based on Da. Under the optimized condition (Da = 0.12), pentamers and hexamers could be produced continuously for a month with a yield of over 35% (7 kg/m(3) in concentration).     

Continuous protein recovery from whey using liquid-solid circulating fluidized bed ion-exchange extraction

A liquid-solid circulating fluidized bed (LSCFB) continuous ion-exchange extraction system has been investigated for total protein recovery from whey solutions under various operating conditions. The effectiveness of a dynamic seal was evaluated between the riser and the downcomer, and the best conditions for the establishment of this seal were established. Start-up studies indicated that the system is robust and stable. Under optimal conditions, a productivity of 8.2 g of total protein removed per hour per kilogram of resin was achieved with a protein removal efficiency of 78.4%. However, higher overall protein recovery of up to 90% was also achieved under other conditions, with lower protein concentration in the effluent and a lower overall productivity.     

A microsatellite-based estimation of clonal diversity and population subdivision in Zostera marina, a marine flowering plant

We examined the genetic population structure in eelgrass (Zostera marina L.), the dominant seagrass species of the northern hemisphere, over spatial scales from 12 km to 10 000 km using the polymorphism of DNA microsatellites. Twelve populations were genotyped for six loci representing a total of 67 alleles. Populations sampled included the North Sea (four), the Baltic Sea (three), the western Atlantic (two), the eastern Atlantic (one), the Mediterranean Sea (one) and the eastern Pacific (one). Microsatellites revealed substantial genetic variation in a plant group with low allozyme diversity. Average expected heterozygosities per population (monoclonal populations excluded) ranged from 0.32 to 0.61 (mean = 0. 48) and allele numbers varied between 3.3 and 6.7 (mean = 4.7). Using the expected frequency of multilocus genotypes within populations, we distinguished ramets from genetic individuals (i.e. equivalent to clones). Differences in clonal diversity among populations varied widely and ranged from maximal diversity (i.e. all ramets with different genotype) to near or total monoclonality (two populations). All multiple sampled ramets were excluded from further analysis of genetic differentiation within and between populations. All but one population were in Hardy-Weinberg equilibrium, indicating that Zostera marina is predominantly outcrossing. From a regression of the pairwise population differentiation with distance, we obtained an effective population size Ne of 2440-5000. The overall genetic differentiation among eelgrass populations, assessed as rho (a standardized estimate of Slatkin's RST) was 0.384 (95% CI 0.34-0.44, P < 0.001). Genetic differentiation was weak among three North Sea populations situated 12-42 km distant from one another, suggesting that tidal currents result in an efficient exchange of propagules. In the Baltic and in Nova Scotia, a small but statistically significant fraction of the genetic variance was distributed between populations (rho = 0.029-0. 053) at scales of 15-35 km. Pairwise genetic differentiation between European populations were correlated with distance between populations up to a distance of 4500 km (linear differentiation-by-distance model, R2 = 0.67). In contrast, both Nova Scotian populations were genetically much closer to North Sea and Baltic populations than expected from their geographical distance (pairwise rho = 0.03-0.08, P < 0.01). A biogeographical cluster of Canadian with Baltic/North Sea populations was also supported using a neighbour-joining tree based on Cavalli-Sforza's chord distance. Relatedness between populations may be very different from predictions based on geographical vicinity.     

Alcoholic fermentation by agar-immobilized yeast cells

Immobilized yeast cells in agar gel beads were used in a packed bed reactor for the production of ethanol from cane molasses at 30°C, pH 4.5. The maximum productivity, 79.5g ethanol/l.h was obtained with 195g/l reducing sugar as feed. Substrate (64.2%) was utilized at a dilution of 1.33h^-1. The immobilized cell reactor was operated continuously at a constant dilution rate of 0.67h^-1 for 100 days. The maximum specific ethanol productivity and specific sugar uptake rate were 0.610g ethanol/g cell.h and 1.275g sugar/g cell.h, respectively.     

用膨胀床金属亲和层析从淡菜匀浆液中分离纯化纤维素酶

研究了一种新的膨胀床金属亲和层析技术,即将金属亲和层析结合膨胀床层析,直接从淡菜(Blue mussel)匀浆液中纯化纤维素酶。研究了金属亲和配基种类、pH、离子强度及流速对酶吸附和解吸的影响,确定了酶洗脱条件和介质再生条件。一步可纯化纤维素酶194倍,酶收率达82%。本方法不需要预先去除细胞碎片,而且处理速率比传统层析技术高3～4倍。     

Pilot scale production of a recombinant human epidermal growth factor, secreted by Bacillus brevis, using expanded bed adsorption

Recombinant Bacillus brevis which carried an expression plasmid encoding the human epidermal growth factor (EGF) gene on a cryptic high-copy number plasmid, pHT926, extracellularly produced EGF in its biologically active form at a concentration of over 1.5 g L⁻¹ in the culture broth in a 30-L jar fermenter. The culture broth also contained some other EGF compounds, which mainly consisted of oligomeric and polymeric forms with disulfide bonds. We developed a simple purification method for EGF, without prior cell removal from the culture broth, comprising cation exchange expanded bed adsorption followed by ultrafiltration with UF 10 000 and 3000 membranes. The EGF compounds were efficiently separated from the EGF in its native form in the expanded bed adsorption step. With this purification method, only EGF in its native form was recovered from the culture broth, with a yield of nearly 80%, and 90% purity. This efficient and economic system has made it possible to use EGF as a pharmaceutical in the livestock industry. Received 10 June 1998/ Accepted in revised form 10 September 1998     

Prepared Bed Land Treatment of Soils Containing Diesel and Crude Oil Hydrocarbons

An ex situ, field-scale, prepared bed land treatment unit (LTU) was used to bio-remediate soils containing petroleum hydrocarbons. Two soils were treated in side-by-side units to compare performance: (1) a clayey silt containing crude oil hydrocarbons from releases 30 to 40 years ago and (2) a silty sand containing diesel fuel hydrocarbons from a leak about three years prior to the bioremediation. The effectiveness of the bioremediation in the LTU was evaluated over a period of 18 months. The results indicated that: (1) prepared bed bioremediation reduced the hydrocarbon concentration, mobility, and relative toxicity in the soil with the diesel fuel, and (2) chemical bioavailability appeared to limit bioremediation of the soil containing the crude oil hydrocarbons. Although the soils containing the crude oil hydrocarbons contained an average of 10,000?mg TPH/kg dry soil, these soils had limited hydrocarbon availability, nontoxic conditions, and low potential for chemical migration. For the soils containing the diesel fuel, active prepared bed bioremediation of about 15 weeks was adequate to reach an environmentally acceptable endpoint. At that time, there was little further TPH loss, no Microtox^TM toxicity, and limited hydrocarbon mobility.     

Separation of fructooligosaccharides using zeolite fixed bed columns

Recent studies have shown that the chromatographic separation of mixtures of monosaccharides and disaccharides may be improved by employing Y zeolites, a procedure which holds promise in the separation of oligosaccharides. In the present study, a column packed with zeolite was employed to study the separation of fructooligosaccharides (FOS). FOS were produced by an enzyme isolated from Rhodotorula sp., which produces GF₂ (kestose), GF₃ (nystose) and GF₄ (frutofuranosyl nystose). The identification and quantification of the sugars were carried out by ion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The separation of fructooligosaccharides was carried out using a fixed bed column packed with Ba²⁺-exchange Y zeolites. The effects of temperature (40–50 °C), injected volume per bed volume (2.55–7.64%), superficial velocity (0.1–0.15 cm min⁻¹) and eluent composition (40–60% ethanol) were investigated using a fractionary factorial design with separation efficiency as the response. The results showed that the most favorable conditions for the separation of the oligosaccharide–glucose mixture were 60% ethanol as eluent, temperature of 50 °C, superficial velocity of 0.1 cm min⁻¹ and 2.55% injection volume per bed volume of injection mixture, using two columns in series. The values for separation efficiency were 0.60 for oligosaccharide–glucose, 1.00 for oligosaccharide–fructose, 0.22 for oligosaccharide–sucrose, 0.43 for glucose–fructose, 0.82 for glucose–sucrose and 1.23 for fructose–sucrose.     

Transport and settlement of organic matter in small streams

1. After it enters streams, terrestrially derived organic matter (OM) rapidly absorbs water. Using field and laboratory experiments, we examined how this process affected the buoyancy, settling velocity, transport distance and retention locations of four types of organic matter typically found in Pacific coastal streams (‘flexible’ red alder leaves and three ‘stiff’ particle types – Douglas‐fir needles, red cedar fronds and Douglas‐fir branch pieces). 2. Immersion in water rapidly changed the physical characteristics of alder leaves, Douglas‐fir needles and red cedar fronds, which all reached constant still‐water settling velocities after only a few days of soaking. In contrast, the settling velocity of branch pieces continued to increase for 13 days, eventually reaching much higher values than any other OM type. Dried alder leaves became negatively buoyant after only two days of immersion, while other types took substantially longer (up to 24 days) before the specific gravity of all particles was >1. 3. We released saturated OM particles in an experimental channel and found that all particle types travelled further in a fast, shallow ‘riffle’ than a slow, deep ‘pool’. Comparisons with a passive settlement null model indicated that leaves were retained more rapidly than expected in the riffle (by large protruding stones), while the three stiff particle types travelled further than expected (probably due to turbulent suspension) and were retained when they settled in deeper water between larger stones. In pools, passive settlement appeared to dominate the retention of all OM types, with leaves travelling furthest. 4. These retention patterns corresponded well with those observed when saturated OM particles collected in the field were released in two pools and two riffles in a second‐order coastal stream. 5. When the experimental channel and in‐stream data were combined, the retention rates of the three stiff OM types were closely related to calculated Rouse numbers (Rouse number = particle settling velocity/shear velocity), whereas the retention rate of alder leaves was not. This suggests that different physical mechanisms are responsible for the retention of leaves and stiff OM types in shallow streams.