Continuous production of ethanol from fructose by immobilized growing cells of Zymomonas mobilis

Immobilized growing cells of Zymomonas mobilis were found to ferment rapidly and efficiently media containing 100 g/L fructose in a continuous reactor. A volumetric ethanol productivity of 94.8 g/L h was achieved at a substrate conversion of 75.5%. With 97% conversion of substrate the productivity was 28.4 g/L h. At fructose concentrations of 150 and 200 g/L substrate and product inhibitions limited the performance of the reactor. Ethanol production was constant over a period of 55 days.     

Ultrasound-enhanced bioprocess. II: Dehydrogenation of hydrocortisone by Arthrobacter simplex

Dehydrogenation of hydrocortisone by agitated suspensions of free and immobilized cells of Arthrobacter simplex ATCC 6946 was investigated under controlled ultrasonic irradiation at a frequency of 20 kH(z). The microbial conversion was optimized first with respect to mechanical agitation and subsequently with respect to an additionally superimposed sonication. The optimization of ultrasound intensity and its mode of application were established at a level which maintained the structural integrity of the cells as well as their biocatalytic activity. Various regimes of ultrasound at power densities of 0.030-0.120 W/mL were applied in systems of soluble (0.4 g/L) and excess (1 g/L) hydrocortisone and only a moderate enhancement of the bioconversion by free cells was observed. This result was explained by a better ultrasound-induced dispersal of microscopic clumps of cells and self-adhering clusters of the steroids. However, a quite significant enhancement effect was obtained in bioconversion systems of soluble substrate by gel-entrapped cells. This enhancement was explained by a phonophoretic effect associated with ultrasound-facilitated diffusion of the substrates-oxygen and hydrocortisone-within the gel beads.     

双菌多轮序列协同转化甾体制氢化可的松

建立了蓝色犁头霉AS 3．65和新月弯孢霉AS 3．4381协同多轮转化17α-羟基孕甾-4-烯-3,20-二酮-21-醋酸酯（RSA）割氢化可的松（hydrocortisone,HC）新工艺。在培养好的AS 3．65和AS 3．4381所组成的协同转化体系中,AS 3．65首先将RSA水解为脱氧皮质酮（RS）,这较AS 3．4381单轮批次转化省去了RSA到RS的化学水解工序。在甾体底物RSA平均投料质量浓度为1．3g/L和1g／L的条件下,所选定的协同转化体系可分别被重复利用3轮和6轮,相应的平均产率能维持在较高水平,分别高达81．6％和85％。另外,该工艺明显减少了底物RSA投料浓度对C11位羟化的影响,并有效抑制了AS 3．4381和AS 3．65单独转化过程中出现的14α—OH—RS和11α-OH—RS副产物．     

Performance of an upflow anaerobic reactor combining a sludge blanket and a filter treating sugar waste

A new hybrid reactor, the upflow blanket filter (UBF), which combined on open volume in the bottom two-thirds of the reactor for a sludge blanket and submerged plastic rings (Flexiring, Koch Inc., 235 m(2)/m(3)) in the upper one-third of the reactor volume, was studied. This UBF reactor was operated at 27 degrees C at loading rates varying from 5 to 51 g chemical oxygen demand (COD)/L d with soluble sugar wastewater (2500 mg COD/L). Maximum removal rates of 34 g COD/L d and CH(4) production rates of 7 vol/vol d [standard temperature and pressure (STP)] were obtained. The biomass activity was about 1.2 g COD/g volatile suspended solids per day. Conversion (based on effluent soluble COD) was over 93% with loading rates up to 26 g COD/L d. At higher loading rates conversion decreased rapidly. The packing was very efficient in retaining biomass.     

Microencapsulation of recombinant Saccharomyces cerevisiae cells with invertase activity in liquid-core alginate capsules

As a means of integrating cell growth and immobilization, recombinant Saccharomyces cerevisiae cells with invertase activity were immobilized in liquid-core alginate capsules and cultured to a high density. S. cerevisiae cells of SEY 2102 (MAT alpha ura3-52 leu2-3, 112 his4-519) harboring plasmid pRB58 with the SUC2 gene coding for invertase were grown to 83 g/L of liquid-core volume inside the capsule on a dry weight basis. The cloned invertase was expressed well in the immobilized cells with slightly higher activity than the free cells in a batch culture. Invertase in the immobilized cells showed slightly more improved thermal stability than in the free cells. Storage in a Na-acetate buffer at 4 degrees C and 10 degrees C for 1 month resulted in 7% and 8% loss in activity, respectively. The sucrose hydrolysis reaction was stably maintained for 25 repeated batches for 7 days at 30 degrees C. Continuous hydrolysis of 0.3 M sucrose was carried out in a packed bed reactor with a conversion of more than 90% at a maximum productivity of 55.5 g glucose/L per hour for 7 days. In a continuous stirred tank reactor, the maximum productivity of 80.8 g glucose/L per hour was achieved at a conversion of 59.1% using 1.0 M sucrose solution, and 0.5 M sucrose solution was hydrolyzed for 1 week with a 95% conversion at a productivity of 48.8 g/L per hour. (c) 1996 John Wiley & Sons, Inc.     

Performance of a down-flow fluidized bed reactor under sulfate reduction conditions using volatile fatty acids as electron donors

The use of a down-flow fluidized bed (DFFB) reactor for the treatment of a sulfate-rich synthetic wastewater was investigated to obtain insight into the outcome of sulfate reduction in a biofilm attached to a plastic support under a down-flow regime. Fine low-density polyethylene particles were used as support for developing a biofilm within the reactor. The reactor treated a volatile fatty acids mixture of acetate or lactate, propionate, and butyrate at different chemical oxygen demand (COD) to sulfate ratios ranging from 1.67 to 0.67 (g/g). Organic loading rate changed from 2.5 to 5 g COD/L x day and sulfate loading rate increased from 1.5 to 7.3 g SO(4) (2-)/L x day. At the beginning of continuous operation, methanogenesis was the predominant process; however, after 187 days, sulfate reduction became the main ongoing biological process. After 369 days, a COD removal of 93% and a sulfate removal of 75% were reached. Total sulfide concentrations in the reactor ranged from 105, when the reactor was mainly methanogenic, to around 1,215 mg/L at the end of the experiment. The high sulfide concentrations did not affect the performance of the reactor. Results demonstrated that the configuration of the DFFB reactor was suitable for the anaerobic treatment of sulfate-rich wastewater.     

Continuous delta 1-hydrocortisone dehydrogenation with in situ product recovery.

A continuous aerated process for delta 1-hydrocortisone dehydrogenation by polyacrylamide-hydrazide (PAAH) bead-entrapped A. simplex cells was developed. The process allows for stable conversion of 1.6 g l-1 hydrocortisone (x 5 the solubility in water), made possible by the incorporation of selected cosolvent [5% (v/v) triethyleneglycol]. A large difference in substrate and product solubilities in the cosolvent-buffer medium allowed for in situ product recovery in an aerated, fluidized-bed, immobilized-cell reactor by the controlled addition of fine product-adsorbing powder (microcrystalline cellulose). The product was recovered at the reactor outlet as a fine complex with the adsorbent. Stable continuous operation of at least 4 weeks was recorded for a prototype reactor configuration, followed by the exhibition of similar operational stability in a modified version of a commercially available 2.5-l airlift reactor. Our results demonstrate that in addition to an obvious desirable cosolvent effect on substrate solubility enhancement, it may also enable easy in situ product recovery by creating a large gap in the solubilities of the substrate and the product in the cosolvent-containing reaction medium.     

High-Rate two-phase process for the anaerobic degradation of cellulose, employing rumen microorganisms for an efficient acidogenesis

A novel two-stage anaerobic process for the microbial conversion of cellulose into biogas has been developed. In the first phase, a mixed population of rumen bacteria and ciliates was used in the hydrolysis and fermentation of cellulose. The volatile fatty acids (VFA) produced in this acidogenic reactor were subsequently converted into biogas in a UASB-type methanogenic reactor.A stepwise increase of the loading rate from 11.9 to 25.8 g volatile solids/L reactor volume/day (g VS/L/day) did not affect the degradation efficiency in the acidogenic reactor, whereas the methanogenic reactor appeared to be overloaded at the highest loading rate. Cellulose digestion was almost complete at all loading rates applied. The two-stage anaerobic process was also tested with a closed fluid circuit. In this instance total methane production was 0.438 L CH(4)g VS added, which is equivalent to 98% of the theoretical value. The application of rumen microorganisms in combination with a high-rate methane reactor is proposed as a means of efficient anaerobic degradation of cellulosic residues to methane. Because this newly developed two-phase system is based on processes and microorganisms from the ruminant, it will be referred to as "Rumen Derived Anaerobic Digestion" (RUDAD-) process.     

Lactose hydrolysis by immobilized beta-galactosidase in capillary bed reactor

The hydrolysis of lactose by immobilized beta-galactosidase was studied in a continuous-flow capillary bed reactor operating at 30 degrees C. Solutions containing 50, 100, and 150 g lactose and 0.5 g sodium acetate/L were fed to the reactor. Lactose conversions ranging from 24% to greater than 99% were achieved at reactor space times ranging from 0.06 to 6.3 min. These conversion data were successfully modeled in terms of a plug flow reactor model and a form of Michaelis-Menten kinetics which included competitive inhibition by both the alpha and beta forms of galactose.     

High ethanol tolerance of the thermophilic anaerobic ethanol producer Thermoanaerobacter BG1L1

The low ethanol tolerance of thermophilic anaerobic bacteria, generally less than 2% (v/v) ethanol, is one of the main limiting factors for their potential use for second generation fuel ethanol production. In this work, the tolerance of thermophilic anaerobic bacterium Thermoanaerobacter BG1L1 to exogenously added ethanol was studied in a continuous immobilized reactor system at a growth temperature of 70°C. Ethanol tolerance was evaluated based on inhibition of fermentative performance e.g. inhibition of substrate conversion. At the highest ethanol concentration tested (8.3% v/v), the strain was able to convert 42% of the xylose initially present, indicating that this ethanol concentration is not the upper limit tolerated by the strain. Long-term strain adaptation to high ethanol concentrations (6–8.3%) resulted in an improvement of xylose conversion by 25% at an ethanol concentration of 5% v/v, which is the concentration required in practice for economically efficient product recovery. For all ethanol concentrations tested, relatively high and stable ethanol yields (0.40–0.42 g/g) were seen. The strain demonstrated a remarkable ethanol tolerance, which is the second highest displayed by thermophilic anaerobic bacteria known to the authors. This appears to be the first study of the ethanol tolerance of these microorganisms in a continuous immobilized reactor system.     

Influence of substrate concentration on the stability and yield of continuous biohydrogen production

The effect of substrate concentration (sucrose) on the stability and yield of a continuous fermentative process producing hydrogen was studied. High substrate concentrations are attractive from an energy standpoint as they would minimise the energy required for heating. The reactor was a CSTR; temperature was maintained at 35 degrees C; pH was controlled between 5.2 and 5.3, and the hydraulic retention time (HRT) was 12 h. Online measurements were taken for ORP, pH, temperature, %CO2, gas output and %H2, and data logged using a MatLAB data acquisition toolbox. Steady-state operation was obtained at 10, 20 and 40 g/L of sucrose in the influent, but a subsequent step change to 50 g/L was unsustainable. The hydrogen content ranged between 50% and 60%. The yield of hydrogen decreased as the substrate concentration increased from 1.7 +/- 0.2 mol/mol hexose added at 10 g/L, to 0.8 +/- 0.1 mol/mol at 50 g/L. Sparging with nitrogen improved the hydrogen yield by at least 35% at 40 g/L and at least 33% at 50 g/L sucrose. Sparging also enabled steady-state operation at 50 g/L sucrose. Addition of an extra 4 g/L of n-butyric acid to the reactor operating at 40 g/L sucrose increased the butyrate concentration from 9,830 to 18,900 mg/L, immediately stopping gas production and initiating the production of propionate, whilst the addition of 2 g/L taking the butyrate concentration to 12,200 mg/L did not do so. It was shown that operation at 50 g/L sucrose in a CSTR in butyrate fermentation is possible.     

Influence of Nitrate and Sulfate on the Anaerobic Treatment of Pharmaceutical Wastewater

Anaerobic treatment of wastewater from the pharmaceutical industry, which contained about 3.2 g/L of sulfate, was carried out in an Upflow Anaerobic Sludge Blanket (UASB) reactor. After a startup period of 120 days, a chemical oxygen demand (COD) removal efficiency of more than 90 % was obtained along with an organic loading rate (OLR) of 1.5 g COD/(L day). During the same period, the sulfate removal was about 90 %. However, the performance of the reactor was affected when the loading rate was increased to 2.09 g COD/(L day). It was found that the accumulation of sulfides, combined with a decrease in the pH, affected the reactor performance. In batch reactor studies with pharmaceutical wastewater it was observed that methane production began only after the initiation of nitrate consumption. The denitrification process can inhibit sulfate reduction at high nitrate concentrations, but compared to reactors without nitrate, the sulfate reduction process and sulfide formation were quickly initiated at low nitrate concentrations. The methanogenic activity was however affected by the presence of more than 2 g/L of sulfate.     

Tagatose production by immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant in a packed-bed bioreactor

Using immobilized recombinant Escherichia coli cells containing Geobacillus stearothermophilus l-arabinose isomerase mutant (Gali 152), we found that the galactose isomerization reaction was maximal at 70 degrees C and pH 7.0. Manganese ion enhanced galactose isomerization to tagatose. The immobilized cells were most stable at 60 degrees C and pH 7.0. The cell and substrate concentrations and dilution rate were optimal at 34 g/L, 300 g/L, and 0.05 h(-1), respectively. Under the optimum conditions, the immobilized cell reactor with Mn2+ produced an average of 59 g/L tagatose with a productivity of 2.9 g/L.h and a conversion yield of 19.5% for the first 20 days. The operational stability of immobilized cells with Mn2+ was demonstrated, and their half-life for tagatose production was 34 days. Tagatose production was compared for free and immobilized enzymes and free and immobilized cells using the same mass of cells. Immobilized cells produced the highest tagatose concentration, indicating that cell immobilization was more efficient for tagatose production than enzyme immobilization.     

Methane production using whole and screened dairy manure in conventional and fixed-film reactors

The technical feasibility of adopting the fixed-film reactor concept for biogas production from screened dairy manure was investigated. The methane production capability of laboratory-scale 4-L anaerobic reactors (conventional and fixed-film) receiving screened dairy manure and operated at 35 degrees C was compared. Dairy manure filtrate with 4.4% total solids (TS) and 3.4% volatile solids (VS) (average value) was prepared from 1:1 manure-water slurry. The feed material was added intermittently at loading rates ranging from 2.34 to 25 and 2.25 to 785 g VS/L d, respectively, for the conventional and fixed-film reactors. Maximum methane production rate (L CH(4)/L d) for the conventional reactor was 0.63 L CH(4)/L d achieved at a 6-day hydraulic retention time (HRT). For the fixed-film reactor the maximum production rate was 3.53 L CH(4)/L d when operated at a loading rate of 262 g VS/L d (3 h HRT). The fixed-film reactor was capable of sustaining a loading of 785 g VS/L d (1 h HRT). The fixed-film reactor performed much better than the conventional reactors. These results indicate that a large reduction of required reactor volume is possible through application of a fixed-film concept combined with a liquid-solid separation pretreatment of dairy manure.     

The effects of cyclosporin and hydrocortisone on human T lymphocyte colony formation

The immunosuppressive activities of two compounds, cyclosporin A and hydrocortisone, were evaluated using a human T lymphocyte colony assay. Both compounds produced a dose-related reduction in colony formation. With cyclosporin, concentrations of 1.0–100 μg/ml virtually abolished PHA-induced lymphocyte growth; as little as 0.01 μg/ml decreased clonal growth by 48%. Suppression was observed as late as 4 days after the addition of PHA. The addition of exogenous IL-2 did not completely restore clonal growth to normal. Similarly, hydrocortisone, in concentrations of 0.4 μg/ml, produced a 96% inhibition in clonal growth. Partial suppression could also be obtained if the drug was added as late as 4 days after PHA. Exogenous IL-2 completely reversed the inhibitory effects of hydrocortisone. By contrast, IL-1 was able to only partially restore growth potential. Parallel studies using TPA indicated that both immunosup-pressants inhibited responses to this mitogen. However, in plates containing both TPA and PHA, hydrocortisone failed to impair clonal growth.     

Characterization of an immobilized cell, trickle bed reactor during long term butanol (ABE) fermentation

Acetone-butanol-ethanol (ABE) fermentation was performed continuously in an immobilized cell, trickle bed reactor for 54 days without, degeneration by maintaining the pH above 4.3. Column clogging was minimized by structured packing of immobilization matrix. The reactor contained two serial glass columns packed with Clostridium acetobutylicum adsorbed on 12- and 20-in.-long polyester sponge strips at total flow rates between 38 and 98.7 mL/h. Cells were initially grown at 20 g/L glucose resulting in low butanol (1.15 g/L) production encouraging cell growth. After the initial cell growth phase a higher glucose concentration (38.7 g/L) improved solvent yield from 13.2 to 24.1 wt%, and butanol production rate was the best. Further improvement in solvent yield and butanol production rate was not observed with 60 g/L of glucose. However, when the fresh nutrient supply was limited to only the first column, solvent yield increased to 27.3 wt% and butanol selectivity was improved to 0.592 as compared to 0.541 when fresh feed was fed to both columns. The highest butanol concentration of 5.2 g/L occurred at 55% conversion of the feed with 60 g/L glucose. Liquid product yield of immobilized cells approached the theoretical value reported in the literature. Glucose and product concentration profiles along the column showed that the columns can be divided into production and inhibition regions. The length of each zone was dependent upon the feed glucose concentration and feed pattern. Unlike batch fermentation, there was no clear distinction between acid and solvent production regions. The pH dropped, from 6.18-6.43 to 4.50-4.90 in the first inch of the reactor. The pH dropped further to 4.36-4.65 by the exit of the column. The results indicate that the strategy for long term stable operation with high solvent yield requires a structured packing of biologically stable porous matrix such as polyester sponge, a pH maintenance above 4.3, glucose concentrations up to 60 g/L and nutrient supply only to the inlet of the reactor.     

Conversion of paper sludge to ethanol in a semicontinuous solids-fed reactor

Conversion of paper sludge to ethanol was investigated with the objective of operating under conditions approaching those expected of an industrial process. Major components of the bleached Kraft sludge studied were glucan (62 wt.%, dry basis), xylan (11.5%), and minerals (17%). Complete recovery of glucose during compositional analysis required two acid hydrolysis treatments rather than one. To avoid the difficulty of mixing unreacted paper sludge, a semicontinuous solids-fed laboratory bioreactor system was developed. The system featured feeding at 12-h intervals, a residence time of 4 days, and cellulase loading of 15 to 20 FPU/g cellulose. Sludge was converted to ethanol using simultaneous saccharification and fermentation (SSF) featuring a -glucosidase-supplemented commercial cellulase preparation and glucose fermentation by Saccharomyces cerevisiea. SSF was carried out for a period of 4 months in a first-generation system, resulting in an average ethanol concentration of 35 g/L. However, steady state was not achieved and operational difficulties were encountered. These difficulties were avoided in a retrofitted design that was operated for two 1-month runs, achieving steady state with good material balance closure. Run 1 with the retrofitted reactor produced 50 g/L ethanol at a cellulose conversion of 74%. Run 2 produced 42 g/L ethanol at a conversion of 92%. For run 2, the ethanol yield was 0.466 g ethanol/g glucose equivalent fermented and >94% of the xylan fed to the reactor was solubilized to a mixture of xylan oligomers and xylose.     

Production of galacto-oligosaccharides from lactose by Aspergillus oryzae beta-galactosidase immobilized on cotton cloth.

The production of galacto-oligosaccharides (GOS) from lactose by A. oryzae beta-galactosidase immobilized on cotton cloth was studied. The total amounts and types of GOS produced were mainly affected by the initial lactose concentration in the reaction media. In general, more and larger GOS can be produced with higher initial lactose concentrations. A maximum GOS production of 27% (w/w) of initial lactose was achieved at 50% lactose conversion with 500 g/L of initial lactose concentration. Tri-saccharides were the major types of GOS formed, accounting for more than 70% of the total GOS produced in the reactions. Temperature and pH affected the reaction rate, but did not result in any changes in GOS formation. The presence of galactose and glucose at the concentrations encountered near maximum GOS greatly inhibited the reactions and reduced GOS yield by as much as 15%. The cotton cloth as the support matrix for enzyme immobilization did not affect the GOS formation characteristics of the enzyme, suggesting no diffusion limitation in the enzyme carrier. The thermal stability of the enzyme increased approximately 25-fold upon immobilization on cotton cloth. The half-life for the immobilized enzyme on cotton cloth was more than 1 year at 40 degrees C and 48 days at 50 degrees C. Stable, continuous operation in a plugflow reactor was demonstrated for 2 weeks without any apparent problem. A maximum GOS production of 21 and 26% (w/w) of total sugars was attained with a feed solution containing 200 and 400 g/L of lactose, respectively, at pH 4.5 and 40 degrees C. The corresponding reactor productivities were 80 and 106 g/L/h, respectively, which are at least several-fold higher than those previously reported.     

Thermophilic high-rate anaerobic treatment of wastewater containing long-chain fatty acids: effect of washed out biomass recirculation

Biomass washed out from an expanded granular sludge bed (EGSB) reactor treating oleate (82%, w/w) was inves-tigated. This biomass had a 50% higher activity compared to granules present in the EGSB reactor. Recirculationof washed out biomass into reactor remarkably improved the treatment performance. The highest methane conversion rate from oleate, 300 mg CH₄-COD/g VSS.d, was achieved at a concentration of 4 g oleate-COD/l and a volumetric loading rate of 8 g COD/l.d. This revised version was published online in November 2006 with corrections to the Cover Date.     

Biological sulphate reduction using gas-lift reactors fed with hydrogen and carbon dioxide as energy and carbon source

Feasibility and engineering aspects of biological sulphate reduction in gas-lift reactors were studied. Hydrogen and carbon dioxide were used as energy and carbon source. Attention was paid to biofilm formation, sulphide toxicity, sulphate conversion rate optimization, and gasliquid mass transfer limitations. Sulphate-reducing bacteria formed stable biofilms on pumice particles. Biofilm formation was not observed when basalt particles were used. However, use of basalt particles led to the formation of granules of sulphate-reducing biomass. The sulphate-reducing bacteria, grown on pumice, easily adapted to free H(2)S concentrations up to 450 mg/L. Biofilm growth rate then equilibrated biomass loss rate. These high free H(2)S concentrations caused reversible inhibition rather than acute toxicity. When free H(2)S concentrations were kept below 450 mg/L, a maximum sulphate conversion rate of 30 g SO(4) (2-)/L . d could be achieved after only 10 days of operation. Gas-to-liquid hydrogen mass transfer capacity of the reactor determined the maximum sulphate conversion rate. (c) 1994 John Wiley & Sons, Inc.