Continuous Potable Alcohol Production by Immobilized Zymomonas mobilis on γ-Alumina Pellets

Experiments were carried out with Z. mobilis immobilized on -γ-alumina pellets in a continuous reactor for potable alcohol production. It was found that -γ-alumina was a suitable support for continuous ethanol production processes. Specifically, an ethanol productivity (calculated on the basis of liquid volume) of 23.8 g/L/hr was achieved at 0.88 hr^?1dilution rate, 159 g/L invert sugar content and 43.7% conversion, using raisin extracts as feed material. Similar productivities were obtained with media containing glucose. Biomass productivity, yield and free cells concentration in glucose and raisin extracts were examined. Ethanol production was constant during the time the system was operated, a period of over 60 days.     

Continuous fermentation of sugar cane syrup using immobilized yeast cells

Several strains of Saccharomyces sp. and commercial Baker's yeast were immobilized by adhesion onto chrysotile, a fibrous magnesium silicate (Mg6Si4O10 (OH)8). The activity of the cells is higher when immobilized, mainly for fermentation of 30 to 50% w/v glucose solutions. In the medium containing 30% w/v glucose, the initial fermentation rate increased 1.2-2.5 times. Yields were in the range of 80.4 to 97.3% for the immobilized cells and 72.7 to 84.5% for the free cells. A packed bed reactor for continuous fermentation was set up using one of the tested strains immobilized onto chrysotile. An average productivity of 20 to 25 g.l(-1).h(-1) was obtained in the first 20 d, and an average of 16 g.l(-1).h(-1) was obtained after 50 d of operation.     

固定化菊粉酶的研究

报道了克鲁维酵母突变株（ｋｌｕｙｖｅｒｏｍｙｃｅｓ－ＵＶ－Ｇ－４０－３）所产菊粉酶的固定化及固定化酶的性质，并用固定化菊粉酶酶解洋姜提取液生产果糖，在分批式反应器中，当底物和固定化酶体积比为３．５∶１时，２．５ｈ水解率达到９２．４％，产率为２８．７ｇ／Ｌｈ，在连续填充床反应器中，在稀释率为０．５ｈ－１，转化率达９３．６％，产率为１５．７ｇ／Ｌｈ，半衰期达１１０ｄ以上。     

Continuous production of lactic acid from molasses by perfusion culture of Lactococcus lactis using a stirred ceramic membrane reactor

A perfusion culture system was used for continuous production of lactic acid by retaining cells at a high density of Lactococcus lactis in a stirred ceramic membrane reactor (SCMR). After the cell concentration increased to 248 g/l, half of the culture broth volume was replaced with the fermentation medium. Subsequently, a substrate solution containing glucose (run 1) or molasses (run 2) was continuously supplied to the cells retained in the SCMR. Simultaneously, the culture supernatant was extracted using a ceramic filter with a pore size of 0.2 mum. The dilution rate was initially set at 0.4 h(-1) and gradually decreased to 0.2 h(-1) due to reduction in the permeability of the filter. The concentration of glucose in the substrate solution was adjusted to 60 g/l for the transition and the first period until 240 h, 90 g/l for the second period from 240 h to 440 h, and 70 g/l for the third period from 440 h to 643 h. The average concentration of lactic acid in the filtrate reached 46 g/l in the first period, 43 g/l in the second period, and 33 g/l for the third period. The productivity obtained for the first period reached 15.8 g.l(-1).h(-1), twice as much as that achieved in repeated batch fermentations. Based on the results obtained in run 1, the substrate solution containing 120 g/l of molasses was continuously supplied for 240 h in run 2. The concentration and productivity of lactic acid reached 40 g/l and 10.6 g.l(-1).h(-1), respectively, by continuously replenishing the culture medium at a dilution rate of 0.26 h(-1). These results demonstrated that the filtration capacity of the SCMR was sufficient for a continuous and rapid replenishment of molasses solution from the dense cell culture and, therefore, the perfusion culture system is considered to provide a low-cost process for continuous production of lactic acid from cheap resources.     

Technical note: Application of immobilized cells of Kluyveromyces marxianus for continuous hydrolysis to fructose of fructans in Jerusalem artichoke extracts

Dead cells of Kluyveromyces marxianus having inulase (β-D-fructo fructanohydrolase E.C.3.2.1.7) activity were immobilized in alginate beads and used as a biocatalyst in a packed bed reactor and a stirred batch reactor. Fructosans of Jerusalem artichoke tubers, after extraction, were utilized for continuous or semi-continuous production of fructose. In a bed reactor packed with 100 ml of beads, a volumetric productivity of 36 g/l/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a half life of 28 days was observed for the biocatalyst. Using artichoke extract containing 20% fructan solution, 98% conversion could be achieved in a batch reactor in 20 hr. Repeated cycling of beads resulted in considerable loss of catalyst from the reactor and subsequent loss in catalytic activity, thus giving a half life of only 14 days.     

Synchronized fresh cell bioreactor system for continuous L-(+)-lactic acid production using Lactococcus lactis IO-1 in hydrolysed sago starch

An efficient bioreactor, termed a 'synchronized fresh cell bioreactor', was developed and consisted of a pH-dependent substrate feed system coupled with cross flow filtration and turbidity control. The effect of high dilution rate and high cell density coupled with high cell viability on the production of l-lactic acid in continuous culture by Lactococcus lactis IO-1 in enzyme-hydrolysed sago starch medium was investigated. For all changes in dilution rate, cells responded in a synchronized way to the addition of glucose by increasing the rate of biomass formation. Consequently, a glucose-free feed solution was required to maintain the cell concentration at a particular pre-set value. This set-up facilitated the maintenance of the cells in a permanent log phase. At a cell concentration of 15 gl(-1) and a feed glucose concentration of 53 gl(-1), volumetric LA productivities of 8.2, 19.3 and 33.1 gl(-1)h(-1) were obtained at dilution rates of 0.21, 0.50 and 1.1 h(-1), respectively. The respective residual glucose concentrations in the spent medium were 1.90, 0.24 and 3.80 gl(-1). By increasing the cell density, the volumetric productivity increased proportionally. At high cell density, higher dilution rates resulted in lower lactate concentrations in the culture medium resulting in higher productivity. This reactor facilitated efficient operation with high cell viability by maintaining the cells in continuous growth phase for long-term fermentation. Therefore, the growth rate (mu) was calculated according to the Monod equation. Using this system, high specific productivities can be obtained which guarantees high commercial productivity at economical cost with only a small investment for setting up the sago industry.     

Continuous Hydrolysis of Fructans in Jerusalem Artichoke Extracts using Immobilized Nonviable Cells of Kluyveromyces marxianus

Non-viable immobilized cells of Kluyveromyces marxianus in alginate beads having inulase (ß-2,1-fructan fructano-hydrolyase E.C.3.2.1.7) acitivity were used as biocatalyst in a packed bed reactor. Extracts of Jerusalem artichoke tubers were contacted with the biocatalyst for continuous conversion of the fructan component to fructose. In a bed reactor packed with 100 mL of beads, a volumetric productivity of 136 g/L/hr total reducing sugars was obtained with 98% substrate conversion. When operated continuously for 30 days, a 55% loss in the original activity was observed, giving a half life for the biocatalyst of 28 days.     

Continuous acetic acid production by a packed bed bioreactor employing charcoal pellets derived from waste mushroom medium

A packed bed bioreactor using charcoal pellets produced from waste mushroom medium by thermal carbonization was developed and applied to continuous acetic acid production. The pellets were characterized by their high specific surface area (200 m2/g) with numerous micropores (2-10 microm). The continuous acetic acid fermentation started up smoothly after seeding and was successfully operated for about 180 d under various retention times. The maximum acetic acid productivity was about 3.9 g/l/h using normal aeration and 6.5 g/l/h using air enriched with 40% O2. The pellets are expected to prove useful as a new packing material for bioreactor in terms of their bacterial affinity, high specific surface area with appropriate pore sizes for bacteria, as well as the operational stability of the system and the low production cost.     

Continuous Production of Glucose from Dextrin by Glucoamylase Immobilized on Porous Silica

Glucoamylase was covalently attached to porous silica particles and investigated for its applicability as an industrial catalyst in the production of glucose from starch hydrolysates. Reactivity at various reaction conditions, as well as enzyme loading, reaction kinetics, diffusional effects, and thermal stability, were determined in laboratory scale experiments. Pilot scale tests on continuous production of glucose were performed in a 1 cubic foot packed column reactor that could produce approximately 1,000 lbs/d glucose at 40°C. The maximum glucose concentration (based on dissolved solids) varied from 87 to 93% depending on the dextrose equivalent (D.E.) and degree of retrogradation of the feed dextrin. In 80 days of continuous operation no appreciable enzyme deactivation was observed. Initial sterilization of the reactor and continuous heat sterilization of the feed stream (120°C, 3–4 min) virtually ensured operation of the immobilized enzyme reactor at low levels of contamination, typical bacteria counts of the product effluent being 30–50/ml. Occasional interruption of the system resulted in much higher levels of microorganisms but did not affect the overall enzyme reactivity.     

Increase of xylitol productivity by cell-recycle fermentation of Candida tropicalis using submerged membrane bioreactor

Candida tropicalis, an osmophilic strain isolated from honeycomb, produced xylitol at a maximal volumetric productivity of 3.5 g l(-1) h(-1) from an initial xylose concentration of 200 g l(-1). Even at a very high xylose concentration, e.g., 350 g l(-1), this strain produced xylitol at a moderate rate of 2.07 g l(-1) h(-1). In a fed-batch fermentation of xylose and glucose, 260 g l(-1) xylose was added, and the xylitol production was 234 g l(-1) for 48 h, corresponding to a rate of 4.88 g l(-1) h(-1). To increase xylitol productivity, cells were recycled in a submerged membrane bioreactor with suction pressure and air sparging. For each recycle round in cell-recycle fermentation, the average concentration of xylitol produced, fermentation time, volumetric productivity, and product yield were 180 g l(-1), 19.5 h, 8.5 g l(-1) h(-1), and 85%, respectively. When cell-recycle fermentation was started with the cell mass concentrated twofold after batch fermentation and performed for 10 recycle rounds, we achieved a very high productivity of 12 g l(-1) h(-1). The productivity and total amount of xylitol in cell-recycle fermentation were 3.4- and 11.0-fold higher than those in batch fermentation, respectively.     

Strategies to remove water formed as by-product on the monoolein synthesis by enzymatic esterification performed on packed bed reactor

Different strategies to remove water generated on the monoolein synthesis by enzymatic esterification of oleic acid with glycerol were evaluated. Esterification reactions were catalyzed by Penicillium camembertii lipase immobilized on SiO₂-PVA and runs carried out on a packed bed reactor (PBR) under continuous mode. The use of molecular sieves was the selected technique to remove the water formed, and for this purpose, two experimental setups were performed: (1) reactor packed with alternate layers of molecular sieves and immobilized lipase and (2) three reactors in series having a water column extractor between two immobilized packed beds, making it possible to replace the saturated molecular sieves by a regenerated one at a given time. Continuous run without applying dehydration techniques to remove the formed water was used as a control. The best performance was obtained using a water column extractor as verified by an increase in the monoolein formation from 22 to 35%. This strategy also provided a good performance of the bed reactor as a result of the high operational stability estimated for the immobilized derivative (half-life = 19 days).     

葡萄糖对光滑球拟酵母发酵生产丙酮酸的影响

在30L发酵罐中研究了初始葡萄糖质量浓度和补料方式对光滑球拟酵母WSH-IP303发酵生产丙酮酸的影响．实验确定116.4g/L左右是较为适宜的初始葡萄糖质量浓度,发酵58h时丙酮酸质量浓度和产率分别为58.0g/L和0.516g/g．采用初始葡萄糖质量浓度为53.4g/L,发酵24h分批补料至葡萄糖总质量浓度为115g/L的培养方式,发酵64h时丙酮酸质量浓度和产率分别为60.2g/L和0.559g/g;采用初始葡萄糖质量浓度为62.6g/L,发酵24h开始连续补料至葡萄糖总质量浓度为115g/L的培养方式,发酵72h时丙酮酸质量浓度和产率分别为63.3g/L和0.586g/g,与葡萄糖总质量浓度相似(115g/L)的分批发酵相比,丙酮酸产量分别提高了3.8%和9.1%．实验结果表明适宜的初始葡萄糖质量浓度能促进光滑球拟酵母发酵生产丙酮酸;尽管葡萄糖补料培养可适度提高丙酮酸的产量及产率,但生产强度却有所下降．     

Ethanol yield and volatile compound content in fermentation of agave must by Kluyveromyces marxianus UMPe-1 comparing with Saccharomyces cerevisiae baker's yeast used in tequila production

In tequila production, fermentation is an important step. Fermentation determines the ethanol productivity and organoleptic properties of the beverage. In this study, a yeast isolated from native residual agave must was identified as Kluyveromyces marxianus UMPe-1 by 26S rRNA sequencing. This yeast was compared with the baker's yeast Saccharomyces cerevisiae Pan1. Our findings demonstrate that the UMPe-1 yeast was able to support the sugar content of agave must and glucose up to 22% (w/v) and tolerated 10% (v/v) ethanol concentration in the medium with 50% cells survival. Pilot and industrial fermentation of agave must tests showed that the K. marxianus UMPe-1 yeast produced ethanol with yields of 94% and 96% with respect to fermentable sugar content (glucose and fructose, constituting 98%). The S. cerevisiae Pan1 baker's yeast, however, which is commonly used in some tequila factories, showed 76% and 70% yield. At the industrial level, UMPe-1 yeast shows a maximum velocity of fermentable sugar consumption of 2.27g·L(-1)·h(-1) and ethanol production of 1.38g·L(-1)·h(-1), providing 58.78g ethanol·L(-1) at 72h fermentation, which corresponds to 96% yield. In addition, the major and minor volatile compounds in the tequila beverage obtained from UMPe-1 yeast were increased. Importantly, 29 volatile compounds were identified, while the beverage obtained from Pan1-yeast contained fewer compounds and in lower concentrations. The results suggest that the K. marxianus UMPe-1 is a suitable yeast for agave must fermentation, showing high ethanol productivity and increased volatile compound content comparing with a S. cerevisiae baker's yeast used in tequila production.     

Continuous production of wine vinegar in bubble column reactors of up to 60-litre capacity

This paper describes the development of a continuous acetic acid fermentation process for the production of wine vinegar in bubble column reactors of up to 60 l capacity. To determine appropriate fermentation conditions a study of the influence of residual ethanol concentration, inlet flow rate and aeration was carried out using a 6-l laboratory reactor, white table wine as fermentation medium, a temperature of 30 °C and an air flow rate of 0.125 min^-1 (vvm). The concentration of acetic acid obtained in the continuous wine vinegar production ranged from 91 g/l at 28.6 ml/h to 28 g/l at 154.1 ml/h by increasing the inlet flow rate. As expected, the biomass decreased as well, from 208 mg/l to 106 mg/l. The maximum acetification rate was observed in the range 85-110 ml/h, corresponding to a value of about 1.1 g/l/h. A further increase in the flow rate produced a slight decrease in the acetification rate. Best yields, between 94.5 and 94.7%, were obtained in the flow rate range of 60-75 ml/h. The acetification rate was improved only by about 10% by increasing the aeration from 0.125 to 0.250 min^-1. The continuous wine vinegar production was scaled up from the laboratory fermentor to a 60-l pilot acetator. During the steady state (residential time >6), with an inlet flow rate of 950 ml/h, temperature of 30 °C and aeration of 0.250 min^-1, the following parameters were obtained: acetic acid concentration 72 g/l, overall productivity 1.41 g/l/h and yield 94.2%.     

Microbial production of inulo-oligosaccharides by an endoinulinase from Pseudomonas sp. expressed in Escherichia coli

In an attempt to utilize the whole cell as a biocatalyst for inulo-oligosaccharide (IOS) production from inulin, the endoinulinase gene (inu1) of Pseudomonas sp. was cloned into the plasmid pBR322 using EcoRI restriction endonuclease and Escherichia coli HB101 as the host strain. The endoinulinase from E. coli HB101/pKMG50 was constitutively expressed, producing a high yield of IOS (78%). In a batchwise reaction, the initial enzyme concentration determined the total oligosaccharide yield, and excess enzyme decreased the total oligosaccharide yield due to the formation of high amounts of free sugars such as glucose and fructose. The recombinant E. coli expressing endoinulinase activity were immobilized on a polystyrene carrier material, resulting in a dramatically enhanced thermal stability of the enzyme. Continuous production of IOS from inulin was also carried out at 50 degrees C using a bioreactor packed with the immobilized cells. Under the optimal operation conditions, continuous production of IOS was achieved with a productivity of 150 g/l.h for 17 d at 50 degrees C without significant loss of initial activity.     

Continuous Synthesis of Glucoamylase by Immobilized Fungal Mycelium of Aspergillus niger

The extracellular glucoamylase enzyme (EC 3.2.1.3) was synthesized continuously by the immobilized mycelial fragments of A. niger. Of the several polymeric matrices attempted for immobilization k-carrageenan and alginate were found to be the most effective. However, the enzyme activity exhibited by the immobilized mycelia (I.M.) was 15-20% lower than that of free cells under batch conditions. The immobilized cells have retained nearly the same enzymatic activity (120IU/g of I.M.) for 6 repeated batches and thereafter decline in activity was noticed. An aerated packed bed reactor with I.M. was operated continuously for 360 h. The volumetric productivity of the reactor was 1600IU/L/h for 192 h and reduced to 25% in 360 h.     

Effective onion vinegar production by a two-step fermentation system

A two-step fermentation system combining a repeated batch process using a flocculating yeast with a charcoal pellet bioreactor was developed for onion vinegar production. Juice from the red onion R-3, which contained 67.3 g/l total sugar, was smoothly converted to onion alcohol containing 30.6 g/l ethanol by repeated batch operation using the flocculating yeast Saccharomyces cerevisiae strain IR-2. Stable operation was possible and the maximum productivity was about 8.0 g/l/h. A packed bed bioreactor containing charcoal pellets produced from waste mushroom medium was then applied to continuous onion vinegar production from the onion alcohol. Onion vinegar was successfully produced, with a maximum productivity and acetic acid concentration of about 3.3 g/l/h and 37.9 g/l, respectively. The total acetic acid yield calculated from the amount of sugar consumed was 0.86. The two-step system was operated for 50 d and proved to be competitive with other systems in terms of its high productivity, high acetic acid yield, operational stability and low production costs.     

Lipase-catalyzed interesterification of high oleic sunflower oil and fully hydrogenated soybean oil comparison of batch and continuous reactor for production of zero trans shortening fats

Lipase-catalyzed interesterification of high oleic sunflower oil (HO) and fully hydrogenated soybean oil (FHSBO) at different weight ratios (55:45, 60:40, 65:35 and 70:30, HO:FHSBO) was carried out in both a batch-type reactor (BA) and a packed-bed reactor (PBR) to produce zero trans shortening. Interesterified products in both PBR and BA consisted of 34-46 g/100 g saturated fatty acids (SFA) (mainly stearic acid) and 54-66 g/100 g unsaturated fatty acid (USFA) (mainly oleic acid). After interesterification the physical characteristics such as melting point and solid fat content (SFC) were changed in each product. The differential scanning calorimetry (DSC) result showed that SFC content in PBR was higher than that in BA at each measured temperature. Decrease in tocopherols was also observed, however, the PBR product contained much higher amounts of tocopherols than BA product.     

Production of Single Cell Protein from Cassava Starch in Air-lift Fermenter by Cephalosporium eichhorniae

Batch and continuous cultivations were used to investigate the single cell protein (SCP) production by Cephalosporium eichhorniae 152 in an air-lift reactor. The fungus was grown in an 8L air-lift fermenter. The culture medium consisted of cassava as a sole-carbon source, (NH₄)₂SO₄ as a nitrogen source and KH₂PO₄ as a buffering agent. The pH of the medium was adjusted to 3.8 and was controlled during fermentation by using an automatic pH-controller via 0.5 N NaOH. In batch fermentation, 1% of cassava gave the highest yield of 0.4. The optimum condition for continuous fermentation was 1% cassava in feed medium at a dilution rate of 0.4 h^-1 (396 ml/h where 0.35 was the final yield. The productivity of continuous culture was found to be higher than that of the batch culture (0.135 g/l.h versus 0.04 g/lh).     

Continuous plant cell perfusion culture: bioreactor characterization and secreted enzyme production

Culture perfusion is widely practiced in mammalian cell processes to enhance secreted antibody production. Here, we report the development of an efficient continuous perfusion process for the cultivation of plant cell suspensions. The key to this process is a perfusion bioreactor that incorporates an annular settling zone into a stirred-tank bioreactor to achieve continuous cell/medium separation via gravitational sedimentation. From washout experiments, we found that under typical operating conditions (e.g., 200 rpm and 0.3 vvm) the liquid phase in the entire perfusion bioreactor was homogeneous despite the presence of the cylindrical baffle. Using secreted acid phosphatase (APase) produced in Anchusa officinalis cell culture as a model we have studied the perfusion cultures under complete or partial cell retention. The perfusion culture was operated under phosphate limitation to stimulate APase production. Successful operation of the perfusion process over four weeks has been achieved in this work. When A. officinalis cells were grown in the perfusion reactor and perfused at up to 0.4 vvd with complete cell retention, a cell dry weight exceeding 20 g/l could be achieved while secreted APase productivity leveled off at approximately 300 units/l/d. The culture became extremely dense with the maximum packed cell volume (PCV) surpassing 70%. In comparison, the maximum cell dry weight and overall secreted APase productivity in a typical batch culture were 10-12 g/l and 100-150 units/l/d, respectively. Operation of the perfusion culture under extremely high PCV for a prolonged period, however, led to declined oxygen uptake and reduced viability. Subsequently, cell removal via a bleed stream at up to 0.11 vvd was tested and shown to stabilize the culture at a PCV below 60%. With culture bleeding, both specific oxygen uptake rate and viability were shown to increase. This also led to a higher cell dry weight exceeding 25 g/l, and further improvement of secreted APase productivity that reached a plateau fluctuating around 490 units/l/d.