Production of long-chain isomaltooligosaccharides from maltotriose using the thermostable amylomaltase and transglucosidase enzymes

Amylomaltase and transglucosidase were combined to produce long-chain isomaltooligosaccharides (IMOs). IMOs are effective prebiotics that stimulate the growth of healthy bacteria in human intestines and thus promote better overall health. In this study, the p17bAMY amylomaltase was expressed from its gene, which had been directly isolated from soil samples, while transglucosidase was purchased and purified by a gel-filtration column. Crude amylomaltase was purified by heat treatment, Q-, and phenyl-sepharose column. The purified amylomaltase had a molecular weight of 57 kDa. Specificity on the substrates of the amylomaltase was also studied and it was found that this enzyme was able to catalyze transglucosylation activity using substrates G₂ to G₇. However, G₃ was the most preferred substrate for the enzyme. Here, K _m-G3 and k _cat/K _m were 23 mM and 1.72 × 10⁸ mM/min, respectively. Amylomaltase and transglucosidase were tested both alone and in combination on a G₃ substrate to study the efficient process for the IMOs production. The obtained products from the enzymatic reactions were monitored using the TLC analytical method and a densitometer. The amylomaltase led to products containing linear maltooligosaccharides, while the transglucosidase produced short-chain IMOs. Interestingly, when amylomaltase and transglucosidase were used in combination, long-chain IMOs with sizes larger than IMO₄ were observed under the determined condition.     

Continuous production of high-purity fructooligosaccharides and ethanol by immobilized Aspergillus japonicus and Pichia heimii

High-purity fructooligosaccharides (FOS) were produced from sucrose by an innovative process incorporating immobilized Aspergillus japonicus and Pichia heimii cells. Intracellular FTase of A. japonicus converted sucrose into FOS and glucose, and P. heimii fermented glucose mainly into ethanol. The continuous production of FOS was carried out using a tanks-in-series bioreactor consisting of three stirred tanks. When a solution composed of 1 g L^?1 yeast extract and 300 g L^?1 sucrose was fed continuously to the bioreactor at a dilution rate of 0.1 h^?1, FOS at a purity of up to 98.2 % could be achieved and the value-added byproduct ethanol at 79.6 g L^?1 was also obtained. One gram of sucrose yielded 0.62 g FOS and 0.27 g ethanol. This immobilized dual-cell system was effective for continuous production of high-purity FOS and ethanol for as long as 10 days.     

Optimization of growth and production of toxins by three dinoflagellates in photobioreactor cultures

A bioreactor system for biotoxin production was appraised against traditional methods of growing dinoflagellate cultures. In an optimised bioreactor culture (5.4?L) operated in batch mode, growth of Karenia selliformis was more efficient than in 15-L bulk carboy culture in terms of growth rate (μ?=?0.07?day^?1 versus 0.05?day^?1) and growth maximum (G _max, 169.10⁶ versus 41.10⁶ cells L^?1). Maximal gymnodimine concentration (1200?μg L^?1) in bioreactor culture was 8-fold higher than in bulk carboy culture, and the yield per cell (pg cell^?1) was 2-fold higher. Similarly the bioreactor batch culture of Alexandrium ostenfeldii performed more efficiently than carboy cultures in terms of growth rate (1.6-fold higher), growth maximum (15-fold higher) and desmethyl C spirolide (SPX-desMe-C) yield (5-fold higher [μg L^?1], though the yield [pg cell^?1basis] was lower). When bioreactor cultures of K. selliformis were operated in continuous mode, the yield of gymnodimine was substantially higher than a carboy or the bioreactor run in batch mode to growth max (793?μg day^?1 over 58?days in continuous culture was achieved versus an average of 60?μg day^?1 [carboy over 40?days] or 249?μg day^?1 [batch mode] over 26?days). Likewise in continuous bioreactor cultures of A. ostenfeldii run over 25?days, the yield of SPX-desMe-C (29?μg day^?1) was substantially higher than in same cultures run in batch mode or carboys (10.2 day^?1 and 7.7?μg day^?1 respectively). Similarly 5.4?L bioreactor batch cultures of K. brevisulcata reached 3.8-fold higher cell densities than carboy cultures, and when operated in continuous mode, the brevisulcatic acids were more efficiently produced than in batch culture (12?μg day^?1 versus 7?μg day^?1). When the bioreactor system was upscaled to 52?L, the maximum cell densities and toxin yields of K. brevisulcata cultures were somewhat less than those achieved in the smaller reactor, which was attributed to reduced light penetration.     

High cell density ethanol fermentation in an upflow packed-bed cell recycle bioreactor

An upflow packed-bed cell recycle bioreactor (IUPCRB) is proposed for obtaining a high cell density. The system is comprised of a stirred tank bioreactor in which cells are retained partially by a packed-bed. A 1.3 cm (ID) × 48 cm long packed-bed was installed inside a 2 L bioreactor (working volume 1 L). Continuous ethanol fermentation was carried out using a 100 g/L glucose solution containing Saccharomyces cerevisiae (ATCC 24858). Cell retention characteristics were investigated by varying the void fraction (VF) of the packed bed by packing it with particles of 0.8∼2.0 mm sized stone, cut hollow fiber pieces, ceramic, and activated carbon particles. The best results were obtained using an activated carbon bed with a VF of 30∼35%. The IUPCRB yielded a maximum cell density of 87 g/L, an ethanol concentration of 42 g/L, and a productivity of 21 g/L/h when a 0.5 h⁻¹ dilution rate was used. A natural bleeding of cells from the filter bed occurred intermittently. This cell loss consisted of an average of 5% of the cell concentration in the bioreactor when a high cell concentration (approximately 80 g/L) was being maintained.     

Itaconic acid production using an air-lift bioreactor in repeated batch culture of Aspergillus terreus

Repeated itaconic acid production using an air-lift bioreactor was carried out by three methods—two with cell recycling by means of centrifugation and filtration by a stainless steel filter set inside the bioreactor and one by repeated batch culture without cell recycling. In a flask culture, repeated itaconic acid production was stable for 9 cycles (45 d) and the production rate was 0.47 g/l/h. However, in the air-lift bioreactor, it was difficult to produce itaconic acid in the repeated batch culture with cell recycling for a long period due to a decrease in fluidity resulting from an increase in mycelium concentration. In the method without cell recycling, however, repeated itaconic acid production was stable for 4 cycles (21 d) and the production rate was 0.37 g/l/h.     

Ethanol production by Saccharomyces cerevisiae in biofilm reactors

Biofilms are natural forms of cell immobilization in which microorganisms attach to solid supports. At ISU, we have developed plastic composite-supports (PCS) (agricultural material (soybean hulls or oat hulls), complex nutrients, and polypropylene) which stimulate biofilm formation and which supply nutrients to the attached microorganisms. Various PCS blends were initially evaluated in repeated-batch culture-tube fermentation with Saccharomyces cerevisiae (ATCC 24859) in low organic nitrogen medium. The selected PCS (40% soybean hull, 5% soybean flour, 5% yeast extract-salt and 50% polypropylene) was then used in continuous and repeated-batch fermentation in various media containing lowered nitrogen content with selected PCS. During continuous fermentation, S. cerevisiae demonstrated two to 10 times higher ethanol production in PCS bioreactors than polypropylene-alone support (PPS) control. S. cerevisiae produced 30 g L⁻¹ ethanol on PCS with ammonium sulfate medium in repeated batch fermentation, whereas PPS-control produced 5 g L⁻¹ ethanol. Overall, increased productivity in low cost medium can be achieved beyond conventional fermentations using this novel bioreactor design. Received 20 May 1997/ Accepted in revised form 29 August 1997     

Optimized endodextranase-epoxy CIM® disk reactor for the continuous production of molecular weight-controlled prebiotic isomalto-oligosaccharides

An epoxy-activated monolithic Convective Interaction Media (CIM^®) disk was used for the immobilization of endodextranase D8144 from Penicillium sp. (EC 3.2.1.11) in order to produce on-line isomalto-oligosaccharides (IMOs) from Dextran T40. Enzymatic parameters, molecular weight of IMOs and performance of the IMmobilized Enzymes Reactor (IMER) were investigated. The immobilization yield of enzymes was about 45.3% (w/w), and the real specific activity close to 3.26 U mg⁻¹. The K_m values did not significantly change between free (12.8 g L⁻¹) and immobilized enzymes (14.2 g L⁻¹), due to the absence of diffusional limitation. The IMER system presented more than 80% of its residual activity after 5000 column volumes, highlighting the high stability of the immobilized endodextranases. Response surface methodology was used to enhance the performance of the IMER. Depending on dextran concentrations and flow rates, specific patterns of IMOs distributions were observed during the enzymatic hydrolysis. Finally, prebiotic activity was also investigated on IMOs produced by medium conditions (flow rate 0.3 mL min⁻¹ and dextran concentrations 4% w/w) against Lactobacillus rhamnosus GG (ATCC 53103). Their scores were at least as good as two commercialized fructo-oligosaccharides (FOS), Fibrulose^® F97 and Orafti^® P95.     

Interactions between Torulaspora delbrueckii and Saccharomyces cerevisiae in wine fermentation: influence of inoculation and nitrogen content

Alcoholic fermentation by an oenological strain of Torulaspora delbrueckii in association with an oenological strain of Saccharomyces cerevisiae was studied in mixed and sequential cultures. Experiments were performed in a synthetic grape must medium in a membrane bioreactor, a special tool designed to study indirect interactions between microorganisms. Results showed that the S. cerevisiae strain had a negative impact on the T. delbrueckii strain, leading to a viability decrease as soon as S. cerevisiae was inoculated. Even for high inoculation of T. delbrueckii (more than 20× S. cerevisiae) in mixed cultures, T. delbrueckii growth was inhibited. Substrate competition and cell-to-cell contact mechanism could be eliminated as explanations of the observed interaction, which was probably an inhibition by a metabolite produced by S. cerevisiae. S. cerevisiae should be inoculated 48 h after T. delbrueckii in order to ensure the growth of T. delbrueckii and consequently a decrease of volatile acidity and a higher isoamyl acetate production. In this case, in a medium with a high concentration of assimilable nitrogen (324 mg L^?1), S. cerevisiae growth was not affected by T. delbrueckii. But in a sequential fermentation in a medium containing 176 mg L^?1 initial assimilable nitrogen, S. cerevisiae was not able to develop because of nitrogen exhaustion by T. delbrueckii growth during the first 48 h, leading to sluggish fermentation.     

Effective conversion of industrial starch waste to L-Lactic acid by Lactococcus lactis in a dialysis sac bioreactor

We describe here a simple technological process based on the direct fermentation of potato starch waste (PSW), an inexpensive agro-processing industrial waste, by a potential probiotic strain, Lactococcus lactis subsp. lactis, for enhancing L-lactic acid production. To maximize bioconversion and increase cell stability, we designed and tested a novel dialysis sac-based bioreactor. Shake flask fermentation (SFF) and fed batch fermentation in the dialysis sac bioreactor were compared for L-lactic acid production efficiency. The results showed that the starch (20 g/L) in the PSW-containing medium was completely consumed within 24 h in the dialysis sac bioreactor, compared with 48 h in the SFF. The maximum lactic acid concentration (18.9 g/L) and lactic acid productivity (0.79 g/L·h) obtained was 1.2- and 2.4-fold higher in the bioreactor than by SFF, respectively. Simultaneous saccharification and fermentation was effected at pH 5.5 and 30 °C. L. lactis cells were viable for up to four cycles in the fed batch fermentation compared to only one cycle in the SFF.     

Comparative analysis of trehalose production by Debaryomyces hansenii and Saccharomyces cerevisiae under saline stress

The comparative analysis of growth, intracellular content of Na⁺ and K⁺, and the production of trehalose in the halophilic Debaryomyces hansenii and Saccharomyces cerevisiae were determined under saline stress. The yeast species were studied based on their ability to grow in the absence or presence of 0.6 or 1.0 M NaCl and KCl. D. hansenii strains grew better and accumulated more Na⁺ than S. cerevisiae under saline stress (0.6 and 1.0 M of NaCl), compared to S. cerevisiae strains under similar conditions. By two methods, we found that D. hansenii showed a higher production of trehalose, compared to S. cerevisiae; S. cerevisiae active dry yeast contained more trehalose than a regular commercial strain (S. cerevisiae La Azteca) under all conditions, except when the cells were grown in the presence of 1.0 M NaCl. In our experiments, it was found that D. hansenii accumulates more glycerol than trehalose under saline stress (2.0 and 3.0 M salts). However, under moderate NaCl stress, the cells accumulated more trehalose than glycerol. We suggest that the elevated production of trehalose in D. hansenii plays a role as reserve carbohydrate, as reported for other microorganisms.     

Bioprocess development for kefiran production by Lactobacillus kefiranofaciens in semi industrial scale bioreactor

Lactobacillus kefiranofaciens is non-pathogenic gram positive bacteria isolated from kefir grains and able to produce extracellular exopolysaccharides named kefiran. This polysaccharide contains approximately equal amounts of glucose and galactose. Kefiran has wide applications in pharmaceutical industries. Therefore, an approach has been extensively studied to increase kefiran production for pharmaceutical application in industrial scale. The present work aims to maximize kefiran production through the optimization of medium composition and production in semi industrial scale bioreactor. The composition of the optimal medium for kefiran production contained sucrose, yeast extract and K₂HPO₄ at 20.0, 6.0, 0.25 g L⁻¹, respectively. The optimized medium significantly increased both cell growth and kefiran production by about 170.56% and 58.02%, respectively, in comparison with the unoptimized medium. Furthermore, the kinetics of cell growth and kefiran production in batch culture of L. kefiranofaciens was investigated under un-controlled pH conditions in 16-L scale bioreactor. The maximal cell mass in bioreactor culture reached 2.76 g L⁻¹ concomitant with kefiran production of 1.91 g L⁻¹.     

Effects of oxygen volumetric mass transfer coefficient on transglutaminase production by Bacillus circulans BL32

The effects of oxygenation in cultures of Bacillus circulans BL32 on transglutaminase (TGase) production and cell sporulation were studied by varying the agitation speed and the volume of aeration. Kinetics of cultivations has been studied in batch systems using a 2 L bioreactor, and the efficiency of agitation and aeration was evaluated through the oxygen volumetric mass transfer coefficient (k_La). It was adopted a two-stage aeration rate control strategy: first stage to induce biomass formation, followed by a second stage, in which cell sporulation was stimulated. A correlation of TGase production, spores formation, and oxygen concentration was established. Under the best conditions (500 rpm; 2 vvm air flow, followed by no air supply during stationary phase; k_La of 33.7 h⁻¹), TGase production reached a volumetric production of 589 U/L after 50 h of cultivation and the enzyme yield was 906 U/g cells. These values are 61% higher than that obtained in shaker cultures and TGase productivity increased 82%, when k_La varied from 4.4 to 33.7 h⁻¹. The maximal cell concentration increased four times in relation to shaker cultures and the cultivation time for the highest TGase activity was reduced from 192 h to just 50 h. These results show the importance of bioprocess design for the production of microbial TGase, especially concerning the oxygen supply of cultures and the induction of cell sporulation.     

Sporulation in Hansenula wingei Is Induced by Nitrogen Starvation in Maltose-Containing Media

The sexually agglutinative yeast Hansenula wingei lives in association with bark beetles that inhabit coniferous trees. This yeast was induced to sporulate by malt extract, which contains a high percentage of maltose (50%) and a low percentage of nitrogen (0.5%). A solution of 1.5% maltose without any growth factors also induced ascosporogenesis in H. wingei. Thus, only a carbon source is required for sporulation as in Saccharomyces. However, potassium acetate did not induce sporulation in H. wingei as it does in S. cerevisiae. Instead, disaccharides (such as maltose, sucrose, or cellobiose) promote sporulation better than either monosaccharides (such as dextrose, fructose, or mannose) or respiratory substrates (such as ethanol or glycerol). The specificity of disaccharides in promoting sporulation in H. wingei may be considered an adaptation since these disaccharides are present in the natural environment of this yeast. In addition, the specificity of disaccharides may be related to the induction of the disaccharidase because cells precultured on dextrose sporulate well on maltose, but cells precultured on maltose sporulate poorly on maltose. When (NH₄)₂SO₄ was added at a low concentration (3 mM) to synthetic sporulation medium (1.5% maltose solution), sporulation was abolished, whereas other salts and nitrogen sources inhibited to a lesser extent and vitamins and trace elements had no effect. Oxygen was required for sporulation, as expected for an obligate aerobe. Maximal sporulation was achieved in 2% malt extract broth at high cell density (10⁹ cells per ml), pH 5, and 25°C. By using these optimal physiological conditions and hybrid strains selected from an extensive genetic breeding program, about 30% asci (10% tetrads) were obtained routinely. Thus, the genetics of cell recognition in this yeast can now be studied.     

可同化阿拉伯糖的木糖还原酿酒酵母菌株构建

[目的] 以秸秆等木质纤维素类生物质为原料生产液体生物燃料乙醇,目前生产成本高,大规模工业化生产尚有较大难度。构建能同化阿拉伯糖进行木糖还原生产木糖醇的重组酿酒酵母菌株,以实现原料中全糖利用、生产高附加值产品,实现产品多元化。[方法] 首先,利用CRISPR/Cas9基因编辑技术依次向出发菌株中导入阿拉伯糖代谢途径和木糖还原酶基因,使菌株获得代谢阿拉伯糖和将木糖转化为木糖醇的能力;其次,通过适应性驯化的进化工程手段,提高重组菌株对阿拉伯糖的利用效率;最后,通过混合糖发酵验证重组菌株利用阿拉伯糖和还原木糖产木糖醇的能力。[结果] 通过导入植物乳杆菌的阿拉伯糖代谢途径,酿酒酵母菌株获得了较好的利用阿拉伯糖生长繁殖的能力;进一步导入假丝酵母的木糖还原酶基因后,重组菌株在葡萄糖作为辅助碳源条件下可高效还原木糖产木糖醇,但阿拉伯糖的利用能力下降。利用以阿拉伯糖为唯一碳源的培养基进行反复批次驯化,阿拉伯糖的利用能力得以恢复和提升,得到表型较好的重组菌株KAX3-2。该菌株在木糖（50 g/L）和阿拉伯糖（20 g/L）混合糖发酵条件下发酵72 h时,对阿拉伯糖和木糖利用率分别达到42.1%和65.9%,木糖醇的收率为64%。[结论] 本研究成功构建了一株能有效利用阿拉伯糖并能将木糖转化为木糖醇的重组酿酒酵母菌株KAX3-2,为后续构建、获得阿拉伯糖代谢能力更强、木糖醇积累效率更高菌株的工作奠定了基础。     

Single cell protein production from mandarin orange peel

Summary As the hydrolysis of mandarin orange peel with macerating enzyme (40°C, 24 h) produced 0.59 g g^–1 reducing sugar per dry peel compared to 0.36 by acid-hydrolysis (15 min at 120°C with 0.8 N H₂SO₄), the production of single cell protein (SCP) from orange peel was studied mostly using enzymatically hydrolyzed orange peel.When the enzymatically hydrolyzed peel media were used, the utilization efficiency of reducing sugars (%) and the growth yield from reducing sugars (g g^–1) were: 63 and 0.51 for Saccharomyces cerevisiae; 56 and 0.48 for Candida utilis; 74 and 0.69 for Debaryomyces hansenii and 64 and 0.70 for Rhodotorula glutinis. SCP production from orange peel by D. hansenii and R. glutinis were further studied. Batch cultures for 24 h at 30°C using 100 g dried orange peel produced 45 g of dried cultivated peel (protein content, 33%) with D. hansenii and 34 g (protein content, 50%) with R. glutinis, and 38 g (protein content, 44%) with a mixture of both yeasts.     

Homo-fermentative production of d-lactic acid by Lactobacillus sp. employing casein whey permeate as a raw feed-stock

Casein whey permeate (CWP), a lactose-enriched dairy waste effluent, is a viable feed stock for the production of value-added products. Two lactic acid bacteria were cultivated in a synthetic casein whey permeate medium with or without pH control. Lactobacillus lactis ATCC 4797 produced d-lactic acid (DLA) at 12.5 g l^?1 in a bioreactor. The values of Leudking–Piret model parameters suggested that lactate was a growth-associated product. Batch fermentation was also performed employing CWP (35 g lactose l^?1) with casein hydrolysate as a nitrogen supplement in a bioreactor. After 40 h, L. lactis produced 24.3 g lactic acid l^?1 with an optical purity >98 %. Thus CWP may be regarded as a potential feed-stock for DLA production.     

Immobilized cell microchannel bioreactor for evaluating fermentation characteristics of mixed substrate consumption and product formation

An immobilized cell microchannel bioreactor was designed to test continuous fermentation. The fermentation set-up included a bottom hydrophilic quartz channel to immobilize cells using 0.4 wt% polyethyleneimine and a top channel designed to continuously remove metabolically generated carbon dioxide using hydrophobic polypropylene. To evaluate fermentation characteristics of immobilized cells, ethanol fermentation was carried out using Saccharomyces cerevisiae and Pichia stipitis. The immobilized cell microchannel bioreactor was used to identify long-term activity of immobilized S. cerevisiae cells. The continuous flow microchannel bioreactor was operated stably over a period of 1 month. The immobilized cell microchannel bioreactor was used to examine the characteristics cells that consumed mixed substrates. The concentration ratio of glucose to xylose for simultaneous utilization of hemicellulosic sugars was evaluated using the microchannel bioreactor and the results were compared with those obtained by using conventional batch fermentation with P. stipitis.     

Oxygen mass transfer and scale-up studies in baffled roller bioreactors

Oxygen mass transfer was studied in conventional, bead mill and baffled roller bioreactors. Using central composite rotational design, impacts of size, rotation speed and working volume on the oxygen mass transfer were evaluated. Baffled roller bioreactor outperformed its conventional and bead mill counterparts, with the highest k _L a obtained in these configurations being 0.58, 0.19, 0.41 min^?1, respectively. Performances of the bead mill and baffled roller bioreactor were only comparable when a high bead loading (40 %) was applied. Regardless of configuration increase in rotation speed and decrease in working volume improved the oxygen mass transfer rate. Increase in size led to enhanced mass transfer and higher k _L a in baffled roller bioreactor (0.49 min^?1 for 2.2 L and 1.31 min^?1 for 55 L bioreactors). Finally, the experimentally determined k _L a in the baffled roller bioreactors of different sizes fit reasonably well to an empirical correlation describing the k _L a in terms of dimensionless numbers.     

Rapid and stable production of 2,3-butanediol by an engineered <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> strain in a continuous airlift bioreactor

Utilization of renewable feedstocks for the production of bio-based bulk chemicals, such as 2,3-butanediol (2,3-BDO), by engineered strains of the non-pathogenic yeast, Saccharomyces cerevisiae, has recently become an attractive option. In this study, to realize rapid production of 2,3-BDO, a flocculent, 2,3-BDO-producing S. cerevisiae strain YPH499/dPdAdG/BDN6-10/FLO1 was constructed from a previously developed 2,3-BDO-producing strain. Continuous 2,3-BDO fermentation was carried out by the flocculent strain in an airlift bioreactor. The strain consumed more than 90 g/L of glucose, which corresponded to 90% of the input, and stably produced more than 30 g/L of 2,3-BDO over 380 h. The maximum 2,3-BDO productivity was 7.64 g/L/h at a dilution rate of 0.200/h, which was higher than the values achieved by continuous fermentation using pathogenic bacteria in the previous reports. These results demonstrate that continuous 2,3-BDO fermentation with flocculent 2,3-BDO-producing S. cerevisiae is a promising strategy for practical 2,3-BDO production.