Simplified feeding strategies for fed-batch cultivation of Kluyveromyces marxianus in cheese whey

The present work reports the effect of simple feeding strategies to obtain high-cell-density cultures of Kluyveromyces marxianus maximizing β-galactosidase productivity using cheese whey as basic medium. Linear and exponential feeding strategies, with feeding times of 20, 25 and 35 h, and three different feeding media concentrations (140 g/L, 210 g/L, and 280 g/L lactose concentration), were tested. Final biomass concentration reached 35 g cells dry weight/L and our results showed that continuous lactose addition to culture were able to produce high specific enzyme activities, consequently improving volumetric activities of β-galactosidase when compared to batch cultivations. The best fed-batch strategy, which was the feeding of three-fold lactose concentration in the cheese whey-medium during 25 h, resulted in β-galactosidase productivity of 291 U/L h, representing an increase of more than 50% compared to batch cultivations.     

Production of poly (3-hydroxybutyrate) from starch by Azotobacter chroococcum

Production of poly (3-hydroxybutyrate) (PHB) from starch was investigated in flask, batch, and fed-batch cultures of Azotobacter chroococcum. In flask culture, PHB content increased up to 74% of dry cell wt with increasing culture volume. In batch culture, PHB content increased to 44% with O2 limitation. In fed-batch culture, cell concentration of 71 g/l with 20% PHB was obtained without O2 limitation, whereas cell concentration of 54 g/l with 46% PHB was obtained with O2 limitation.     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Large-scale production of endotoxin-free plasmids for transient expression in mammalian cell culture

Transient expression of recombinant proteins in mammalian cell culture in a 100-L scale requires a large quantity of plasmid that is very labour intensive to achieve with shake flask cultures and commercially available plasmid purification kits. In this paper we describe a process for plasmid production in 100-mg scale. The fermentation is carried out in a 4-L fed-batch culture with a minimal medium. The detection of the end of batch and triggering the exponential (0.1 h(-1)) feed profile was unattended and controlled by Multi-fermenter Control System. A restricted specific growth rate in fed-batch culture increased the specific plasmid yield compared to batch cultures with minimal and rich media. This together with high biomass concentration (68-107 g L(-1) wet weight) achieves high volumetric yields of plasmid (95-277 mg L(-1) depending on the construct). The purification process consisted of alkaline lysis, lysate clarification and ultrafiltration, two-phase extraction with Triton X-114 for endotoxin removal, anion-exchange chromatography as a polishing step, ultrafiltration and sterile filtration. Both fermentation and purification processes were used without optimisation for production of four plasmids yielding from 39 to 163 mg of plasmids with endotoxin content of 2.5 EU mg(-1) or less.     

过量表达自身hemA基因的重组大肠杆菌发酵生产5-氨基乙酰丙酸的研究

研究了优化重组大肠杆菌产5-氨基乙酰丙酸（ALA）的条件,提高大肠杆菌发酵生产AL气的产量。在测定重组大肠杆菌GT48的生长曲线的基础上,确定诱导时间,优化摇瓶发酵条件。然后,进一步在5L发酵罐上进行间歇和流加发酵研究。摇瓶实验表明,细胞培养最佳初始pH为6．5,最佳诱导时间为稳定期前期,最佳接种量为2％,过高的葡萄糖浓度对细胞生长和产物合成均有一定的抑制作用。在5L发酵罐间歇发酵中,重组菌产ALA能力达到47．8mg／L。采用流加发酵可以进一步将产物产量提高到63．8mg／L。构建的过量表达自身的hemA基因的大肠杆菌具有较高的产ALA能力,通过发酵条件优化和采用流加发酵可以提高AL气产量。     

High cell density cultivation of <Emphasis Type="Italic">Escherichia coli</Emphasis> with surface anchored transglucosidase for use as whole-cell biocatalyst for α-arbutin synthesis

A fed-batch culture strategy for the production of recombinant Escherichia coli cells anchoring surface-displayed transglucosidase for use as a whole-cell biocatalyst for α-arbutin synthesis was developed. Lactose was used as an inducer of the recombinant protein. In fed-batch cultures, dissolved oxygen was used as the feed indicator for glucose, thus accumulation of glucose and acetate that affected the cell growth and recombinant protein production was avoided. Fed-batch fermentation with lactose induction yielded a biomass of 18 g/L, and the cells possessed very high transglucosylation activity. In the synthesis of α-arbutin by hydroquinone glucosylation, the whole-cell biocatalysts showed a specific activity of 501 nkat/g cell and produced 21 g/L of arbutin, which corresponded to 76% molar conversion. A sixfold increased productivity of whole cell biocatalysts was obtained in the fed-batch culture with lactose induction, as compared to batch culture induced by IPTG.     

Production of β-carotene by recombinant Escherichia coli with engineered whole mevalonate pathway in batch and fed-batch cultures

Recombinant Escherichia coli engineered to contain the whole mevalonate pathway and foreign genes for β-carotene biosynthesis, was utilized for production of β-carotene in bioreactor cultures. Optimum culture conditions were established in batch and pH-stat fed-batch cultures to determine the optimal feeding strategy thereby improving production yield. The specific growth rate and volumetric productivity in batch cultures at 37°C were 1.7-fold and 2-fold higher, respectively, than those at 28°C. Glycerol was superior to glucose as a carbon source. Maximum β-carotene production (titer of 663 mg/L and overall volumetric productivity of 24.6 mg/L × h) resulted from the simultaneous addition of 500 g/L glycerol and 50 g/L yeast extract in pH-stat fed-batch culture.     

一株法夫酵母虾青素高产菌株的生产性能

为了评价虾青素高产菌株-法夫酵母JMU-MVP14的生产性能及建立虾青素高产发酵技术,通过测定糖、生物量、虾青素产量、总类胡萝卜素产量等发酵参数,用摇瓶试验对比了法夫酵母JMU-MVP14和出发菌株的差异,用7 L罐试验对比了pH值调控方式及补料培养基成分对发酵的影响,用1 m3罐试验评估了法夫酵母JMU-MVP14高密度发酵虾青素的产量水平。摇瓶发酵结果表明,法夫酵母JMU-MVP14虾青素及总类胡萝卜素的细胞产率分别达到6.01 mg/g及10.38 mg/g;7 L罐分批发酵试验结果表明,自动流加调     

Uricase production by a recombinant <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> strain harboring <Emphasis Type="Italic">Candida utilis</Emphasis> uricase gene

Uricase is an important medical enzyme which can be used to determine urate in clinical analysis, to therapy gout, hyperuricemia, and tumor lysis syndrome. Uricase of Candida utilis was successfully expressed in Hansenula polymorpha under the control of methanol oxidase promoter using Saccharomyces cerevisiae alpha-factor signal peptide as the secretory sequence. Recombinant H. polymorpha MU200 with the highest extracellular uricase production was characterized with three copies of expression cassette and selected for process optimization for the production of recombinant enzyme. Among the parameters investigated in shaking flask cultures, the pH value of medium and inoculum size had great influence on the recombinant uricase production. The maximum extracellular uricase yield of 2.6 U/ml was obtained in shaking flask culture. The yield of recombinant uricase was significantly improved by the combined use of a high cell-density cultivation technique and a pH control strategy of switching culture pH from 5.5 to 6.5 in the induction phase. After induction for 58 h, the production of recombinant uricase reached 52.3 U/ml (about 2.1 g/l of protein) extracellularly and 60.3 U/ml (about 2.4 g/l) intracellularly in fed-batch fermentation, which are much higher than those expressed in other expression systems. To our knowledge, this is the first report about the heterologous expression of uricase in H. polymorpha.     

Kinetic Analyses of Desulfurization of Dibenzothiophene by Rhodococcus erythropolis in Batch and Fed-Batch Cultures

The DbtS(sup+) phenotype (which confers the ability to oxidize selectively the sulfur atom of dibenzothiophene [DBT] or dibenzothiophene sulfone [DBTO(inf2)]) of Rhodococcus erythropolis N1-36 was quantitatively characterized in batch and fed-batch cultures. In flask cultures, production of the desulfurization product, monohydroxybiphenyl (OH-BP), was maximal at pH 6.0, while specific productivity (OH-BP cell(sup-1)) was maximal at pH 5.5. Quantitative measurements in fermentors (in both batch and fed-batch modes) demonstrated that DBTO(inf2) as the sole sulfur source yielded a greater amount of product than did DBT. Specifically, 100 (mu)M DBT maximally yielded (apprx=)40 (mu)M OH-BP, while 100 (mu)M DBTO(inf2) yielded (apprx=)60 (mu)M OH-BP. Neither maintaining the pH at 6.0 nor adding an additional carbon source increased the yield of OH-BP. The presence of SO(inf4)(sup2-) in growth media repressed expression of desulfurization activity, but SO(inf4)(sup2-) added to suspensions of cells grown in DBT or DBTO(inf2) did not inhibit desulfurization activity.     

Xylanase production by Trichoderma reesei Rut C-30 grown on L-arabinose-rich plant hydrolysates

The suitability of L-arabinose-rich plant hydrolysates as carbon sources and inducers of xylanase production in Trichoderma reesei Rut C-30 was tested. Significantly higher xylanase activities were obtained in cultures on oat husk and sugar beet pulp hydrolysates than on lactose. In batch culture with oat husk hydrolysate and lactose, the xylanase activity was about 9 times higher ( approximately 510 IU/ml) than in lactose ( approximately 60 IU/ml). Even higher xylanase activity ( approximately 630 IU/ml) was obtained when the batch cultivations were done on sugar beet pulp hydrolysate and lactose. In a fed-batch culture using oat husk hydrolysate-lactose the xylanase activity was as high as 1350 IU/ml in 4 days. The cellulase production clearly decreased when T. reesei was cultured on both hydrolysates compared to the cultivation on lactose. Moreover, the relative amounts of the xylanases I-III were similar regardless the used carbon source.     

Fed-batch cultivation of Bordetella pertussis: Metabolism and Pertussis Toxin production

The production of acellular pertussis in comparison with whole cell pertussis vaccines demands 5-25 times the amount of Bordetella pertussis' virulence factors, such as Pertussis Toxin (PT), to produce the same number of vaccine doses. An increase in the volumetric productivity by employing fed-batch rather than the currently used batch cultivations of B. pertussis could reduce the cost price of acellular pertussis vaccines. This study defined the conditions that enable fed-batch cultivations at high specific PT production. A solution containing lactate and glutamate was fed to the cultures at various rates. The feed rate and whether or not the fed substrates were completely consumed, significantly influenced cellular metabolism. If lactate was detectable in the culture broth while glutamate was not, poly-hydroxy-butyrate (PHB) was formed. Any PHB present was metabolized when glutamate became detectable again in the culture liquid. At higher lactate and glutamate concentrations, free fatty acids were produced. Though toxic, free fatty acids were not the reason the cultures stopped growing. By choosing appropriate conditions, a cell density of 6.5 g/L dry weight was reached, i.e. a 7-fold increase compared to batch culture. The metabolic mechanisms behind the formation of PHB and fatty acids are discussed, as well as how to increase the cell density further. The PT production stopped at 12 mg/L, well before growth stopped, indicating that regulatory mechanisms of PT production may be involved.     

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (AcNPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield.     

Improvement of Panax notoginseng cell culture for production of ginseng saponin and polysaccharide by high density cultivation in pneumatically agitated bioreactors

A Panax notoginseng cell culture was successfully scaled up from shake flask to 1.0-L bubble column reactor and concentric-tube airlift reactor. High-density bioreactor batch cultivation was carried out using a modified MS medium. The maximum cell density in batch cultures reached 20.1, 21.0 and 24.1 g/L in the shake flask, bubble column and airlift reactors, respectively, and their corresponding biomass productivity was 950, 1140 and 1350 mg/(L x d) for each. The productivity of ginseng saponin was 70, 96 and 99 mg/(L x d) in the flask, bubble column and airlift reactors, respectively; and the polysaccharide productivity reached 104, 119 and 151 mg/(L x d) for each. Furthermore, a fed-batch cultivation strategy was developed on the basis of specific oxygen uptake rate (SOUR), i.e., sucrose feeding before a sharp decrease of SOUR, and the highest cell density of 29.7 g/L was successfully achieved in the airlift bioreactor on day 17 with a very high biomass productivity of 1520 mg/(L x d). The concentrations of ginseng saponin and polysaccharide reached about 2.1 and 3.0 g/L, respectively, and their productivity was 106 (saponin) and 158 mg/(L x d) (polysaccharide). This work successfully demonstrated the high-density bioreactor cultivation of P. notoginseng cells in pneumatically agitated bioreactors and the reproduction of the shake flask culture results in bioreactors. The cell density, biomass productivity, production titer and productivity of both ginseng saponin and polysaccharide obtained here were the highest that have been reported on a reactor scale for all the ginseng species.     

Production of cellulase using a mutant strain of Trichoderma reesei growing on lactose in batch culture

The production of cellulases in batch culture was studied using a mutant strain of Trichoderma reesei C-5 growing on lactose. Growth kinetic parameters on 2% lactose were studied and the comparative results for growth and enzyme productivities at two different lactose levels are discussed. The cellulase synthesis rate depended on the lactose concentration in the medium. Although growth was favoured at a higher lactose level, the volumetric enzyme productivity did not increase in proportion and the specific enzyme productivity decreased to a certain extent, indicating that partial catabolic inhibition at higher lactose concentrations may be possible. However, it was noted that the mutant strain was highly depressed and capable of synthesising active cellulases on lactose.     

RNA recovery and detection of mRNA by RT-PCR from preserved prokaryotic samples

Levan fructotransferase (LFTase) from Arthrobacter ureafaciens K2032 was expressed with N-terminal fusion of a LacZ-derived secretion motif (TMITNSSSVP) using the lac promoter system in recombinant Escherichia coli JM109 [pUDF-A81]. In flask cultures, recombinant enzyme activity was detected in culture media, and sequence analysis of N-terminal residues showed that about 40% of the extracellular recombinant LFTase had an authentic N-terminus. In a fed-batch bioreactor containing recombinant E. coli at high cell concentrations (OD(600)>200), the extracellular LFTase accumulated to 46000 U ml(-1) (approximately 2.0 g l(-1)) which was almost 40% of total (intra- and extracellular) recombinant LFTase. The synthesized recombinant enzyme was secreted soon after gene expression was induced by IPTG. Prolonged high secretion caused cell lysis and growth inhibition during the production phase in fed-batch cultures. When lactose was added by continuous feed mode, the secretion of recombinant LFTase and hence the cell lysis were significantly delayed in spite of the increased synthesis level. Therefore the induced cell culture of recombinant E. coli could grow up to a much higher cell concentration with continuing recombinant enzyme synthesis. In the case of the controlled feed of lactose, the maximum activities (U ml(-1)) of total and extracellular LFTase were nearly 100% and 70% higher, respectively.     

Codon optimized Thermobifida fusca hydrolase secreted by Bacillus megaterium

Production and secretion of a 28,172 Da hydrolase from Thermobifida fusca (TFH) in Bacillus megaterium MS941 and WH323 was investigated in shake flask and pH controlled bioreactors. Successful production of heterologous TFH was achieved by adapting the original tfh gene to the optimal codon usage of B. megaterium. A codon adaption index close to one was reached. The codon optimized tfh was cloned into an open reading frame with DNA sequence for the N-terminal signal peptide of B. megaterium lipase A and a C-terminal His(6)-tag, all under the control of a xylose inducible promoter. Successful TFH production and secretion were observed using batch reactor cultivations with complex medium. Expression of the tfh gene from the P(xylA) promoter and secretion of produced TFH were compared in detail to batch reactor cultivations with semi-defined growth medium. For the first time, significant TFH secretion was achieved using a semi-defined medium in glucose limited fed batch cultivations yielding 10-fold higher cell densities compared to LB medium cultivation. Comparable volumetric TFH activities were obtained for both cultivation strategies. Surprisingly, measured specific TFH activities exhibited drastic discrepancies between preparations from LB and semi-defined medium grown B. megaterium. TFH recovery by Ni-chelate affinity chromatography resulted in higher purification factors when LB medium was used. These results indicated that secreted TFH is favorably produced by batch cultures of B. megaterium WH323 in LB medium.     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

Highly efficient strategy for enhancing taxoid production by repeated elicitation with a newly synthesized jasmonate in fed-batch cultivation of Taxus chinensis cells

A highly efficient bioprocessing strategy was developed for enhancing the production of plant secondary metabolites by repeatedly eliciting a fed-batch culture with a newly synthesized powerful jasmonate analog, 2,3-dihydroxypropyl jasmonate (DHPJA). In suspension cultures of a high taxuyunnanine C (Tc)-producing cell line of Taxus chinensis, 100 microM DHPJA was added on day 7 to fed-batch cultures with feeding of 20 g L(-1) sucrose on the same day. The synergistic effect of elicitation and substrate feeding on Tc biosynthesis was observed, which resulted in higher Tc accumulation than that by elicitation or sucrose feeding alone. More interestingly, both specific Tc yield (i.e., Tc content) and volumetric yield was further improved by a second addition of 100 microM DHPJA (on day 12) to the fed-batch cultures. In particular, with repeated elicitation and sucrose feeding the Tc volumetric yield was increased to 827 +/- 29 mg L(-1), which was 5.4-fold higher than that of the nonelicited batch culture. Furthermore, the above novel strategy was successfully applied from shake flask to a 1-L airlift bioreactor. A high Tc production and productivity of 738 +/- 41 mg L(-1) and 33.2 +/- 1.9 mg L(-1) d(-1), respectively, was achieved, which is higher than previous reports on Tc production in bioreactors. The results suggest that the aforementioned bioprocessing strategy may potentially be applied to other cell culture systems for efficient production of plant secondary metabolites.     

Protein-free fed-batch culture of non-GS NS0 cell lines for production of recombinant antibodies

Presented is a novel antibody production platform based on the fed-batch culture of recombinant, NS0-derived cell lines. A standardized fed-batch cell culture process was developed for five non-GS NS0 cell lines using enriched and optimized protein-free, cholesterol-free, and chemically defined basal and feed media. The process performed reproducibly and scaled faithfully from the 2-L to the 100-L bioreactor scale achieving a volumetric productivity of > 120 mg/L per day. Fed-batch cultures for all five cell lines exhibited significant lactate consumption when the cells entered the stationary or death phase. Peak and final lactate concentrations were low relative to a previously developed fed-batch process (FBP). Such low lactate production and high lactate consumption rates were unanticipated considering the fed-batch culture basal medium has an unconventionally high initial glucose concentration of 15 g/L, and an overall glucose consumption in excess of 17 g/L. The potential of this process platform was further demonstrated through additional media optimization, which has resulted in a final antibody concentration of 2.64 +/- 0.19 g/L and volumetric productivity of > 200 mg/L per day in a 13-day FBP for one of the five production cell lines. Use of this standardized protein-free, cholesterol-free NS0 FBP platform enables consistency in development time and cost effectiveness for manufacturing of therapeutic antibodies.