In situ cell retention of a CHO culture by a reverse‐flow diafiltration membrane bioreactor

Heterogeneities occur in various bioreactor designs including cell retention devices. Whereas in external devices changing environmental conditions cannot be prevented, cells are retained in their optimal environment in internal devices. Conventional reverse‐flow diafiltration utilizes an internal membrane device, but pulsed feeding causes temporal heterogeneities. In this study, the influence of conventional reverse‐flow diafiltration on the yeast Hansenula polymorpha is investigated. Alternating 180 s of feeding with 360 s of non‐feeding at a dilution rate of 0.2 h^?1 results in an oscillating DOT signal with an amplitude of 60%. Thereby, induced short‐term oxygen limitations result in the formation of ethanol and a reduced product concentration of 25%. This effect is enforced at increased dilution rate. To overcome this cyclic problem, sequential operation of three membranes is introduced. Thus, quasi‐continuous feeding is achieved reducing the oscillation of the DOT signal to an amplitude of 20% and 40% for a dilution rate of 0.2 h^?1 and 0.5 h^?1, respectively. Fermentation conditions characterized by complete absence of oxygen limitation and without formation of overflow metabolites could be obtained for dilution rates from 0.1 h^?1 – 0.5 h^?1. Thus, sequential operation of three membranes minimizes oscillations in the DOT signal providing a nearly homogenous culture over time. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1348–1355, 2014     

The simultaneous utilization of kinetic analysis and flow cytometry in the assessment of Lactobacillus rhamnosus ATCC 7469 physiological states produced by increasing oxygen limitation levels and lactic acid accumulation

Carbon limited continuous cultures of Lactobacillus rhamnosus ATCC 7469 were grown at dilution rates between 0.1 h⁻¹ and 0.6 h⁻¹. At 0.45 h⁻¹, oxygen uptake decreases producing a deficiency in the production of cell energy, lowering the concentration of biomass and finally accumulating glucose in the broth. Under the lack of energy pressure, L. rhamnosus ATCC 7469 triggers the production of lactic acid from pyruvate freeing NAD⁺ and stimulates glycolysis to continue, producing extra ATP from substrate-level phosphorylation. The 12-fold growing concentration of lactic acid and the 2-fold increase of succinic acid are in parallel with the steep 4-fold decrease of acetic acid production and small concentration changes of formic and propionic acids.The way the cells balance the available energy between the growing dilution rate and detoxification produces a stress within the culture, detected and described by flow cytometry. As the dilution rate increased, the proportion of L. rhamnosus ATCC 7469 cells with depolarized membrane steadily increased (1% at D = 0.20 h⁻¹, 8% at D = 0.30 h⁻¹, 14% at D = 0.45 h⁻¹ and 26% for D = 0.62 h⁻¹, respectively). Only a low level of 3.7% of the population did not recover from the demanding growth rates in the acidic environment.     

Continuous succinic acid fermentation by Actinobacillus succinogenes

Fermentations were performed in an external recycle bioreactor using CO₂ and d-glucose at feed concentrations of 20 and 40 g L⁻¹. Severe biofilm formation prevented kinetic analysis of suspended cell (‘chemostat’) fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells. The highest productivity of 6.35 g L⁻¹ h⁻¹ was achieved at a dilution rate of 0.56 h⁻¹. A constant succinic acid yield of 0.69 ± 0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm build-up – approximately 56% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h⁻¹ when the product concentrations were low (succinic acid < 10 g L⁻¹), while minimal growth was observed at succinic acid concentrations above this threshold.     

High cell density fed-batch fermentation for the production of a microbial lipase

Extracellular lipase of the yeast Candida rugosa was produced via high cell density fed-batch fermentations using palm oil as the sole source of carbon and energy. Feeding strategies consisted of a pH-stat operation, foaming-dependent control and specific growth rate control in different experiments. Compared to foaming-dependent feeding and the pH-stat operation, the specific growth rate control of feeding proved to be the most successful. At the specific growth rate control set at 0.05 h⁻¹, the final lipase activity in the culture broth was the highest at ∼700 U L⁻¹. This was 2.6-fold higher than the final enzyme activity obtained at a specific growth rate control set at 0.15 h⁻¹. The peak enzyme concentration achieved using the best foaming-dependent control of feeding was around 28% of the peak activity attained using the specific growth rate control of feeding at 0.05 h⁻¹. Similarly, the peak enzyme concentration attained using the pH-stat feeding operation was a mere 9% of the peak activity attained by specific growth rate control of feeding at a set-point of 0.05 h⁻¹. Fed-batch fermentations were performed in a 2 L stirred-tank bioreactor (30 °C, pH 7) with the dissolved oxygen level controlled at 30% of air saturation.     

Over-production of human interferon-γ by HCDC of recombinant Escherichia coli

A simple fed-batch process was developed using a modified variable specific growth rate feeding strategy for high cell density cultivation of Escherichia coli BL21 (DE3) expressing human interferon-gamma (hIFN-γ). The feeding rate was adjusted to achieve the maximum attainable specific growth rate during fed-batch cultivation. In this method, specific growth rate was changed from a maximum value of 0.55 h⁻¹ at the beginning of feeding and then it was reduced to 0.4 h⁻¹ at induction time.The final concentration of biomass and IFN-γ was reached to ∼115 g l⁻¹ (DCW) and 42.5 g(hIFN-γ) l⁻¹ after 16.5 h, also the final specific yield and overall productivity of recombinant hIFN-γ (rhIFN-γ) were obtained 0.37 g(hIFN-γ) g⁻¹ DCW and 2.57 g(hIFN-γ) l⁻¹ h⁻¹, respectively. According to available data this is the highest specific yield and productivity that has been reported for recombinant proteins production yet.     

Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming

A thermo-alkaline pectate lyase (BliPelA) gene from an alkaliphilic Bacillus licheniformis strain was cloned and overexpressed in Escherichia coli. Mature BliPelA exhibited maximum activity at pH 11 and 70 °C, and demonstrated cleavage capability on a broad range of substrates such as polygalacturonic acid, pectins, and methylated pectins. The highest specific activity, of 320 U mg⁻¹, was towards polygalacturonic acid. Significant ramie (Boehmeria nivea) fiber weight loss (21.5%) was obtained following enzyme treatment and combined enzyme-chemical treatment (29.3%), indicating a high ramie degumming efficiency of BliPelA. The total activity of recombinant BliPelA reached 1450.1 U ml⁻¹ with a productivity of 48.3 U ml⁻¹ h⁻¹ under high-cell-density cultivation with a glycerol exponential feeding strategy for 30 h in 1-l fed-batch fermenter, and 1380.1 U ml⁻¹ with a productivity of 57.5 U ml⁻¹ h⁻¹ after 24 h under constant glucose feeding in a 20-l fermenter using E. coli as the host. The enzyme yields reached 4.5 and 4.3 g l⁻¹ in 1-l and 20-l fed-batch fermenters, respectively, which are higher than those of most reported alkaline Pels. Based on these promising properties and high-level production, BliPelA shows great potential for application in ramie degumming in textile industry.     

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol

The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, and l-tryptophan at a constant feeding rate of 16 mL h⁻¹, the final concentration of violacein reached 4.13 g L⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34 g DCW L⁻¹ and 82.6 mg L⁻¹ h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.     

High production of poly-β-hydroxybutyrate (PHB) by an Azotobacter vinelandii mutant altered in PHB regulation using a fed-batch fermentation process

A mixed fermentation strategy based on exponentially fed-batch cultures (EFBC) and nutrient pulses with sucrose and yeast extract was developed to achieve a high concentration of PHB by Azotobacter vinelandii OPNA, which carries a mutation on the regulatory systems PTSNtr and RsmA-RsmZ/Y, that negatively regulate the synthesis of PHB. Culture of the OPNA strain in shake flaks containing PY-sucrose medium significantly improved growth and PHB production with respect to the results obtained from the cultures with the parental strain (OP). When the OPNA strain was cultured in a batch fermentation keeping constant the DOT at 4%, the maximal growth rate (0.16 h⁻¹) and PHB yield (0.30 g_PHB g_Suc⁻¹) were reached. Later, in EFBC, the OPNA strain increased three fold the biomass and 2.2 fold the PHB concentration in relation to the values obtained from the batch cultures. Finally, using a strategy of exponential feeding coupled with nutrient pulses (with sucrose and yeast extract) the production of PHB increased 7-fold to reach a maximal PHB concentration of 27.3 ± 3.2 g L⁻¹ at 60 h of fermentation. Overall, the use of the mutant of A. vinelandii OPNA, impaired in the PHB regulatory systems, in combination with a mixed fermentation strategy could be a feasible strategy to optimize the PHB production at industrial level.     

Hydrogen sulphide removal from air by biotrickling filter using open-pore polyurethane foam as a carrier

A study was conducted on H₂S removal in a biotrickling filter packed with open-pore polyurethane foam. Thiobacillus denitrificans was used as inoculum and a mixed culture population was developed during the process. The inhibitory effect of sulphate concentration (1.8–16.8 g L⁻¹), pH (6.9–8.6), trickling liquid velocity (TLV, 9.1–22.8 m h⁻¹), H₂S inlet concentration (20–157 ppmv) and the empty bed residence time (EBRT, 9–57 s) on the H₂S removal efficiency (RE) were thoroughly investigated. An increase in pH from 6.9 to 8.5 led to a corresponding increase in H₂S removal. In addition, an inhibitory effect of sulphate concentration was observed from 16.8 g L⁻¹ and the maximum elimination capacity was found to be 22 gS m⁻³ h⁻¹ (RE 98%). The RE was constant (98.8 ± 0.30%) for EBRT ≥ 16 s, but a decrease in the EBRT from 16 to 9 s led to a corresponding decrease in RE from 98.2 to 89.6% for a TLV of 9.1 m h⁻¹ and from 97.9 to 94.9% for a TLV of 22.8 m h⁻¹ (inlet load of 11.0 ± 0.2 gS m⁻³ h⁻¹). The sulphur oxidation capacity in the biotrickling filter was not diminished by the presence of other bacteria.     

Osmoregulation in Dunaliella,Part II: Photosynthesis and starch contribute carbon for glycerol synthesis during a salt stress in Dunaliella tertiolecta

In response to an osmotic stress, Dunaliella tertiolecta osmoregulates by metabolizing intracellular glycerol as compatible solute. Upon the application of a salt stress to 0.17 M or 0.7 M NaCl grown D. tertiolecta cells, rates of total glycerol synthesis were substantially higher than that arising from photosynthetic ¹⁴CO₂ fixation into glycerol. The source of this extra carbon is the reserve starch pool. The contribution of carbon from the starch breakdown to glycerol synthesis was estimated from the difference between the total glycerol synthesized and that arising from ¹⁴CO₂ fixation. The maximum observed flux of carbon from ¹⁴CO₂ to glycerol from photosynthesis was of the order of 15–20 μmol ¹⁴C-glycerol mg⁻¹ Chl h⁻¹, whereas the total glycerol synthesis reached about 70 μmol glycerol mg⁻¹ Chl h⁻¹. The contribution of products of starch breakdown to glycerol synthesis increased progressively with increasing salt stress. In light, contrary to prevailing assumptions, both the photosynthesis and the starch breakdown contribute carbon to glycerol biosynthesis. The relative contributions of these two processes in the light, while cells were actively photosynthesizing, depended on the magnitude of the salt stress. On application of dilution stress, the flux of carbon from newly photosynthetically fixed ¹⁴CO₂ into glycerol was reduced progressively with increasing dilution stress that was also accompanied by a decline in total glycerol contents of the cell. The maximum observed rate of glycerol dissimilation was about 135 μmol glycerol mg⁻¹ Chl h⁻¹.     

Enhanced ethanol production by continuous fermentation in a two-tank system with cell recycling

An experimental method for producing ethanol continuously was designed and tested with a cell-recycling two-tank system, which was composed of two fermentors, each of which was individually equipped with a settler for recycling flocculent yeast. This system was effective for the continuous fermentation of ethanol from sucrose at high cell-recycling (r = 0.8–0.9) and dilution (up to 0.48 h^?1) rates. The system has several advantages; the high cell concentration in the fermentors and relief of substrate and product inhibition. Thus, the enhanced productivity using this continuous fermentation with the two-tank cell-recycling system was significantly higher compared with that of the batch fermentation. The results indicate that increased recycling ratios caused an increase in biomass concentration and subsequently, product concentration in the tank. The ethanol productivity increased with the dilution rate, but higher dilution rates could render increasing amounts of sugar unconverted. Continuous fermentation with the sugar feed concentration of 160 g/l at r = 0.9 and dilution rate of 0.2 h^?1 achieved the highest productivity with less than 2% of the unconverted sugar in the product steam. Under the same cell recycling ratios a productivity range of 6.9–7.5 g/l h^?1 could be achieved with feeding concentrations of 80–200 g/l, while batch fermentation at these sugar concentrations led to productivities of 3.85–4.48 g/l h^?1.     

Escherichia coli HGT: Engineered for high glucose throughput even under slowly growing or resting conditions

Aerobic production-scale processes are constrained by the technical limitations of maximum oxygen transfer and heat removal. Consequently, microbial activity is often controlled via limited nutrient feeding to maintain it within technical operability. Here, we present an alternative approach based on a newly engineered Escherichia coli strain. This E. coli HGT (high glucose throughput) strain was engineered by modulating the stringent response regulation program and decreasing the activity of pyruvate dehydrogenase. The strain offers about three-fold higher rates of cell-specific glucose uptake under nitrogen-limitation (0.6 g_Glc g_CDW⁻¹ h⁻¹) compared to that of wild type, with a maximum glucose uptake rate of about 1.8 g_Glc g_CDW⁻¹ h⁻¹ already at a 0.3 h⁻¹ specific growth rate. The surplus of imported glucose is almost completely available via pyruvate and is used to fuel pyruvate and lactate formation. Thus, E. coli HGT represents a novel chassis as a host for pyruvate-derived products.     

Ability of a perfluoropolymer membrane to tolerate by-products of ethanol fermentation broth from dilute acid-pretreated rice straw

A perfluoropolymer (PFP) membrane has been prepared for use in vapor permeation to separate aqueous ethanol mixtures produced from rice straw with xylose-assimilating recombinant Saccharomyces cerevisiae. PFP membranes commonly have been used for dehydration process and possess good selectivity and high permeances. The effects of by-products during dilute acid pretreatment, addition of yeast extract, and ethanol fermentation on PFP membrane performance were investigated. While feeding mixtures of ethanol (90 wt%) in water, to which individual by-products (0.1–2 g/L) were added, the PFP membrane demonstrated no clear change in permeation rate (439–507 g m⁻² h⁻¹) or separation factor (14.9–23.5) from 2 to 4 h of the process. The PFP membrane also showed no clear change in permeation rate (751–859 g m⁻² h⁻¹) or separation factor (12.5–13.8) while feeding the mixture (final ethanol conc.: 61 wt%) of ethanol and distillation of the fermentation broth using a suspended fraction of dilute acid-pretreated rice straw for 20 h. These results suggest that the PFP membrane can tolerate actual distillation liquids from ethanol fermentation broth obtained from lignocellulosic biomass pretreated with dilute acid.     

Fed-batch culture of recombinant Saccharomyces cerevisiae for glucose 6-phosphate dehydrogenase production

We examined glucose 6-phosphate dehydrogenase (G6PD) production by fed-batch cultivation, using a recombinant strain of Saccharomyces cerevisiae W303-181 overexpressing this enzyme. The cultivations were carried out in a 3 L fermenter at pH 5.7, 30 °C, 2.0 vvm aeration, 200 rpm agitation and an inoculum concentration of 1.0 g/L. The volume of the culture medium in the fed-batch process varied from 1.333 to 2.0 L, due to the addition of 15.0 g/L glucose solution during 5 h. Different feeding rates were studied (exponentially increasing and decreasing feeding rates), and the feeding profile was determined by values of the parameter K (time constant), namely: 0.2, 0.5 and 0.8 h⁻¹. The best enzyme production (847 U/L) was obtained with an exponentially increasing feeding rate and K = 0.2 h⁻¹. The results attained also showed that this process is promising for G6PD production.     

Continuous 2-keto-gluconic acid (2KGA) production from corn starch hydrolysate by Pseudomonas fluorescens AR4

A continuous fermentation process for 2-keto-gluconic acid (2KGA) production from cheap raw material corn starch hydrolysate was developed using the strain Pseudomonas fluorescens AR4. The dilution rate and feeding glucose concentration had a significant effect on the cell concentrations, glucose utilization and 2KGA production performance. The optimal operating factors were obtained as: 0.065 h⁻¹ of dilution rate, 180 g/L of feeding glucose concentration, and 16 h of batch fermentation time as the starting point. Under these conditions, the steady state had the 135.92 g/L of produced 2KGA concentration, 8.83 g/L.h of average volumetric productivity, and 0.9510 g/g of yield. In conclusion, the proposed efficient and stable continuous fermentation process for 2KGA production by the strain P. fluorescens AR4 is potentially competitive for industrial production from corn starch hydrolysate in terms of 2KGA productivity and yield.     

d- and l-lactic acid production from fresh sweet potato through simultaneous saccharification and fermentation

The aim of this study was to develop a bioprocess for l- and d-lactic acid production from raw sweet potato through simultaneous saccharification and fermentation by Lactobacillus paracasei and Lactobacillus coryniformis, respectively. The effects of enzyme and nitrogen source concentrations as well as of the ratio of raw material to medium were investigated. At dried material concentrations of 136.36–219.51 g L⁻¹, yields of 90.13–91.17% (w/w) and productivities of 3.41–3.83 g L⁻¹ h⁻¹ were obtained with lactic acid concentrations as high as 198.32 g L⁻¹ for l-lactic acid production. In addition, d-lactic acid was produced with yields of 90.11–84.92% (w/w) and productivities of 2.55–3.11 g L⁻¹ h⁻¹ with a maximum concentration of 186.40 g L⁻¹ at the same concentrations of dried material. The simple and efficient process described in this study will benefit the tuber and root-based lactic acid industries without requiring alterations in plant equipment.     

Seasonal variations in photosynthetic irradiance response curves of macrophytes from a Mediterranean coastal lagoon

The main photosynthesis and respiration parameters (dark respiration rate, light saturated production rate, saturation irradiance, photosynthetic efficiency) were measured on a total of 23 macrophytes of the Thau lagoon (2 Phanerogams, 5 Chlorophyceae, 10 Rhodophyceae and 6 Phaeophyceae). Those measurements were performed in vitro under controlled conditions, close to the natural ones, and at several seasons. Concomitantly, measurements of pigment concentrations, carbon, phosphorous and nitrogen contents in tissues were performed. Seasonal intra-specific variability of photosynthetic parameters was found very high, enlightening an important acclimatation capacity. The highest photosynthetic capacities were found for Chlorophyceae (e.g. Monostroma obscurum thalli at 17 °C, 982 μmol O₂ g⁻¹ dw h⁻¹ and 9.1 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹, respectively for light saturated net production rate and photosynthetic efficiency) and Phanerogams (e.g. Nanozostera noltii leaves at 25 °C, 583 μmol O₂ g⁻¹ dw h⁻¹ and 2.6 μmol O₂ g⁻¹ dw h⁻¹/μmol photons m⁻² s⁻¹ respectively for light saturated net production rate and photosynthetic efficiency). As expected, species with a high surface/volume ratio were found to be more productive than coarsely branched thalli and thick blades shaped species. Contrary to R_d (ranging 6.7–794 μmol O₂ g⁻¹ dw h⁻¹, respectively for Rytiphlaea tinctoria at 7 °C and for Dasya sessilis at 25 °C) for which a positive relationship with water temperature was found whatever the species studied, the evolution of P/I curves with temperature exhibited different responses amongst the species. The results allowed to show summer nitrogen limitation for some species (Gracilaria bursa-pastoris and Ulva spp.) and to propose temperature preferences based on the photosynthetic parameters for some others (N. noltii, Zostera marina, Chaetomorpha linum).     

Repeatability of a running heat tolerance test

At present there is no standardised heat tolerance test (HTT) procedure adopting a running mode of exercise. Current HTTs may misdiagnose a runner's susceptibility to a hyperthermic state due to differences in exercise intensity. The current study aimed to establish the repeatability of a practical running test to evaluate individual's ability to tolerate exercise heat stress. Sixteen (8M, 8F) participants performed the running HTT (RHTT) (30 min, 9 km h⁻¹, 2% elevation) on two separate occasions in a hot environment (40 °C and 40% relative humidity). There were no differences in peak rectal temperature (RHTT1: 38.82±0.47 °C, RHTT2: 38.86±0.49 °C, Intra-class correlation coefficient (ICC)=0.93, typical error of measure (TEM)=0.13 °C), peak skin temperature (RHTT1: 38.12±0.45, RHTT2: 38.11±0.45 °C, ICC=0.79, TEM=0.30 °C), peak heart rate (RHTT1: 182±15 beats min⁻¹, RHTT2: 183±15 beats min⁻¹, ICC=0.99, TEM=2 beats min⁻¹), nor sweat rate (1721±675 g h⁻¹, 1716±745 g h⁻¹, ICC=0.95, TEM=162 g h⁻¹) between RHTT1 and RHTT2 (p>0.05). Results demonstrate good agreement, strong correlations and small differences between repeated trials, and the TEM values suggest low within-participant variability. The RHTT was effective in differentiating between individuals physiological responses; supporting a heat tolerance continuum. The findings suggest the RHTT is a repeatable measure of physiological strain in the heat and may be used to assess the effectiveness of acute and chronic heat alleviating procedures.     

Enhancement of polysaccharide production in suspension cultures of protocorm-like bodies from Dendrobium huoshanense by optimization of medium compositions and feeding of sucrose

A strategy that optimization of medium compositions for maximum biomass followed by feeding of sucrose for maximum polysaccharide synthesis was developed for enhancing polysaccharide production in suspension culture of protocorm-like bodies (PLBs) of Dendrobium huoshanense C.Z. Tang et S.J. Cheng. In growth stage, the original half-strength MS medium was optimized with carbon sources, nitrogen sources and metal ion combinations. The effects of different carbon sources on PLBs growth were remarkable and sucrose at 35 g l⁻¹ was the most suitable. Sole nitrate nitrogen of 30 mmol l⁻¹ was the best for PLBs growth. Metal ions (Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) showed different influences on PLBs growth. The optimal concentration of Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ was 4.5 mmol l⁻¹, 0.1 mmol l⁻¹, 0.5 mmol l⁻¹ and 0.06 mmol l⁻¹, respectively. In the optimized medium (sucrose, nitrate, Ca²⁺, Fe²⁺, Mn²⁺ and Zn²⁺ concentration as described above, the other component concentration seen in half-strength MS), 33.9 g DW l⁻¹ PLBs were harvested after 30 days of culture and biomass increase was improved 245% as compared with that in the original medium. In production stage, polysaccharide synthesis was significantly improved by the feeding sucrose. The maximum polysaccharide production (22 g l⁻¹) was obtained in the case of 50 g l⁻¹ sucrose feeding at day 30 of culture, which was about 109-fold higher than that in the original medium without feeding of sucrose.     

Galactose-limited fed-batch cultivation of Escherichia coli for the production of lacto-N-tetraose

Lacto-N-tetraose (Gal(β1-3)GlcNAc(β1-3)Gal(β1-4)Glc) is one of the most abundant oligosaccharide structures in human milk. We recently described the synthesis of lacto-N-tetraose by a whole-cell biotransformation with recombinant Escherichia coli cells. However, only about 5% of the lactose was converted into lacto-N-tetraose by this approach. The major product obtained was the intermediate lacto-N-triose II (GlcNAc(β1-3)Gal(β1-4)Glc).In order to improve the bioconversion of lactose to lacto-N-tetraose, we have investigated the influence of the carbon source on the formation of lacto-N-tetraose and on the intracellular availability of the glycosyltransferase substrates, UDP-N-acetylglucosamine and UDP-galactose. By growth of the recombinant E. coli cells on D-galactose, the yield of lacto-N-tetraose (810.8 mg L⁻¹ culture) was 3.6-times higher compared to cultivation on D-glucose.Using fed-batch cultivation with galactose as sole energy and carbon source, a large-scale synthesis of lacto-N-tetraose was demonstrated. During the 26 h feeding phase the growth rate (μ = 0.05) was maintained by an exponential galactose feed. In total, 16 g L⁻¹ lactose were fed and resulted in final yields of 12.72 ± 0.21 g L⁻¹ lacto-N-tetraose and 13.70 ± 0.10 g L⁻¹ lacto-N-triose II. In total, 173 g of lacto-N-tetraose were produced with a space-time yield of 0.37 g L⁻¹ h⁻¹.