Fed-batch Cultivation Strategy Research Articles

Development of Fed-Batch Strategy to Produce Bacteria Cellulose

Bacteria Cellulose (BC) is a type of biopolymer widely used in areas such as biomedicine and paper manufacturing industries. The batch fermentation mode of BC is less cost-effective as the yield is low. This study aims to determine the best fed-batch cultivation strategy to produce BC of high yield and quality. First, batch fermentation mode was carried out to determine the glucose consumption profile of Gluconacetobacter xylinus using Yeast Extract Glucose Chloramphenicol Medium (YGC). By using the information, the fed-batch strategies with feeding frequencies of 1 time and 3 times per 14-day total fermentation period and varied feed composition (YGC medium and pure glucose solution) were set up. The yield, water holding capacity, morphology, and strength of BC were monitored. It is found that BC produced from fed-batch modes has a higher yield (3.75 g/L for low feed frequency and 4.79 g/L for high feed frequency) compared to batch mode (2.24 g/L). The BC produced with pure glucose solution has the highest water holding capacity (87.34 g water/g sample) among the different modes. The FE-SEM micrographs showed that BC produced in the high feeding frequency with YGC medium fed-batch mode has the highest microfibril density and, thus, the best mechanical strength from this mode. To conclude, the fed-batch mode with higher feeding frequency(3 days intervals for a total of 14 days) using YGC medium has the best BC yield and quality.

Comprehensive Exploration of the Growth and Lipid Synthesis Phases of T. oleaginosus Cultures Implementing Design of Experiments and Response Surface Methodology.

Trichosporon oleaginosus is an unconventional oleaginous yeast distinguished by its remarkable capacity to accumulate lipids in excess of 70% of its dry weight, particularly when cultivated in nitrogen-restricted conditions with ample carbon sources. A pivotal question that arises pertains to the nutrient dynamics in the culture medium, which give rise to both the excessive lipid content and corresponding lipid concentration. While previous research has predominantly focused on evaluating the impact of the initial carbon-to-nitrogen (C/N) ratio on lipid production, the precise critical thresholds of glucose and ammonium sulfate ((NH4)2SO4) at which growth and intracellular lipid production are either stimulated or impeded remain inadequately defined. This study employs an experimental design and response surface methodology to investigate the complex mechanism of lipid accumulation and its interaction with cellular growth. Application of the aforementioned methodologies resulted in the production of 10.6 g/L of microbial oil in batch cultures under conditions that correspond to a C/N ratio of 76. However, the primary objective is to generate knowledge to facilitate the development of efficient fed-batch cultivation strategies that optimize lipid production exclusively employing inorganic nitrogen sources by finely adjusting carbon and nitrogen levels. The intricate interaction between these levels is comprehensively addressed in the present study, while it is additionally revealed that as glucose levels rise within a non-inhibitory range, lipid-free biomass production decreases while lipid accumulation simultaneously increases. These findings set the stage for further exploration and the potential development of two-stage cultivation approaches, aiming to fully decouple growth and lipid production. This advancement holds the promise of bringing microbial oil production closer to commercial viability.

Scale-up and fed-batch cultivation strategy for the enhanced co-production of microbial lipids and carotenoids using renewable waste feedstock

Agro-industrial by-product valorization as a feedstock for the bioproduction of high-value products has demonstrated a feasible alternative to handle the environmental impact of waste. Oleaginous yeasts are promising cell factories for the industrial production of lipids and carotenoids. Since oleaginous yeasts are aerobic microorganisms, studying the volumetric mass transfer (kLa) could facilitate the scale-up and operation of bioreactors to grant the industrial availability of biocompounds. Scale-up experiments were performed to assess the simultaneous production of lipids and carotenoids using the yeast Sporobolomyces roseus CFGU-S005 and comparing the yields in batch and fed-batch mode cultivation using agro-waste hydrolysate in a 7 L bench-top bioreactor. The results indicate that oxygen availability in the fermentation affected the simultaneous production of metabolites. The highest production of lipids (3.4 g/L) was attained using the kLa value of 22.44 h−1, while higher carotenoid accumulation of 2.58 mg/L resulted when agitation speed was increased to 350 rpm (kLa 32.16 h−1). The adapted fed-batch mode in the fermentation increased the production yields two times. The fatty acid profile was affected according to supplied aeration and after the fed-batch cultivation mode. This study showed the scale-up potential of the bioprocess using the strain S. roseus in the obtention of microbial oil and carotenoids by the valorization of agro-industrial byproducts as a carbon source.

Effect of Fed-batch Culturing on the Growth and Lipid Production of Chlorella vulgaris fo. tertia Applying pH-auxostat Acetic Acid and Predefined Exponential Glucose Feeding

The techniques of heterotrophic microalgae cultivation used to be resulting in higher productivity and better yield than autotrophic culturing. Batch cultivation strategy is commonly used with high glucose concentration, but its potential is limited for biomass production at an industrial scale. Usually, the best productivity can obtain at lower glucose concentration. Moreover, other carbon sources can cause inhibition at higher concentrations. Therefore, the fed-batch cultivation strategy is an obvious choice, as it can maintain the optimal amount of carbon source can be maintained throughout the fermentation by automating the feeding. Such self-regulatory automation is provided by the pH-auxostat addition of acetic acid, which was investigated in this study for Chlorella vulgaris fo. tertia. The pH-auxostat fermentation was upscaled, then the feeding profile was modelled and transformed to another fermentation where glucose was used as a carbon source instead of acetic acid. Thus, the preferred carbon sources were compared under the same circumstances. It was found that the tested strain consumes dissolved oxygen very fast on both carbon substrates. It favored the acetic acid at high nitrogen and phosphorus concentrations. The final biomass concentration was 29.2 g/L under pH-auxostat fed-batch strategy with acetic acid and 18.8 g/L with glucose, respectively. The highest lipid content (393 mg/g) was measured from the biomass in the case of acetic acid. The fermentation settings need further optimization, but the results concluded that pH-auxostat acetic acid feeding has a great potential for scale-up of Chlorella fermentation.

A highly productive mixotrophic fed-batch strategy for enhanced microalgal cultivation

A highly productive fed-batch cultivation strategy is presented, that consists of intermittent pulses of organic carbon that promotes mixotrophic microalgal growth whilst favouring starch and lipid formation.

Development of a High-Titer Culture Medium for the Production of Cholesterol by Engineered Saccharomyces cerevisiae and Its Fed-Batch Cultivation Strategy.

Steroids are a class of compounds with cyclopentane polyhydrophenanthrene as the parent nucleus, and they usually have unique biological and pharmacological activities. Most of the biosynthesis of steroids is completed by a series of enzymatic reactions starting from cholesterol. Synthetic biology can be used to synthesize cholesterol in engineered microorganisms, but the production of cholesterol is too low to further produce other high-value steroids from cholesterol as the raw material and precursor. In this work, combinational strategies were established to increase the production of cholesterol in engineered Saccharomyces cerevisiae RH6829. The basic medium for high cholesterol production was selected by screening 8 kinds of culture media. Single-factor optimization of the carbon and nitrogen sources of the culture medium, and the addition of calcium ions, zinc ions and citric acid, further increased the cholesterol production to 192.53 mg/l. In the 5-L bioreactor, through the establishment of strategies for glucose and citric acid feeding and dissolved oxygen regulation, the cholesterol production was further increased to 339.87 mg/l, which was 734% higher than that in the original medium. This is the highest titer of cholesterol produced by microorganisms currently reported. The fermentation program has also been conducted in a 50-L bioreactor to prove its stability and feasibility.

Biodegradation of waste cooking oil and simultaneous production of rhamnolipid biosurfactant by Pseudomonas aeruginosa P7815 in batch and fed-batch bioreactor.

Biosurfactants are non-toxic, surface-active biomolecules capable of reducing surface tension (ST) and emulsifying interface at a comparably lower concentration than commercial surfactants. Yet, poor yield, costlier substrates, and complex cultivation processes limit their commercial applications. This study focuses on producing biosurfactants by Pseudomonas aeruginosa P7815 in batch and fed-batch bioreactor systems using waste cooking oil (WCO) as the sole carbon source. The batch study showed a 92% of WCO biodegradation ability of P. aeruginosa producing 11g L-1 of biosurfactant. To enhance this biosurfactant production, a fed-batch oil feeding strategy was opted to extend the stationary phase of the bacterium and minimize the effects of substrate deprivation. An enhanced biosurfactant production of 16g L-1 (i.e. 1.5 times of batch study) was achieved at a feed rate of 5.7g L-1d-1 with almost 94% of WCO biodegradation activity. The biosurfactant was characterized as rhamnolipid using Fourier transform infrared spectroscopy (FTIR), and its interfacial characterization showed ST reduction to 29 ± 1 mN m-1 and effective emulsification stability at pH value of 4, temperature up to 40°C and salinity up to 40g L-1. The biosurfactant exhibited antibacterial activity with minimum inhibitory concentration (MIC) values of 100µgmL-1 and 150µgmL-1 for pathogenic E. hirae and E. coli, respectively. These findings suggest that biodegradation of WCO by P. aeruginosa in a fed-batch cultivation strategy is a potential alternative for the economical production of biosurfactants, which can be further explored for biomedical, cosmetics, and oil washing/recovery applications.

Fermentation strategies for improving the production of bacteriocin‐like inhibitory substances by Lactobacillus brevis C23 with nutrient supplementation, pH, and temperature variations

In this study, fermentation strategies were carried out for improving the production of bacteriocin-like inhibitory substances by Lactobacillus brevis C23 with nutrient supplementation, pH, and temperature variations, as well as fed-batch cultivation. The BLIS activity was highest (91.52%) in the MRS medium supplemented with 1.5% (w/v) lactose and 1% (w/v) meat extract. pH 5 augmented both the cell biomass and BLIS activity, but incubation temperature only affected growth at 37°C. Fed-batch cultivation strategy that utilized meat extract feeding would positively affect cell growth, whereas BLIS activity was enhanced by lactose feeding. The highest BLIS activity from L. brevis C23 was obtained at the 24 hr mark by feeding the culture medium with lactose (77.73%). The BLIS production of L. brevis C23 fed with meat extract followed a similar trend to lactose (74.20%). Increased BLIS activity in the final solution would reduce the downstream step such as concentrating the purified product. Novelty impact statement Synergic effect of lactose and meat extract enhanced BLIS activity (91.52%) of CFS by Lactobacillus brevis C23 as compared to the control (69.29%) Temperature at 40℃ did not affect the BLIS activity, but it inhibited the cell growth. Fed-batch cultivation strategy that utilized meat extract feeding would positively affect cell growth, whereas BLIS activity was enhanced by lactose feeding.

Glucose-Limited Fed-Batch Cultivation Strategy to Mimic Large-Scale Effects in Escherichia coli Linked to Accumulation of Non-Canonical Branched-Chain Amino Acids by Combination of Pyruvate Pulses and Dissolved Oxygen Limitation

Insufficient mixing in large-scale bioreactors provokes gradient zones of substrate, dissolved oxygen (DO), pH, and other parameters. E. coli responds to a high glucose, low oxygen feeding zone with the accumulation of mixed acid fermentation products, especially formate, but also with the synthesis of non-canonical amino acids, such as norvaline, norleucine and β-methylnorleucine. These amino acids can be mis-incorporated into recombinant products, which causes a problem for pharmaceutical production whose solution is not trivial. While these effects can also be observed in scale down bioreactor systems, these are challenging to operate. Especially the high-throughput screening of clone libraries is not easy, as fed-batch cultivations would need to be controlled via repeated glucose pulses with simultaneous oxygen limitation, as has been demonstrated in well controlled robotic systems. Here we show that not only glucose pulses in combination with oxygen limitation can provoke the synthesis of these non-canonical branched-chain amino acids (ncBCAA), but also that pyruvate pulses produce the same effect. Therefore, we combined the enzyme-based glucose delivery method Enbase® in a PALL24 mini-bioreactor system and combined repeated pyruvate pulses with simultaneous reduction of the aeration rate. These cultivation conditions produced an increase in the non-canonical branched chain amino acids norvaline and norleucine in both the intracellular soluble protein and inclusion body fractions with mini-proinsulin as an example product, and this effect was verified in a 15 L stirred tank bioreactor (STR). To our opinion this cultivation strategy is easy to apply for the screening of strain libraries under standard laboratory conditions if no complex robotic and well controlled parallel cultivation devices are available.

Engineering oleaginous yeast Rhodotorula toruloides for overproduction of fatty acid ethyl esters

BackgroundProduction of biofuels and green chemicals by microbes is currently of great interest due to the increasingly limited reserves of fossil fuels. Biodiesel, especially fatty acid ethyl esters (FAEEs), is considered as an attractive alternative because of its similarity with petrodiesel and compatibility with existing infrastructures. Cost-efficient bio-production of FAEEs requires a highly lipogenic production host that is suitable for large-scale fermentation. As a non-model oleaginous yeast that can be cultured to an extremely high cell density and accumulate over 70% cell mass as lipids, Rhodotorula toruloides represents an attractive host for FAEEs production.ResultsWe first constructed the FAEE biosynthetic pathways in R. toruloides by introducing various wax ester synthase genes from different sources, and the bifunctional wax ester synthase/acyl-CoA-diacyglycerol acyltransferase (WS/DGAT) gene from Acinetobacter baylyi was successfully expressed, leading to a production of 826 mg/L FAEEs through shake-flask cultivation. We then mutated this bifunctional enzyme to abolish the DGAT activity, and further improved the titer to 1.02 g/L. Finally, to elevate the performance of Δku70-AbWS* in a bioreactor, both batch and fed-batch cultivation strategies were performed. The FAEEs titer, productivity and yield were 4.03 g/L, 69.5 mg/L/h and 57.9 mg/g (mg FAEEs/g glucose) under batch cultivation, and 9.97 g/L, 90.6 mg/L/h, and 86.1 mg/g under fed-batch cultivation. It is worth mentioning that most of the produced FAEEs were secreted out of the cell, which should greatly reduce the cost of downstream processing.ConclusionWe achieved the highest FAEEs production in yeast with a final titer of 9.97 g/L and demonstrated that the engineered R. toruloides has the potential to serve as a platform strain for efficient production of fatty acid-derived molecules.

Model-based Optimization of Biopolymer Production from Glycerol

The present study focuses on sustainable production of biodegradable polymers by <br /> Cupriavidus necator DSMZ 545 using glycerol as substrate. The batch growth and biopolymer production kinetics were established in a 7-L bioreactor, which resulted in a <br /> total biomass of 8.88 g L–1 and poly(3-hydroxybutyrate) (PHB) accumulation of 6.76 g L–1. The batch kinetic and independently acquired substrate inhibition data were then used to develop a mathematical model for PHB production process. This was eventually used to design different nutrient feeding strategies under constant feed rate, decreasing feed rate, and pseudo steady state of substrate (glycerol) to optimize the PHB production during fed-batch cultivation. Among all the fed-batch cultivation strategies, the highest PHB accumulation and productivity of 13.12 g L–1 and 0.27 g L–1 h–1, respectively, was <br /> achieved in fed-batch bioreactor cultivation where a pseudo steady state with respect to <br /> glycerol was maintained.

Modification Wettability and Interfacial Tension of Heavy Crude Oil by Green Bio-surfactant Based on Bacillus licheniformis and Rhodococcus erythropolis Strains under Reservoir Conditions: Microbial Enhanced Oil Recovery

Oil spill contamination in soil is still problematic. At the same time, petroleum-contaminated soil in oil reservoirs contain various microbes, which have the ability for biosurfactant production. Extracting these biosurfactants is a very promising and cost-effective strategy for the microbial enhanced oil recovery process. Biosurfactants production using Bacillus licheniformis AnBa7 and Rhodococcus erythropolis sp., isolated from Egyptian crude oils, was enhanced using various carbon sources. The best biosurfactant characteristics were observed when 1% of crude oil was used as a carbon source. The production was further improved by using a developed fed-batch cultivation strategy depends on using 1% Glucose as a single addition at the beginning of the culture. Then 1% of crude oil was added three times during the production process. This strategy enhanced surfactin and trehalose productivity by 1.8 and 4.7 fold higher than the normal conditions, respectively. The surface-active and thermodynamic properties were studied. The results indicated that the calculated values of ΔGmic for surfactin complex, and trehalose complex were −18.47 and −18.28 kJ/mol at 60 °C, respectively while ΔGads values were −30.42 and −29.46 kJ/mol at 60 °C. The interfacial tension (IFT) values of surfactin complex and trehalose complex systems were ranging from 0.75 to 0.19 mN m–1 and from 0.93 to 0.26 mN m–1 at 60 °C, respectively. However, the (IFT) for the blank solution was ∼11.57 mN m–1, and the wettability was changed to an excellent water-wet state (θ = ∼ 17.42–24.0°). The core-flooding studies showed that the enhanced oil recovery for surfactin complex and trehalos complex, at maximum concentration 6 g/L, were 59.21% and 51.83%, respectively. A predicted mechanism was illustrating through the text.

Scale-up of two-step acid-catalysed glycerol pretreatment for production of oleaginous yeast biomass from sugarcane bagasse by Rhodosporidium toruloides

Two-step dilute acid and acid-catalysed glycerol pretreatment was developed to maximise sugar yield from sugarcane bagasse. At the laboratory scale, dilute acid pretreatment at 130 °C followed by acid-catalysed glycerol pretreatment at 170 °C led to a total sugar (C5 + C6) yield of 82%, 31% higher than that from one-step acid-catalysed glycerol pretreatment. At the pilot scale, the two-step dilute acid and acid-catalysed glycerol pretreatment led to a maximum sugar yield of 74%, 13% higher than that from one-step pretreatment with 52% reduction in glycerol usage. The enzymatic hydrolysate containing glucose and residual glycerol were used to produce microbial oils by a Rhodosporidium toruloides strain. A fed-batch cultivation strategy led to the production of 44.8 g/L cell mass, including 26.6 g/L oil, 8.6 g/L protein and 12.7 mg/L carotenoid. The cell mass and oil yields were 19% higher than those from batch cultivation as feedstock inhibition and catabolite repression were alleviated.

A novel fed-batch strategy to boost cyst cells production based on the understanding of intracellular carbon and nitrogen metabolism in Haematococcus pluvialis

Haematococcus pluvialis is a prominent feedstock of astaxanthin. The ratio of carbon to nitrogen (C/N) strongly influences the metabolic pathways of mixotrophic-grown microalgae, however, its role involved in astaxanthin biosynthesis is still not fully understood. In this study, integrative metabolic and physiologic profiles were analyzed in elucidating how C/N affected carbon and nitrogen assimilation and thereby exerted influence on astaxanthin biosynthesis. It was demonstrated that high C/N up-regulated the activities of acetate kinase by increase of 5.76 folds in early logarithmic phase, leading a significant increase of acetyl-CoA. The increased carbon skeletons were then funneled into astaxanthin biosynthesis. Additionally, high C/N increased the proportion of carotenoid-intermediates in cytoplasm from chloroplast. Finally, a fed-batch cultivation strategy based on C/N gradient was developed. Biomass attained 9.18 g L−1 in 100% type of immotile cyst cells, which presented astaxanthin productivity at 15.45 mg L−1 d−1 afterward, exhibiting a promising paradigm in commercial production.

Assessment of fed-batch cultivation strategies for an inducible CHO cell line

In order to maximize cell growth and productivity for an inducible CHO cell line expressing rituximab, various fed-batch culture strategies were investigated. In each case, the performance was evaluated for cultures induced at moderate and high cell density conditions (4 × 106 and 10 × 106 cells/mL) to assess the impact of the timing of induction. We first demonstrate the importance of starting the feeding process during the growth phase, as this translated into significantly improved integral of viable cells and antibody concentration, when compared to post-induction feeding only. Secondly, we investigated the impact of the feed rate by maintaining different levels of glucose (25, 35 and 50 mM) via a dynamic feeding strategy. The highest antibody concentrations were achieved under a moderate feeding regime for both cell densities at induction, highlighting the risks of under- or over-feeding the cultures. We then evaluated the impact of performing a temperature shift at induction by testing different mild hypothermia conditions. At small-scale, the highest production yields (1.2 g/L) were achieved when the temperature was reduced from 37 to 30 °C during the production phase of a culture induced at high cell density. When the strategy was applied in bioreactor, the better controlled conditions led to even greater product concentrations (1.8 g/L). Furthermore, this production protocol was shown to promote a more galactosylated glycan profile than a bioreactor culture initiated at 34 °C during growth and downshifted to 30 °C during the production phase.

High PHA density fed-batch cultivation strategies for 4HB-rich P(3HB-co-4HB) copolymer production by transformant Cupriavidus malaysiensis USMAA1020

P(3HB-co-4HB) with a high 4HB monomer composition was previously successfully produced using the transformant Cupriavidus malaysiensis USMAA1020 containing an additional copy of the PHA synthase gene. In this study, high PHA density fed-batch cultivation strategies were developed for such 4HB-rich P(3HB-co-4HB). The pulse, constant and mixed feeding strategies resulted in high PHA accumulation, with a PHA content of 74–92 wt% and 4HB monomer composition of 92–99 mol%. The pulse-feed of carbon and nitrogen resulted in higher PHA concentration (30.7 g/L) than carbon alone (22.3 g/L), suggesting that a trace amount of nitrogen is essential to support cell density for PHA accumulation. Constant feeding was found to be a more feasible strategy than mixed feeding, since the latter caused a drastic fluctuation in the C/N ratio, as evidenced by higher biomass formation indicating more carbon flux towards the competitive TCA pathway. A two-times carbon and nitrogen pulse feeding was the most optimal strategy achieving 92 wt% accommodation of the total biomass, with the highest PHA concentration (46 g/L) and yield (Yp/x) of 11.5 g/g. The strategy has kept the C/N at optimal ratio during the active PHA-producing phase. This is the first report of the production of high PHA density for 4HB-rich P(3HB-co-4HB).

Fed-batch mixotrophic cultivation of Chlamydomonas reinhardtii for high-density cultures

The green algae Chlamydomonas reinhardtii is a model organism that has been proposed as a potential production platform for high-value recombinant products, but to date, there has been little development of a commercially viable cultivation process. Here we demonstrate a novel mixotrophic fed-batch cultivation strategy in which average biomass density, productivity, and total amount of recombinant GFP significantly increases. Systematic feeding of acetic acid and nutrients into a bioreactor resulted in culture densities increasing from an initial 0.45 ± 0.03 to 23.69 ± 0.5 g L−1 AFDW after 168 h at an average productivity of 181.01 ± 8.8 mg L−1 h−1, a 10-fold increase in comparison to traditional batch cultures. GFP expression was low under both conditions, but fed-batch cultivation resulted in a 2.5-fold increase in total GFP upon culture termination. The effect of fed-batch cultivation on lipid composition and primary metabolites was investigated and elevated levels of the osmoregulatory molecules proline and glycerol were found, suggesting that salt accumulation may have increased over time in fed-batch cultures and eventually limited growth.

Improved fed-batch production of high-purity PHB (poly-3 hydroxy butyrate) by Cupriavidus necator (MTCC 1472) from sucrose-based cheap substrates under response surface-optimized conditions.

Experimental investigations were carried out for Cupriavidus necator (MTCC 1472)-based improved production of poly-3 hydroxy butyrate (PHB) through induced nitrogen limiting fed-batch cultivation strategies. Initially Plackett-Burman design and response surface methodology were implemented to optimize most influencing process parameters. With optimized process parameter values, continuous feeding strategies ware applied in a 5-l fermenter with table sugar concentration of 100g/l, nitrogen concentration of 0.12g/l for fed-batch fermentation with varying dilution rates of 0.02 and 0.0461/h. To get enriched production of PHB, concentration of the sugar was further increased to 150 and 200g/l in feeding. Maximum concentrations of PHB achieved were 22.35 and 23.07g/l at those dilution rates when sugar concentration maintains at 200g/l in feeding. At maximum concentration of PHB (23.07g/l), productivity of 0.58g/lh was achieved with maximum PHB accumulation efficiency up to 64% of the dry weight of biomass. High purity of PHB, close to medical grade was achieved after surfactant hypochlorite extraction method, and it was further confirmed by SEM, EDX, and XRD studies.

Model-Based Nutrient Feeding Strategies for the Increased Production of Polyhydroxybutyrate (PHB) by Alcaligenes latus

Polyhydroxyalkanoates (PHAs) are biodegradable polymers which are considered as an effective alternative for conventional plastics due to their mechanical properties similar to the latter. However, the widespread use of these polymers is still hampered due to their higher cost of production as compared to plastics. The production cost could be overcome by obtaining high yields and productivity. The goal of the present research was to enhance the yield of polyhydroxybutyrate (PHB) with the help of two simple fed-batch cultivation strategies. In the present study, average batch kinetic and substrate limitation/inhibition study data of Alcaligenes latus was used for the development of PHB model which was then adopted for designing various off-line nutrient feeding strategies to enhance PHB accumulation. The predictive ability of the model was validated by experimental implementation of two fed-batch strategies. One such dynamic strategy of fed-batch cultivation under pseudo-steady state with respect to nitrogen and simultaneous carbon feeding strategy resulted in significantly high biomass and PHB concentration of 39.17 g/L and 29.64g/L, respectively. This feeding strategy demonstrated a high PHB productivity and PHB content of 0.6g/Lh and 75%, respectively, which were remarkably high in comparison to batch cultivation. The mathematical model can also be employed for designing various other nutrient feeding strategies.

Improving the productivity of S-adenosyl-l-methionine by metabolic engineering in an industrial Saccharomyces cerevisiae strain

S-Adenosyl-l-methionine (SAM) is an important metabolite having prominent roles in treating various diseases. In order to improve the production of SAM, the regulation of three metabolic pathways involved in SAM biosynthesis were investigated in an industrial yeast strain ZJU001. GLC3 encoded glycogen-branching enzyme (GBE), SPE2 encoded SAM decarboxylase, as well as ERG4 and ERG6 encoded key enzymes in ergosterol biosynthesis, were knocked out in ZJU001 accordingly. The results indicated that blocking of either glycogen pathway or SAM decarboxylation pathway could improve the SAM accumulation significantly in ZJU001, while single disruption of either ERG4 or ERG6 gene had no obvious effect on SAM production. Moreover, the double mutant ZJU001-GS with deletion of both GLC3 and SPE2 genes was also constructed, which showed further improvement of SAM accumulation. Finally, SAM2 was overexpressed in ZJU001-GS to give the best SAM-producing recombinant strain ZJU001-GS-SAM2, in which 12.47g/L SAM was produced by following our developed pseudo-exponential fed-batch cultivation strategy, about 81.0% increase comparing to its parent strain ZJU001. The present work laid a solid base for large-scale SAM production with the industrial Saccharomyces cerevisiae strain.