Effect of the size of yeast flocs and zinc supplementation on continuous ethanol fermentation performance and metabolic flux distribution under very high concentration conditions

Taking continuous ethanol fermentation with the self‐flocculating yeast SPSC01 under very high concentration conditions as an example, the fermentation performance of the yeast flocs and their metabolic flux distribution were investigated by controlling their average sizes at 100, 200, and 300 µm using the focused beam reflectance online measurement system. In addition, the impact of zinc supplementation was evaluated for the yeast flocs at the size of 300 µm grown in presence or absence of 0.05 g L^?1 zinc sulfate. Among the yeast flocs with different sizes, the group with the average size of 300 µm exhibited highest ethanol production (110.0 g L^?1) and glucose uptake rate (286.69 C mmol L^?1 h^?1), which are in accordance with the increased flux from pyruvate to ethanol and decreased flux to glycerol. And in the meantime, zinc supplementation further increased ethanol production and cell viability comparing with the control. Zinc addition enhanced the carbon fluxes to the biosynthesis of ergosterol (28.6%) and trehalose (43.3%), whereas the fluxes towards glycerol, protein biosynthesis, and tricarboxylic acid cycle significantly decreased by 37.7%, 19.5%, and 27.8%, respectively. This work presents the first report on the regulation of metabolic flux by the size of yeast flocs and zinc supplementation, which provides the potential for developing engineering strategy to optimize the fermentation system. Biotechnol. Bioeng. 2010;105: 935–944. © 2009 Wiley Periodicals, Inc.     

Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient

Aims: To improve the yield and productivity of docosahexaenoic acid (DHA) by Schizochytrium sp. in terms of the analysis of microbial physiology. Methods and Results: A two‐stage oxygen supply control strategy, aimed at achieving high concentration and high productivity of DHA, was proposed. At the first 40 h, K_La was controlled at 150·1 h^?1 to obtain high μ for cell growth, subsequently K_La was controlled at 88·5 h^?1 to maintain high q_p for high DHA accumulation. Finally, the maximum lipid, DHA content and DHA productivity reached 46·6, 17·7 g l^?1 and 111 mg l^?1 h^?1, which were 43·83%, 63·88% and 32·14% over the best results controlled by constant K_La. Conclusions: This paper described a two‐stage oxygen supply control strategy based on the kinetic analysis for efficient DHA fermentation by Schizochytrium sp. Significance and Impact of the study: This study showed the advantage of two‐stage control strategy in terms of microbial physiology. As K_La is a scaling‐up parameter, the idea developed in this paper could be scaled‐up to industrial process and applied to other industrial biotechnological processes to achieve both high product concentration and high productivity.     

Different fermentation strategies by Schizochytrium mangrovei strain pq6 to produce feedstock for exploitation of squalene and omega‐3 fatty acids

Schizochytrium mangrovei strain PQ 6 was investigated for coproduction of docosahexaenoic acid (C22: 6ω‐3, DHA ) and squalene using a 30‐L bioreactor with a working volume of 15 L under various batch and fed‐batch fermentation process regimes. The fed‐batch process was a more efficient cultivation strategy for achieving higher biomass production rich in DHA and squalene. The final biomass, total lipid, unsaponifiable lipid content, and DHA productivity were 105.25 g · L^?1, 43.40% of dry cell weight, 8.58% total lipid, and 61.66 mg · g^?1 · L^?1, respectively, after a 96 h fed‐batch fermentation. The squalene content was highest at 48 h after feeding glucose (98.07 mg · g^?1 of lipid). Differences in lipid accumulation during fermentation were correlated with changes in ultrastructure using transmission electron microscopy and Nile Red staining of cells. The results may be of relevance to industrial‐scale coproduction of DHA and squalene in heterotrophic marine microalgae such as Schizochytrium .     

A C6/C5 co‐fermenting Saccharomyces cerevisiae strain with the alleviation of antagonism between xylose utilization and robustness

During second‐generation bioethanol production from lignocellulosic biomass, the desired traits for fermenting microorganisms, such as Saccharomyces cerevisiae, are high xylose utilization and high robustness to inhibitors in lignocellulosic hydrolysates. However, as observed previously, these two traits easily showed the antagonism, one rising and the other falling, in the C6/C5 co‐fermenting S. cerevisiae strain. In this study, LF1 obtained in our previous study is an engineered budding yeast strain with a superior co‐fermentation capacity of glucose and xylose, and was then mutated by atmospheric and room temperature plasma (ARTP) mutagenesis to improve its robustness. The ARTP‐treated cells were grown in 50% (v/v) leachate from lignocellulose pretreatment with high inhibitors content for adaptive evolution. After 30 days, the generated mutant LF1‐6 showed significantly enhanced tolerance, with a six‐fold increase in cell density in the above leachate. Unfortunately, its xylose utilization dropped markedly, indicating the recurrence of the negative correlation between xylose utilization and robustness. To alleviate this antagonism, LF1‐6 cells were iteratively mutated with ARTP mutagenesis and then anaerobically grown using xylose as the sole carbon source, and xylose utilization was restored in the resulting strain 6M‐15. 6M‐15 also exhibited increased co‐fermentation performance of xylose and glucose with the highest ethanol productivity reported to date (0.525 g g^?1 h^?1) in high‐level mixed sugars (80 g L^?1 glucose and 40 g L^?1 xylose) with no inhibitors. Meanwhile, its fermentation time was shortened by 8 h compared to that of LF1. During the fermentation of non‐detoxified lignocellulosic hydrolysate with high inhibitor concentrations at pH ~3.5, 6M‐15 can efficiently convert glucose and xylose with an ethanol yield of 0.43 g g^?1. 6M‐15 is also regarded as a potential chassis cell for further design of a customized strain suitable for production of second‐generation bioethanol or other high value‐added products from lignocellulosic biomass.     

Heterotrophic production of Chlorella sp. TISTR 8990—biomass growth and composition under various production conditions

The green microalga Chlorella sp. TISTR 8990 was grown heterotrophically in the dark using various concentrations of a basal glucose medium with a carbon‐to‐nitrogen mass ratio of 29:1. The final biomass concentration and the rate of growth were highest in the fivefold concentrated basal glucose medium (25 g L^?1 glucose, 2.5 g L^?1 KNO₃) in batch operations. Improving oxygen transfer in the culture by increasing the agitation rate and decreasing the culture volume in 500‐mL shake flasks improved growth and glucose utilization. A maximum biomass concentration of nearly 12 g L^?1 was obtained within 4 days at 300 rpm, 30°C, with a glucose utilization of nearly 76% in batch culture. The total fatty acid (TFA) content of the biomass and the TFA productivity were 102 mg g^?1 and 305 mg L^?1 day^?1, respectively. A repeated fed‐batch culture with four cycles of feeding with the fivefold concentrated medium in a 3‐L bioreactor was evaluated for biomass production. The total culture period was 11 days. A maximum biomass concentration of nearly 26 g L^?1 was obtained with a TFA productivity of 223 mg L^?1 day^?1. The final biomass contained (w/w) 13.5% lipids, 20.8% protein and 17.2% starch. Of the fatty acids produced, 52% (w/w) were saturated, 41% were monounsaturated and 7% were polyunsaturated (PUFA). A low content of PUFA in TFA feedstock is required for producing high quality biodiesel. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1589–1600, 2017     

d‐lactic acid production from renewable lignocellulosic biomass via genetically modified Lactobacillus plantarum

d ‐lactic acid is of great interest because of increasing demand for biobased poly‐lactic acid (PLA). Blending poly‐l ‐lactic acid with poly‐d ‐lactic acid greatly improves PLA's mechanical and physical properties. Corn stover and sorghum stalks treated with 1% sodium hydroxide were investigated as possible substrates for d ‐lactic acid production by both sequential saccharification and fermentation and simultaneous saccharification and cofermentation (SSCF). A commercial cellulase (Cellic CTec2) was used for hydrolysis of lignocellulosic biomass and an l ‐lactate‐deficient mutant strain Lactobacillus plantarum NCIMB 8826 ldhL1 and its derivative harboring a xylose assimilation plasmid (ΔldhL1‐pCU‐PxylAB) were used for fermentation. The SSCF process demonstrated the advantage of avoiding feedback inhibition of released sugars from lignocellulosic biomass, thus significantly improving d ‐lactic acid yield and productivity. d ‐lactic acid (27.3 g L^?1) and productivity (0.75 g L^?1 h^?1) was obtained from corn stover and d ‐lactic acid (22.0 g L^?1) and productivity (0.65 g L^?1 h^?1) was obtained from sorghum stalks using ΔldhL1‐pCU‐PxylAB via the SSCF process. The recombinant strain produced a higher concentration of d ‐lactic acid than the mutant strain by using the xylose present in lignocellulosic biomass. Our findings demonstrate the potential of using renewable lignocellulosic biomass as an alternative to conventional feedstocks with metabolically engineered lactic acid bacteria to produce d ‐lactic acid. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:271–278, 2016     

Chlorella vulgaris as a lipid source: Cultivation on air and seawater‐simulating medium in a helicoidal photobioreactor

The freshwater microalga Chlorella vulgaris was cultured batchwise on the seawater‐simulating Schlösser medium either in a 1.1‐L‐working volume helicoidal photobioreactor (HeP) or Erlenmeyer flask (EF) as control and continuously supplying air as CO₂ source. In these systems, maximum biomass concentration reached 1.65 ± 0.17 g L^?1 and 1.25 ± 0.06 g L^?1, and maximum cell productivity 197.6 ± 20.4 mg L^?1 day^?1 and 160.8 ± 12.2 mg L^?1 day^?1, respectively. Compared to the Bold's Basal medium, commonly employed to cultivate this microorganism on a bench‐scale, the Schlösser medium ensured significant increases in all the growth parameters, namely maximum cell concentration (268% in EF and 126% in HeP), maximum biomass productivity (554% in EF and 72% in HeP), average specific growth rate (67% in EF and 42% in HeP), and maximum specific growth rate (233% in EF and 22% in HeP). The lipid fraction of biomass collected at the end of runs was analyzed in terms of both lipid content and fatty acid profile. It was found that the seawater‐simulating medium, despite of a 56–63% reduction of the overall biomass lipid content compared to the Bold's Basal one, led in HeP to significant increases in both the glycerides‐to‐total lipid ratio and polyunsaturated fatty acid content compared to the other conditions taken as an average. These results as a whole suggest that the HeP configuration could be a successful alternative to the present means to cultivate C. vulgaris as a lipid source. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:279–284, 2016     

Semi-industrial scale (30 m<Superscript>3</Superscript>) fed-batch fermentation for the production of <Emphasis Type="SmallCaps">d</Emphasis>-lactate by <Emphasis Type="Italic">Escherichia coli</Emphasis> strain HBUT-D15

d(?)-lactic acid is needed for manufacturing of stereo-complex poly-lactic acid polymer. Large scale d-lactic acid fermentation, however, has yet to be demonstrated. A genetically engineered Escherichia coli strain, HBUT-D, was adaptively evolved in a 15% calcium lactate medium for improved lactate tolerance. The resulting strain, HBUT-D15, was tested at a lab scale (7 L) by fed-batch fermentation with up to 200 g L^?1 of glucose, producing 184–191 g L^?1 of d-lactic acid, with a volumetric productivity of 4.38 g L^?1 h^?1, a yield of 92%, and an optical purity of 99.9%. The HBUT-D15 was then evaluated at a semi-industrial scale (30 m³) via fed-batch fermentation with up to 160 g L^?1 of glucose, producing 146–150 g L^?1 of d-lactic acid, with a volumetric productivity of 3.95–4.29 g L^?1 h^?1, a yield of 91–94%, and an optical purity of 99.8%. These results are comparable to that of current industrial scale l(+)-lactic acid fermentation.     

Acetone–butanol–ethanol production with high productivity using Clostridium acetobutylicum BKM19

Conventional acetone–butanol–ethanol (ABE) fermentation is severely limited by low solvent titer and productivities. Thus, this study aims at developing an improved Clostridium acetobutylicum strain possessing enhanced ABE production capability followed by process optimization for high ABE productivity. Random mutagenesis of C. acetobutylicum PJC4BK was performed by screening cells on fluoroacetate plates to isolate a mutant strain, BKM19, which exhibited the total solvent production capability 30.5% higher than the parent strain. The BKM19 produced 32.5 g L^?1 of ABE (17.6 g L^?1 butanol, 10.5 g L^?1 ethanol, and 4.4 g L^?1 acetone) from 85.2 g L^?1 glucose in batch fermentation. A high cell density continuous ABE fermentation of the BKM19 in membrane cell‐recycle bioreactor was studied and optimized for improved solvent volumetric productivity. Different dilution rates were examined to find the optimal condition giving highest butanol and ABE productivities. The maximum butanol and ABE productivities of 9.6 and 20.0 g L^?1 h^?1, respectively, could be achieved at the dilution rate of 0.85 h^?1. Further cell recycling experiments were carried out with controlled cell‐bleeding at two different bleeding rates. The maximum solvent productivities were obtained when the fermenter was operated at a dilution rate of 0.86 h^?1 with the bleeding rate of 0.04 h^?1. Under the optimal operational condition, butanol and ABE could be produced with the volumetric productivities of 10.7 and 21.1 g L^?1 h^?1, and the yields of 0.17 and 0.34 g g^?1, respectively. The obtained butanol and ABE volumetric productivities are the highest reported productivities obtained from all known‐processes. Biotechnol. Bioeng. 2013; 110: 1646–1653. © 2013 Wiley Periodicals, Inc.     

Semicontinuous sophorolipid fermentation using a novel bioreactor with dual ventilation pipes and dual sieve‐plates coupled with a novel separation system

Sophorolipids (SLs) are biosurfactants with widespread applications. The yield and purity of SLs are two important factors to be considered during their commercial large‐scale production. Notably, SL accumulation causes an increase in viscosity, decrease in dissolved oxygen and product inhibition in the fermentation medium. This inhibits the further production and purification of SLs. This describes the development of a novel integrated system for SL production using Candida albicans O‐13‐1. Semicontinuous fermentation was performed using a novel bioreactor with dual ventilation pipes and dual sieve‐plates (DVDSB). SLs were separated and recovered using a newly designed two‐stage separation system. After SL recovery, the fermentation broth containing residual glucose and oleic acid was recycled back into the bioreactor. This novel approach considerably alleviated the problem of product inhibition and accelerated the rate of substrate utilization. Production of SLs achieved was 477 g l^?1, while their productivity was 1.59 g l^?1 h^?1. Purity of SLs improved by 23.3%, from 60% to 74%, using DVDSB with the separation system. The conversion rate of carbon source increased from 0.5 g g^?1 (in the batch fermentation) to 0.6 g g^?1. These results indicated that the integrated system could improve the efficiency of production and purity of SLs.     

Integrated production of lactic acid and biomass on distillery stillage

The possibilities of parallel lactic acid and biomass production in batch and fed-batch fermentation on distillery stillage from bioethanol production were studied. The highest lactic acid yield and productivity of 92.3 % and 1.49 g L^?1 h^?1 were achieved in batch fermentation with initial sugar concentration of 55 g L^?1. A significant improvement of the process was achieved in fed-batch fermentation where the concentration of lactic acid was increased to 47.6 % and volumetric productivity for 21 % over the batch process. A high number of Lactobacillus rhamnosus ATCC 7469 viable cells of 10⁹ CFU ml^?1 was attained at the end of fed-batch fermentation. The survival of 92.9 % of L. rhamnosus cells after 3 h of incubation at pH 2.5 validated that the fermentation media remained after lactic acid removal could be used as a biomass-enriched animal feed thus making an additional value to the process.     

Productivity of Chlorella sorokiniana in a short light‐path (SLP) panel photobioreactor under high irradiance

Maximal productivity of a 14 mm light‐path panel photobioreactor under high irradiance was determined. Under continuous illumination of 2,100 µmol photons m^?2 s^?1 with red light emitting diodes (LEDs) the effect of dilution rate on photobioreactor productivity was studied. The light intensity used in this work is similar to the maximal irradiance on a horizontal surface at latitudes lower than 37°. Chlorella sorokiniana, a fast‐growing green microalga, was used as a reference strain in this study. The dilution rate was varied from 0.06 to 0.26 h^?1. The maximal productivity was reached at a dilution rate of 0.24 h^?1, with a value of 7.7 g dw m^?2 h^?1 (m² of illuminated photobioreactor surface) and a volumetric productivity of 0.5 g dw L^?1 h^?1. At this dilution rate the biomass concentration inside the reactor was 2.1 g L^?1 and the photosynthetic efficiency was 1.0 g dw mol photons. This biomass yield on light energy is high but still lower than the theoretical maximal yield of 1.8 g mol photons^?1 which must be related to photosaturation and thermal dissipation of absorbed light energy. Biotechnol. Bioeng. 2009; 104: 352–359 © 2009 Wiley Periodicals, Inc.     

Effects of initial population density (IPD) on growth and lipid composition of Nannochloropsis sp.

The microalga Nannochloropsis sp. was cultured under different initial population densities (IPDs) ranging from 0.11 to 9.09 g L^?1. The IPD affected the biomass and lipid accumulation significantly. The algal cultured with higher IPD resulted higher biomass concentration (up to 13.07 g L^?1) in 10 days growth. The biomass productivity with 0.98 g L^?1 IPD was 0.75 g L^?1 d^?1 which was higher than that of other IPDs. For IPDs ranging from 0.11 to 0.98 g L^?1, with the increase of IPD, the biomass productivity increased, while for IPD over 0.98 g L^?1, the biomass productivity decreased. Lipid content of the algal culture started with 0.11 g L^?1 IPD reached to 42 % of dry weight. But with the increase of IPD, the lipid content decreased. Lipid composition was analyzed using thin layer chromatography coupled with flame ionization detection (TLC/FID). Seven lipid classes were identified and quantified. The main reserve lipid, triacylglyceride (TAG), accumulated under all different IPD conditions. However, with the increasing IPD values, TAG content decreased from 59.1 to 23.5 % of the total lipids. Based on these results, to obtain the maximal biomass productivity and lipid productivity of Nannochloropsis sp. in mass cultivation systems, it is necessary to select an appropriate IPD.     

Nisin production of Lactococcus lactis N8 with hemin‐stimulated cell respiration in fed‐batch fermentation system

In this study, nisin production of Lactococcus lactis N8 was optimized by independent variables of glucose, hemin and oxygen concentrations in fed‐batch fermentation in which respiration of cells was stimulated with hemin. Response surface model was able to explain the changes of the nisin production of L. lactis N8 in fed‐batch fermentation system with high fidelity (R² 98%) and insignificant lack of fit. Accordingly, the equation developed indicated the optimum parameters for glucose, hemin, and dissolved oxygen were 8 g L^?1 h^?1, 3 μg mL^?1 and 40%, respectively. While 1711 IU mL^?1 nisin was produced by L. lactis N8 in control fed‐batch fermentation, 5410 IU mL^?1 nisin production was achieved within the relevant optimum parameters where the respiration of cell was stimulated with hemin. Accordingly, nisin production was enhanced 3.1 fold in fed‐batch fermentation using hemin. In conclusion the nisin production of L. lactis N8 was enhanced extensively as a result of increasing the biomass by stimulating the cell respiration with adding the hemin in the fed‐batch fermentation. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:678–685, 2015     

Application of a membrane recycle bioreactor for continuous ethanol production

Summary A series of continuous fermentations were carried out with a production strain of the yeast Saccharomyces cerevisiae in a membrane bioreactor. A membrane separation module composed of ultrafiltration tubular membranes retained all biomass in a fermentation zone of the bioreactor and allowed continuous removal of fermentation products into a cell-free permeate. In a system with total (100%) cell recycle the impact of fermentation conditions [dilution rate (0.03–0.3 h^–1); substrate concentration in the feed (50–300 g·1^–1); biomass concentration (depending on the experimental conditions)] was studied on the behaviour of the immobilized cell population and on ethanol formation. Maximum ethanol productivity (15 g·1^–1·h^–1) was attained at an ethanol concentration of 81 g·1^–1. The highest demands of cells for maintenance energy were found at the maximum feed substrate concentration (300 g·1^–1) and at very low concentrations of cells in the broth.     

Cell recycling during repeated very high gravity bio-ethanol fermentations using the industrial Saccharomyces cerevisiae strain PE-2

A very high gravity (VHG) repeated-batch fermentation system using an industrial strain of Saccharomyces cerevisiae PE-2 (isolated from sugarcane-to-ethanol distillery in Brazil) and mimicking industrially relevant conditions (high inoculation rates and low O₂ availability) was successfully operated during fifteen consecutive fermentation cycles, attaining ethanol at 17.1 ± 0.2% (v/v) with a batch productivity of 3.5 ± 0.04 g l⁻¹ h⁻¹. Moreover, this innovative operational strategy (biomass refreshing step) prevented critical decreases on yeast viability levels and promoted high accumulation of intracellular glycerol and trehalose, which can provide an adaptive advantage to yeast cells under harsh industrial environments. This study contributes to the improvement of VHG fermentation processes by exploring an innovative operational strategy that allows attaining very high ethanol titres without a critical decrease of the viability level thus minimizing the production costs due to energy savings during the distillation process.     

Enhancing ethanol production from cellulosic sugars using <Emphasis Type="Italic">Scheffersomyces (Pichia) stipitis</Emphasis>

Studies were performed on the effect of CaCO₃ and CaCl₂ supplementation to fermentation medium for ethanol production from xylose, glucose, or their mixtures using Scheffersomyces (Pichia) stipitis. Both of these chemicals were found to improve maximum ethanol concentration and ethanol productivity. Use of xylose alone resulted in the production of 20.68 ± 0.44 g L^?1 ethanol with a productivity of 0.17 ± 0.00 g L^?1 h^?1, while xylose plus 3 g L^?1 CaCO₃ resulted in the production of 24.68 ± 0.75 g L^?1 ethanol with a productivity of 0.21 ± 0.01 g L^?1 h^?1. Use of xylose plus glucose in combination with 3 g L^?1 CaCO₃ resulted in the production of 47.37 ± 0.55 g L^?1 ethanol (aerobic culture), thus resulting in an ethanol productivity of 0.39 ± 0.00 g L^?1 h^?1. These values are 229 % of that achieved in xylose medium. Supplementation of xylose and glucose medium with 0.40 g L^?1 CaCl₂ resulted in the production of 44.84 ± 0.28 g L^?1 ethanol with a productivity of 0.37 ± 0.02 g L^?1 h^?1. Use of glucose plus 3 g L^?1 CaCO₃ resulted in the production of 57.39 ± 1.41 g L^?1 ethanol under micro-aerophilic conditions. These results indicate that supplementation of cellulosic sugars in the fermentation medium with CaCO₃ and CaCl₂ would improve economics of ethanol production from agricultural residues.     

Efficient Open Fermentative Production of Polymer-Grade L-Lactate from Sugarcane Bagasse Hydrolysate by Thermotolerant Bacillus sp. Strain P38

Lactic acid is one of the top 30 potential building-block chemicals from biomass, of which the most extensive use is in the polymerization of lactic acid to poly-lactic-acid (PLA). To reduce the cost of PLA, the search for cheap raw materials and low-cost process for lactic acid production is highly desired. In this study, the final titer of produced L-lactic acid reached a concentration of 185 g·L⁻¹ with a volumetric productivity of 1.93 g·L⁻¹·h⁻¹ by using sugarcane bagasse hydrolysate as the sole carbon source simultaneously with cottonseed meal as cheap nitrogen sources under the open fed-batch fermentation process. Furthermore, a lactic acid yield of 0.99 g per g of total reducing sugars was obtained, which is very close to the theoretical value (1.0 g g⁻¹). No D-isomer of lactic acid was detected in the broth, and thereafter resulted in an optical purity of 100%, which exceeds the requirement of lactate polymerization process. To our knowledge, this is the best performance of fermentation on polymer-grade L-lactic acid production totally using lignocellulosic sources. The high levels of optically pure l-lactic acid produced, combined with the ease of handling and low costs associated with the open fermentation strategy, indicated the thermotolerant Bacillus sp. P38 could be an excellent candidate strain with great industrial potential for polymer-grade L-lactic acid production from various cellulosic biomasses.     

Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp. for producing docosahexaenoic acid

Different fermentation processes, including batch, fed-batch and repeated fed-batch processes by Schizochytrium sp., were studied and compared for the effective DHA-rich microbial lipids production. The comparison between different fermentation processes showed that fed-batch process was a more efficient cultivation strategy than the batch process. Among the four different feeding strategies, the glucose concentration feed-back feeding strategy had achieved the highest fermentation results of final cell dry weight, total lipids content, DHA content and DHA productivity of 72.37, 48.86, 18.38 g l^?1 and 138.8 mg l^?1 h^?1, respectively. The repeated fed-batch process had the advantages of reducing the time and cost for seed culture and inoculation between each fermentation cycles. The results of fermentation characteristics and lipid characterization of the repeated fed-batch process indicated that this repeated fed-batch process had promising industrialization prospect for the production of DHA-rich microbial lipids.