Simultaneous single-cell protein production and COD removal with characterization of residual protein and intermediate metabolites during whey fermentation by K. marxianus

Cheese whey fermentation with Kluyveromyces marxianus was carried out at 40 °C and pH 3.5 to examine simultaneous single-cell protein production and chemical oxygen demand (COD) removal, determine the fate of soluble whey protein and characterize intermediate metabolites. After 36 h of batch fermentation, the biomass concentration increased from 2.0 to 6.0 g/L with 55 % COD reduction (including protein), whereas soluble whey protein concentration decreased from 5.6 to 4.1 g/L. It was confirmed through electrophoresis (SDS-PAGE) that the fermented whey protein was different from native whey protein. HPLC and GC–MS analysis revealed a change in composition of organic compounds post-fermentation. High inoculum concentration in batch fermentation resulted in an increase in biomass concentration from 10.3 to 15.9 g/L with 80 % COD reduction (including protein) within 36 h with residual protein concentration of 4.5 g/L. In third batch fermentation, the biomass concentration increased from 7.3 to 12.4 g/L with 71 % of COD removal and residual protein concentration of 4.3 g/L after 22 h. After 22 h, the batch process was shifted to a continuous process with cell recycle, and the steady state was achieved after another 60 h with biomass yield of 0.19 g biomass/g lactose and productivity of 0.26 g/L h. COD removal efficiency was 78–79 % with residual protein concentration of 3.8–4.2 g/L. The aerobic continuous fermentation process with cell recycle could be applied to single-cell protein production with substantial COD removal at low pH and high temperature from cheese whey.     

Comparative metabolic profiling-based improvement of rapamycin production by Streptomyces hygroscopicus

Rapamycin is a clinically important macrocyclic polyketide with immunosuppressive activity produced by Streptomyces hygroscopicus. To rationally guide the improvement of rapamycin production, comparative metabolic profiling analysis was performed in this work to investigate the intracellular metabolic changes in S. hygroscopicus U1-6E7 fermentation in medium M1 and derived medium M2. A correlation between the metabolic profiles and rapamycin accumulation was revealed by partial least-squares to latent structures analysis, and 16 key metabolites that most contributed to the metabolism differences and rapamycin production were identified. Most of these metabolites were involved in tricarboxylic acid cycle, fatty acids, and shikimic acid and amino acids metabolism. Based on the analysis of key metabolites changes in the above pathways, corresponding exogenous addition strategies were proposed as follows: 1.0 g/L methyl oleate was added at 0 h; 1.0 g/L lysine was added at 12 h; 0.5 g/L shikimic acid was added at 24 h; 0.5 g/L sodium succinate, 0.1 g/L phenylalanine, 0.1 g/L tryptophan, and 0.1 g/L tyrosine were added at 36 h, successively, and a redesigned fermentation medium (M3) was obtained finally on the basis of M2. The production of rapamycin in M3 was increased by 56.6 % compared with it in M2, reaching 307 mg/L at the end of fermentation (120 h). These results demonstrated that metabolic profiling analysis was a successful method applied in the rational guidance of the production improvement of rapamycin, as well as other industrially or clinically important compounds.     

应用响应面法优化发酵培养基提高达托霉素产量

[背景]达托霉素来自玫瑰孢链霉菌NRRL 11379的发酵产物,是重要的临床用抗生素.其原始产生菌发酵周期长,影响达托霉素的生产效率.本实验室前期在天蓝色链霉菌中重构了达托霉素的生物合成途径,有效地缩短了发酵周期,但重组菌株K10中达托霉素发酵产量很低,制约了后续的研究和开发.[目的]利用响应面法优化产达托霉素的重组菌...     

Improvement of mannitol production by Lactobacillus brevis mutant 3-A5 based on dual-stage pH control and fed-batch fermentations

Lactobacillus brevis 3-A5 was isolated and expected to produce mannitol efficiently by regulating pH in batch and fed-batch fermentations. In 48 h batch fermentations with free and constant pH, the optimal pH for cell growth and mannitol production in the first 24 h of incubation was 5.5, whereas that for mannitol production in the second 24 h of incubation was 4.5. To achieve high cell density and mannitol yield simultaneously, a dual-stage pH control strategy was proposed based on the kinetic analysis of mannitol production. The pH value was controlled at 5.5 for the first 12 h of fermentation and subsequently shifted to 4.5 until the fermentation was completed. Under dual-stage pH control fermentation, a 103 g/L yield of mannitol with a volumetric production rate of 3.7 g/L/h was achieved after 28 h. The dual-stage pH control fed-batch fermentation strategy was further developed to improve mannitol yield, wherein the yield increased by 109 % to 215 g/L after 98 h of fermentation. This value is the highest yield of mannitol ever reported using L. brevis.     

Utilization of by-products derived from bioethanol production process for cost-effective production of lactic acid

The by-products of bioethanol production such as thin stillage (TS) and condensed distillers solubles (CDS) were used as a potential nitrogen source for economical production of lactic acid. The effect of those by-products and their concentrations on lactic acid fermentation were investigated using Lactobacillus paracasei CHB2121. Approximately, 6.7 g/L of yeast extract at a carbon source to nitrogen source ratio of 15 was required to produce 90 g/L of lactic acid in the medium containing 100 g/L of glucose. Batch fermentation of TS medium resulted in 90 g/L of lactic acid after 48 h, and the medium containing 10 % CDS resulted in 95 g/L of lactic acid after 44 h. Therefore, TS and CDS could be considered as potential alternative fermentation medium for the economical production of lactic acid. Furthermore, lactic acid fermentation was performed using only cassava and CDS for commercial production of lactic acid. The volumetric productivity of lactic acid [2.94 g/(L·h)] was 37 % higher than the productivity obtained from the medium with glucose and CDS.     

Efficient production of l-lactic acid by newly isolated thermophilic Bacillus coagulans WCP10-4 with high glucose tolerance

A thermophilic Bacillus coagulans WCP10-4 with tolerance to high concentration of glucose was isolated from soil and used to produce optically pure l-lactic acid from glucose and starch. In batch fermentation at pH?6.0, 240 g/L of glucose was completely consumed giving 210 g/L of l-lactic acid with a yield of 95 % and a productivity of 3.5 g/L/h. In simultaneous saccharification and fermentation at 50 °C without sterilizing the medium, 200 g/L of corn starch was completely consumed producing 202.0 g/L of l-lactic acid. To the best of our knowledge, this strain shows the highest osmotic tolerance to glucose among the strains ever reported for lactic acid production. This is the first report of simultaneous saccharification and fermentation of starch for lactic acid production under a non-sterilized condition.     

Precursor engineering and cell physiological regulation for high level rapamycin production by Streptomyces hygroscopicus

A process for high level production of rapamycin by Streptomyces hygroscopicus using statistical designs and feeding strategy was developed. The amino acids (i.e. Lys, Tyr, and Gln) for precursor supply were screened out in the initial phase of fermentation. The optimum levels determined with Box-Behnken design were Lys 20, Tyr 4, and Gln 3 g/l. In the rapamycin biosynthesis phase, the important component, ammonium sulphate, was also identified. A novel two-stage feeding strategy was developed successfully to increase the flux of rapamycin biosynthesis, in which the optimized amino acid components were fed in the initial phase of fermentation, and then switched to feed 2 g/l ammonium sulphate at 72 h. The maximal rapamycin production reached 860.6 mg/l in a 7 l fermentor, which was 182 % higher than that of the control. This was the first report to integrate precursor engineering and cell physiological regulation methods to optimize rapamycin production.     

Enhancement of ascomycin production in Streptomyces hygroscopicus var. ascomyceticus by combining resin HP20 addition and metabolic profiling analysis

Combinatorial approach of adsorbent resin HP20 addition and metabolic profiling analysis were carried out to enhance ascomycin production. Under the optimal condition of 5 % m/v HP20 added at 24 h, ascomycin production was increased to 380 from 300 mg/L. To further rationally guide the improvement of ascomycin production, metabolic profiling analysis was employed to investigate the intracellular metabolite changes of Streptomyces hygroscopicus var. ascomyceticus FS35 in response to HP20 addition. A correlation between the metabolic profiles and ascomycin accumulation was revealed by partial least-squares to latent structures discriminant analysis, and 11 key metabolites that most contributed to metabolism differences and ascomycin biosynthesis were identified. Based on the analysis of metabolite changes together with their pathways, the potential key factors associated with ascomycin overproduction were determined. Finally, rationally designed fermentation strategies based on HP20 addition were performed as follows: 2 % v/v n-hexadecane was added at 24 h; 1.0 g/L valine was supplemented at 48 h; 1.0 g/L lysine was added at 72 h. The ascomycin production was ultimately improved to 460 mg/L, a 53.3 % enhancement compared with that obtained in initial condition. These results demonstrated that the combination of HP20 addition and metabolic profiling analysis could be successfully applied to the rational guidance of production improvement of ascomycin, as well as other clinically important compounds.     

In silico aided metabolic engineering of Klebsiella oxytoca and fermentation optimization for enhanced 2,3-butanediol production

Klebsiella oxytoca naturally produces a large amount of 2,3-butanediol (2,3-BD), a promising bulk chemical with wide industrial applications, along with various byproducts. In this study, the in silico gene knockout simulation of K. oxytoca was carried out for 2,3-BD overproduction by inhibiting the formation of byproducts. The knockouts of ldhA and pflB genes were targeted with the criteria of maximization of 2,3-BD production and minimization of byproducts formation. The constructed K. oxytoca ΔldhA ΔpflB strain showed higher 2,3-BD yields and higher final concentrations than those obtained from the wild-type and ΔldhA strains. However, the simultaneous deletion of both genes caused about a 50 % reduction in 2,3-BD productivity compared with K. oxytoca ΔldhA strain. Based on previous studies and in silico investigation that the agitation speed during 2,3-BD fermentation strongly affected cell growth and 2,3-BD synthesis, the effect of agitation speed on 2,3-BD production was investigated from 150 to 450 rpm in 5-L bioreactors containing 3-L culture media. The highest 2,3-BD productivity (2.7 g/L/h) was obtained at 450 rpm in batch fermentation. Considering the inhibition of acetoin for 2,3-BD production, fed-batch fermentations were performed using K. oxytoca ΔldhA ΔpflB strain to enhance 2,3-BD production. Altering the agitation speed from 450 to 350 rpm at nearly 10 g/L of acetoin during the fed-batch fermentation allowed for the production of 113 g/L 2,3-BD, with a yield of 0.45 g/g, and for the production of 2.1 g/L/h of 2,3-BD.     

Efficient Acetone–Butanol–Ethanol Production from Corncob with a New Pretreatment Technology—Wet Disk Milling

Acetone–butanol–ethanol (ABE) production from corncob was achieved using an integrated process combining wet disk milling (WDM) pretreatment with enzymatic hydrolysis and fermentation by Clostridium acetobutylicum SE-1. Sugar yields of 71.3 % for glucose and 39.1 % for xylose from pretreated corncob were observed after enzymatic hydrolysis. The relationship between sugar yields and particle size of the pretreated corncob was investigated, suggesting a smaller particle size benefits enzymatic hydrolysis with the WDM pretreatment approach. Analysis of the correlation between parameters representing particle size and efficiency of enzymatic hydrolysis predicted that frequency 90 % is the best parameter representing particle size for the indication of the readiness of the material for enzymatic hydrolysis. ABE production from corncob was carried out with both separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes using C. acetobutylicum SE-1. Interestingly, when considering the time for fermentation as the time for ABE production, a comparable rate of sugar consumption and ABE production in the SHF process (0.55 g/l·h sugar consumption and 0.20 g/l·h ABE production) could be observed when glucose (0.50 g/l·h sugar consumption and 0.17 g/l·h ABE production) or a mixture of glucose and xylose (0.68 g/l·h sugar consumption and 0.22 g/l·h ABE production) mimicking the corncob hydrolysate was used as the substrate for fermentation. This result suggested that the WDM is a suitable pretreatment method for ABE production from corncob owing to the mild conditions. A higher ABE production rate could be observed with the SSF process (0.15 g/l·h) comparing with SHF process (0.12 g/l·h) when combining the time for saccharification and fermentation and consider it as the time for ABE production. This is possibly a result of low sustained sugar level during fermentation. These investigations lead to the suggestion that this new WDM pretreatment method has the potentials to be exploited for efficient ABE production from corncob.     

Low-cost effective culture medium optimization for <Emphasis Type="SmallCaps">d</Emphasis>-lactic acid production by <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> subsp. <Emphasis Type="Italic">torquens</Emphasis> under oxygen-deprived condition

Lactic acid is considered a commodity and its production is boosted by the synthesis of polylactic acid. d-lactic acid (DLA) isomer offers greater flexibility and biodegradability and it can only be obtained in its pure form through fermentation. The lactate dehydrogenase is stereospecific for homofermentative production of DLA isomer in the metabolic pathway of Lactobacillus coryniformis subsp. torquens, with optical purity of ≥?99.9% under oxygen-deprived condition. A simple culture medium that increases DLA production and reduces fermentation costs is fundamental for industrial applicability. A central composite rotatable design was used to evaluate significant components influencing the DLA production. Concentrations were adjusted using the Design-Expert 7.0 optimization tool with a desirability coefficient of 0.693 and the best concentrations of each component were determined. Finally, an assay in the bioreactor with the modified culture medium resulted in a product yield of 0.95 g/g, volumetric productivity of 0.85 g/L.h and 95% of efficiency.     

Effect of caffeine concentration on biomass production, caffeine degradation, and morphology of Aspergillus tamarii

The aim of the present study was to evaluate the effect of the initial caffeine concentration (1–8 g/L) on growth and caffeine consumption by Aspergillus tamarii as well as pellet morphology, in submerged fermentation. Caffeine was used as sole nitrogen source. At 1 g/L of initial caffeine concentration, caffeine degradation was not affected, resulting in a production of 8.7 g/L of biomass. The highest biomass production (12.4–14.8 g/L) was observed within a range of 2 to 4 g/L of initial caffeine concentration. At these initial caffeine concentrations, after 96 h of fermentation, 41–51 % of the initial caffeine was degraded. Using an initial caffeine concentration of 2–3 g/L, the highest specific growth rate was observed (μ?=?0.069 1/h). Biomass production decreased at 8 g/L of initial caffeine concentration. A. tamarii formed mainly pellets at all concentrations tested. The size of the pellet decreased at a caffeine concentration of 8 g/L.     

Ethanol production from galactose by a newly isolated Saccharomyces cerevisiae KL17

A wild-type yeast strain with a good galactose-utilization efficiency was newly isolated from the soil, and identified and named Saccharomyces cerevisiae KL17 by 18s RNA sequencing. Its performance of producing ethanol from galactose was investigated in flask cultures with media containing various combination and concentrations of galactose and glucose. When the initial galactose concentration was 20 g/L, it showed 2.2 g/L/h of substrate consumption rate and 0.63 g/L/h of ethanol productivity. Although they were about 70 % of those with glucose, such performance of S. cerevisiae KL17 with galactose was considered to be quite high compared with other strains reported to date. Its additional merit was that its galactose metabolism was not repressed by the existence of glucose. Its capability of ethanol production under a high ethanol concentration was demonstrated by fed-batch fermentation in a bioreactor. A high ethanol productivity of 3.03 g/L/h was obtained with an ethanol concentration and yield of 95 and 0.39 g/L, respectively, when the cells were pre-cultured on glucose. When the cells were pre-cultured on galactose instead of glucose, fermentation time could be reduced significantly, resulting in an improved ethanol productivity of 3.46 g/L/h. The inhibitory effects of two major impurities in a crude galactose solution obtained from acid hydrolysis of galactan were assessed. Only 5-Hydroxymethylfurfural (5-HMF) significantly inhibited ethanol fermentation, while levulinic acid (LA) was benign in the range up to 10 g/L.     

Screening of Ganoderma strains with high polysaccharides and ganoderic acid contents and optimization of the fermentation medium by statistical methods

Polysaccharides and ganoderic acids (GAs) are the major bioactive constituents of Ganoderma species. However, the commercialization of their production was limited by low yield in the submerged culture of Ganoderma despite improvement made in recent years. In this work, twelve Ganoderma strains were screened to efficiently produce polysaccharides and GAs, and Ganoderma lucidum 5.26 (GL 5.26) that had been never reported in fermentation process was found to be most efficient among the tested stains. Then, the fermentation medium was optimized for GL 5.26 by statistical method. Firstly, glucose and yeast extract were found to be the optimum carbon source and nitrogen source according to the single-factor tests. Ferric sulfate was found to have significant effect on GL 5.26 biomass production according to the results of Plackett–Burman design. The concentrations of glucose, yeast extract and ferric sulfate were further optimized by response surface methodology. The optimum medium composition was 55 g/L of glucose, 14 g/L of yeast extract, 0.3 g/L of ferric acid, with other medium components unchanged. The optimized medium was testified in the 10-L bioreactor, and the production of biomass, IPS, total GAs and GA-T enhanced by 85, 27, 49 and 93 %, respectively, compared to the initial medium. The fermentation process was scaled up to 300-L bioreactor; it showed good IPS (3.6 g/L) and GAs (670 mg/L) production. The biomass was 23.9 g/L in 300-L bioreactor, which was the highest biomass production in pilot scale. According to this study, the strain GL 5.26 showed good fermentation property by optimizing the medium. It might be a candidate industrial strain by further process optimization and scale-up study.     

Production of salidroside and tyrosol in cell suspension cultures of Rhodiola crenulata

Salidroside and its aglycone tyrosol are important compounds found in Rhodiola plants. In this study, callus derived from Rhodiola crenulata was induced and grown when explants were incubated on a Murashige and Skoog (MS) medium containing various concentrations of 6-benzyaldenine (BA), naphthalene acetic acid (NAA) and thidiazuron (TDZ). Callus was easily initiated from juvenile leaves in half strength MS medium supplemented with 0.5 mg/L BA and 3.0 mg/L NAA, while full strength MS containing 0.5 mg/L TDZ and 0.5 mg/L NAA was the best for callus subculture and subsequent cell suspension culture. The activities of l-phenylalanine ammonia lyase (PAL) and β-d-glucosidase, two key enzymes in salidroside synthesis, increased at first and subsequently decreased in cell suspension cultures. The salidroside and tyrosol levels in the cell suspension cultures were determined using high-performance liquid chromatography. High levels of salidroside and tyrosol were detected in cell suspension cultures of R. crenulata extracted with 75 % methanol, demonstrating that the biotechnological production of these compounds using plant cell suspension cultures derived from R. crenulata may be an attractive alternative to harvest-based production.     

Enhanced isopropanol and <Emphasis Type="Italic">n</Emphasis>-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate

The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production.     

Production of succinic acid and lactic acid by Corynebacterium crenatum under anaerobic conditions

A new succinic acid and lactic acid production bioprocess by Corynebacterium crenatum was investigated in mineral medium under anaerobic conditions. Corynebacterium crenatum cells with sustained acid production ability and high acid volumetric productivity harvested from the glutamic acid fermentation broth were used to produce succinic acid and lactic acid. Compared with the first cycle, succinic acid production in the third cycle increased 120% and reached 43.4 g/L in 10 h during cell-recycling repeated fermentations. The volumetric productivities of succinic acid and lactic acid could maintain above 4.2 g/(L·h) and 3.1 g/(L·h), respectively, for at least 100 h. Moreover, wheat bran hydrolysates could be used for succinic acid and lactic acid production by the recycled C. crenatum cells. The final succinic acid concentration reached 43.6 g/L with a volumetric productivity of 4.36 g/(L·h); at the same time, 32 g/L lactic acid was produced.     

Conversion of acid hydrolysate of oil palm empty fruit bunch to L-lactic acid by newly isolated Bacillus coagulans JI12

Cost-effective conversion of lignocellulose hydrolysate to optically pure lactic acid is commercially attractive but very challenging. Bacillus coagulans JI12 was isolated from natural environment and used to produce L-lactic acid (optical purity?>?99.5 %) from lignocellulose sugars and acid hydrolysate of oil palm empty fruit bunch (EFB) at 50 °C and pH 6.0 without sterilization of the medium. In fed-batch fermentation with 85 g/L initial xylose and 55 g/L xylose added after 7.5 h, 137.5 g/L lactic acid was produced with a yield of 98 % and a productivity of 4.4 g/L?h. In batch fermentation of a sugar mixture containing 8.5 % xylose, 1 % glucose, and 1 % L-arabinose, the lactic acid yield and productivity reached 98 % and 4.8 g/L?h, respectively. When EFB hydrolysate was used, 59.2 g/L of lactic acid was produced within 9.5 h at a yield of 97 % and a productivity of 6.2 g/L?h, which are the highest among those ever reported from lignocellulose hydrolysates. These results indicate that B. coagulans JI12 is a promising strain for industrial production of L-lactic acid from lignocellulose hydrolysate.     

Enhanced fed-batch production of pyrroloquinoline quinine in <Emphasis Type="Italic">Methylobacillus</Emphasis> sp. CCTCC M2016079 with a two-stage pH control strategy

The effects of pH control strategy and fermentative operation modes on the biosynthesis of pyrroloquinoline quinine (PQQ) were investigated systematically with Methylobacillus sp. CCTCC M2016079 in the present work. Firstly, the shake-flask cultivations and benchtop fermentations at various pH values ranging from 5.3 to 7.8 were studied. Following a kinetic analysis of specific cell growth rate (μ _x ) and specific PQQ formation rate (μ _p ), the discrepancy in optimal pH values between cell growth and PQQ biosynthesis was observed, which stimulated us to develop a novel two-stage pH control strategy. During this pH-shifted process, the pH in the broth was controlled at 6.8 to promote the cell growth for the first 48 h and then shifted to 5.8 to enhance the PQQ synthesis until the end of fermentation. By applying this pH-shifted control strategy, the maximum PQQ production was improved to 158.61 mg/L in the benchtop fermenter, about 44.9% higher than that under the most suitable constant pH fermentation. Further fed-batch study showed that PQQ production could be improved from 183.38 to 272.21 mg/L by feeding of methanol at the rate of 11.5 mL/h in this two-stage pH process. Meanwhile, the productivity was also increased from 2.02 to 2.84 mg/L/h. In order to support cell growth during the shifted pH stage, the combined feeding of methanol and yeast extract was carried out, which brought about the highest concentration (353.28 mg/L) and productivity (3.27 mg/L/h) of PQQ. This work has revealed the potential of our developed simple and economical strategy for the large-scale production of PQQ.     

Impact of phosphate concentration on docosahexaenoic acid production and related enzyme activities in fermentation of Schizochytrium sp.

Docosahexaenoic acid (DHA) is an important and widely used infant food additive. In this study, the effects of phosphate concentration on lipid and especially DHA synthesis in the oleaginous fungi Schizochytrium sp. HX-308 have been investigated in batch cultures. The maximum DHA yield (8.9 g/L) and DHA productivity (148.3 mg/L h) in 0.1 g/L KH₂PO₄ concentration were higher than the DHA yield (6.2 g/L) and DHA productivity (86.1 mg/L h) in 4 g/L KH₂PO₄ concentration. Furthermore, differences in related enzyme activities (malic enzyme, glucose-6-phosphate dehydrogenase and NAD⁺-isocitrate dehydrogenase) between phosphate-sufficient and phosphate-limitation conditions were assayed. The results showed that the phosphate-limitation condition could maintain higher activities of malic enzyme and glucose-6-phosphate dehydrogenase in addition to lower activity of NAD⁺-isocitrate dehydrogenase. In addition, glucose-6-phosphate dehydrogenase might be the main supplier of NADPH at the early stage of fermentation while malic enzyme might be the provider at the late stage. This information might explain the regulation mechanism of phosphate limitation for lipid production and be useful for further DHA production enhancement.