Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp.

The effect of aeration on the performance of docosahexaenoic acid (DHA) production by Schizochytrium sp. was investigated in a 1,500-L bioreactor using fed-batch fermentation. Six parameters, including specific growth rate, specific glucose consumption rate, specific lipid accumulation rate, cell yield coefficient, lipid yield coefficient, and DHA yield coefficient, were used to understand the relationship between aeration and the fermentation characteristics. Based on the information obtained from the parameters, a stepwise aeration control strategy was proposed. The aeration rate was controlled at 0.4 volume of air per volume of liquid per minute (vvm) for the first 24 h, then shifted to 0.6 vvm until 96 h, and then switched back to 0.4 vvm until the end of the fermentation. High cell density (71 g/L), high lipid content (35.75 g/L), and high DHA percentage (48.95%) were achieved by using this strategy, and DHA productivity reached 119 mg/L h, which was 11.21% over the best results obtained by constant aeration rate.     

Lipid accumulation in <Emphasis Type="Italic">Schizochytrium</Emphasis> G13/2S produced in continuous culture

Lipid and docosahexaenoic acid (DHA) accumulation into Schizochytrium G13/2S was studied under batch and continuous culture. Different glucose and glutamate concentrations were supplemented in a defined medium. During batch cultivation, lipid accumulation, 35% total fatty acids (TFA) occurred at the arithmetic growth phase but ceased when cell growth stopped. When continuous culture was performed under different glutamate concentrations, nitrogen-growth-limiting conditions induced the accumulation of 30–28% TFA in Schizochytrium. As the dilution rate decreased from 0.08 to 0.02 h⁻¹, both cell dry weight and TFA content of the cell increased. Under a constant dilution rate of 0.04 h⁻¹, carbon-limiting conditions decreased the TFA to 22%. Fatty acid profile was not affected by the different nutrient concentrations provided during continuous culture. Consequently, lipid accumulation can be induced through the carbon and nitrogen source concentration in the medium to maximise the TFA and subsequently DHA productivity by this microorganism.     

Study of a two-stage growth of DHA-producing marine algae <Emphasis Type="Italic">Schizochytrium limacinum</Emphasis> SR21 with shifting dissolved oxygen level

The culture protocol of Schizochytrium limacinum SR 21, a known docosahexaenoic acid (DHA) producing marine algae was modified in this study to better fit fermentation parameters, particularly control of dissolved oxygen (DO) to the known reproductive and growth biology of the microorganism. The cultures controlled at 50% DO saturation produced a cell density of 181 million cells/ml, whereas cultures with 10% DO produced only 98.4 million cells/ml. A fixed-agitation rate of 150 rpm resulted in an even lower density of 22.5 million cells/ml. Fifty percent DO saturation level led to a decreased pH, as well as a negative correlation with lipid accumulation, while low oxygen concentration was obligatory for lipid accumulation. This study indicated that high DO was preferred for the cells’ reproduction via release of zoospores. Thus, the culture of S. limacinum SR21 should be best divided into two stages: (1) a cell-number-increasing stage in which cell reproduction and cell number increase with little increase in the size and weight of each cell; and (2) a cell-size-increasing stage in which cells stop reproduction but cell size enlarges due to lipids accumulation. With such a protocol, the production of algae biomass and DHA was improved to levels of 37.9 g/L and 6.56 g/L, respectively. The two-stage culture process could be potentially used not only for omega-3 PUFA production, but also in other single cell oil (SCO)-producing processes, including biodiesel production from algae.     

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.     

Improvement of docosahexaenoic acid fermentation from Schizochytrium sp. AB‐610 by staged pH control based on cell morphological changes

Schizochytrium sp. AB‐610 accumulates relatively higher amount of DHA‐rich lipid in the cells, and it was found that DHA yield was closely related to the cell morphology and pH value during fermentation period. DHA production from Schizochytrium sp. AB‐610 in fed‐batch fermentation was investigated and four growth stages were clarified as lag stage, balanced growth stage, lipid accumulation stage, and lipid turnover stage, based on the morphologic observation and key parameters changes. Then a simple strategy of two‐stage pH control was developed, in which pH 7.0 was kept until 12 h after the end of balanced growth stage, and then shifted to 5.0 for the rest period in fermentation. A maximal DHA production of 11.44g/L was achieved. This approach has advantage of easy scaling up for industrial DHA fermentation from Schizochytrium sp. cells.     

Direct fermentation of potato starch in wastewater to lactic acid by<Emphasis Type="Italic">Rhizopus oryzae</Emphasis>

The fungal species ofRhizopus oryzae 2062 has the capacity to carry out a single stage fermentation process for lactic acid production from potato starch wastewater. Starch hydrolysis, reducing sugar accumulation, biomass formation, and lactic acid production were affected with variations in pH, temperature, and starch source and concentration. A growth condition with starch concentration approximately 20 g/L at pH 6.0 and 30°C was favourable for starch fermentation, resulting in a lactic acid yield of 78.3%–85.5% associated with 1.5–2.0 g/L fungal biomass produced in 36 h of fermentation.     

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 .     

种子培养基成分对裂殖壶菌发酵产DHA的影响

考察种子培养基中谷氨酸钠质量浓度和NaCl质量浓度（盐度）对Schizochytrium sp. HX-308菌种质量及发酵性能的影响。结果表明：40 g/L谷氨酸钠条件下培养菌种,发酵后DHA质量分数达到（53.25±0.48）%,而其油产量、油脂质量分数和DHA产量分别为（24.9±0.4）g/L、（45.2±0.6）%、（8.6±0.35）g/L; 利用不含谷氨酸钠的种子培养基发酵,其油产量、油脂质量分数和DHA产量分别为（30.1±0.8）g/L、（54.3±0.5）%、（12.1±0.5）g/L,比含40 g/L谷氨酸钠时分别提高了20.88%、20.13%和40.70%,为菌种驯化提供了新的思路和方向。此外,低盐度培养条件有利于菌种生产性能的发挥,6.25 g/L NaCl条件下发酵后油质量分数、DHA产量及DHA质量分数最高,分别为（62.1±0.8）%、（10.0±0.3）g/L、（48.97±0.42）%。     

A recombinant <Emphasis Type="Italic">E. coli</Emphasis> bioprocess for hyaluronan synthesis

An Escherichia coli strain, JM109, was successfully engineered into an efficient hyaluronic acid (HA) producer by co-expressing the only known class-II HA synthase from a Gram-negative bacterium (Pasteurella multocida) and uridine diphosphate-glucose dehydrogenase from E. coli K5 strain. The engineered strain produced about 0.5 g/L HA in shake flask culture and about 2.0–3.8 g/L in a fed-batch fermentation process in a 1-L bioreactor. The sharp increase in viscosity associated with HA accumulation necessitated pure oxygen supplement to maintain fermentation in aerobic regime. Precursor supply during HA synthesis was probed by glucosamine supplement, which shortens biosynthesis pathway and eliminates one step requiring ATP. HA synthesis was increased with glucosamine supplement from 2.7 to 3.7 g/L (37%), which was mirrored with a concomitant 42% decrease in pure oxygen input, suggesting a close connection between energy metabolism and precursor supply. Decoupling HA synthesis from cell growth by using fosfomycin (an inhibitor for cell wall synthesis) led to a 70% increase in HA synthesis, suggesting detrimental effects on HA synthesis from cell growth via precursor competition. This study demonstrates a potentially viable process for HA based on a recombinant E. coli strain. In addition, the precursor supply limitation identified in this study suggests new engineering targets in subsequent metabolic engineering efforts.     

A New Labyrinthulid Isolate That Produces Only Docosahexaenoic Acid

We show here that a new labyrinthulid strain, L72, isolated from a fallen leaf in the Seto Inland Sea of Japan, produced only docohexaenoic acid (DHA) among all the long-chain polyunsaturated fatty acids (LCPUFAs). The main fatty acid composition was 16:0 (28.9%), 18:0 (7.2%), 18:1 (5.7%), 18:2 (10.4%), and DHA (45.9%) without any other LCPUFA. The lipid content of the strain was 27.4%. The cells had many lipid bodies, which were densely located in all of the cells. On phylogenetic analysis using the 18S rDNA sequence, the strain was located in the labyrinthulids group, forming a monophyletic group with Labyrinthula sp. (strain s) and Labyrinthuila sp. (strain L59). We further tested the culture optimization of strain L72 to evaluate the ability of the strain to produce DHA. The optimum salt concentration and the temperature of the strain were 100% of artificial seawater and 20°C. Strain L72 could grow well on soybean oil (SBO) or soybean lecithin (SBL) as the carbon source. When 20 g/l of SBL was added to the medium, DHA production reached the maximum amount at 0.67 g/l for 14 d. The two important facts, that the strain can use SBL as the main nutrient and contains only DHA among the LCPUFAs, will be of great advantage for industry.     

Influences of Culture Temperature on the Growth, Lipid Content and Fatty Acid Composition of Aurantiochytrium sp. Strain mh0186

The growth, lipid content, and fatty acid composition of Aurantiochytrium sp. strain mh0186 at different temperatures were investigated. Strain mh0186 grew well at 15–30°C, but weakly at 10°C. The biomass at 15–30°C was significantly higher than at 10 and 35°C, and the total lipid at 15–35°C was significantly higher than that at 10°C. The amount of DHA in the total fatty acid was highest at 10°C and decreased in response to temperature increase. The content of DHA (mg/g-dry cell weight) at 15–30°C were significantly higher than those at 35°C and those at 15–25°C were significantly higher than those at 10 and 35°C. The DHA yield at 15–35°C was significantly higher than those at 10 and 35°C. Unsaturation of fatty acid was regulated by temperature and was enhanced in response to temperature decrease. The ratio of DHA to DPA varied at different temperatures.     

Improvement of nutritional quality of cultured sobaity sea bream,Sparidentex hasta (Valenciennes) muscle by preharvest feeding of finisher feeds

A 6‐month long study was conducted to improve the nutritional quality of the cultured sobaity bream, Sparidentex hasta by feeding them finisher feeds containing high docosahexaenoic acid (DHA) at the last two months of the grow‐out stage so that the muscle DHA level be increased at par to the wild. A grow‐out feed used from the beginning until the end of the trial was considered as the control (Diet 1). Experimental diets 2 and 3 were formulated to contain 9.0% DHA (e.g. 1.68 g DHA/100 g feed) and 10.5% DHA (2.20 g DHA/100 g feed), by incorporating high DHA tuna oil into a sea bream grow‐out diet. For comparison, a commercial finisher feed (Diet 4) from Skretting, Italy was also used. The results of this study demonstrated that fish fed DHA enriched finisher diets resulted in significantly (p < .05) better growth, feed utilization and higher muscle eicosapentaenoic acid (EPA) and DHA content compared to those fed grow‐out diet. The muscle DHA and EPA of fish fed finisher diets were also higher than those of the whole year average DHA and EPA content of wild sobaity. An organoleptic evaluation showed no significant (p > .05) differences between sensory attributes of muscle from cultured and wild sea bream. The results of the study demonstrated that feeding finisher feed enriched with DHA at the later part of the grow‐out operation, the n‐3 PUFA levels of cultured sobaity can cost‐effectively be increased at par to the wild.     

Single cell oil production by <Emphasis Type="Italic">Gordonia</Emphasis> sp. DG using agro-industrial wastes

Lipid accumulation by Gordonia sp. DG using sodium gluconate as carbon source in comparison with Rhodococcus opacus PD630 was studied. Maximum lipid content 80% was observed at the beginning of the stationary phase for R. opacus and 72% at the end of stationary phase for Gordonia sp. Different agro-industrial wastes were used as carbon source. The cells of the two organism accumulated lipid more than 50% of the biomass with most tested agro-industrial wastes. The maximum value was in presence of sugar cane molasses (93 and 96%) for R. opacus and Gordonia sp. respectively. Maximum triacyglycerols (TAGs), 88.9 and 57.8 mg/l, was obtained using carob and orange waste by R. opacus and Gordonia sp. respectively. The use of orange waste as carbon source by R. opacus, increased lipid unsaturation with C18:3 as the major unsaturated fatty acid. On the other hand, C22:0 and C6:0 were the dominant fatty acids (54.5% of the total identified fatty acids) produced by Gordonia sp. in presence of orange waste as carbon source. Statistical optimization of the medium revealed that maximum lipid content was achieved with 60% orange waste, 0.05 g/l ammonium chloride and 0.2 g/l magnesium sulphate.     

Microalga <Emphasis Type="Italic">Scenedesmus obliquus</Emphasis> as a potential source for biodiesel production

Biodiesel from microalgae seems to be the only renewable biofuel that has the potential to completely replace the petroleum-derived transport fuels. Therefore, improving lipid content of microalgal strains could be a cost-effective second generation feedstock for biodiesel production. Lipid accumulation in Scenedesmus obliquus was studied under various culture conditions. The most significant increase in lipid reached 43% of dry cell weight (dcw), which was recorded under N-deficiency (against 12.7% under control condition). Under P-deficiency and thiosulphate supplementation the lipid content also increased up to 30% (dcw). Application of response surface methodology in combination with central composite rotary design (CCRD) resulted in a lipid yield of 61.3% (against 58.3% obtained experimentally) at 0.04, 0.03, and 1.0 g l⁻¹ of nitrate, phosphate, and sodium thiosulphate, respectively for time culture of 8 days. Scenedesmus cells pre-grown in glucose (1.5%)-supplemented N 11 medium when subjected to the above optimized condition, the lipid accumulation was boosted up to 2.16 g l⁻¹, the value ~40-fold higher with respect to the control condition. The presence of palmitate and oleate as the major constituents makes S. obliquus biomass a suitable feedstock for biodiesel production.     

高产DHA破囊壶菌Aurantiochytrium sp. PKU#SW7诱变株的筛选

【目的】对野生菌株Aurantiochytrium sp.PKU#SW7诱变育种,筛选高产DHA突变株。【方法】采用UV诱变和化学药物胁迫筛选方式,以菌株的生物量、油脂产量、DHA产量作为筛选指标,获得高产DHA突变株。【结果】经鉴定获得一株DHA高产突变株PKU#PM003,该菌株传代4次后仍保持较好的遗传稳定性。摇瓶发酵后,PKU#PM003生物量产量高达6.62 g/L,比原始菌株5.95 g/L提高了11.26%,脂肪酸含量高达4.01 g/L,比原始菌株3.18 g/L提高了26.1%,DHA在脂肪酸中所占比例由29.97%增加到33.43%,产量提高了41.01%,油脂突变效果显著。【结论】突变株PKU#PM003可作为性状优良的工业化发酵生产菌种,并在DHA产量提升上仍具有巨大的空间。     

Efficient docosahexaenoic acid production by Schizochytrium sp. via a two-phase pH control strategy using ammonia and citric acid as pH regulators

The cell growth and docosahexaenoic acid (DHA) synthesis of Schizochytrium sp. are closely related to the culture pH. A two-phase pH control strategy based on nitrogen consumption was developed in which pH 7.0 was used for biomass accumulation and pH 5.0 for DHA synthesis. Using this strategy, the cell dry weight and DHA content reached 98.07 and 25.85 g/L, respectively. Furthermore, ammonia and citric acid were used as pH regulators. Application of citric acid further resulted in 7.88 and 4.87% improvements of total lipids and the ratio of DHA to total fatty acids, respectively. Ammonia, as a suitable nitrogen source, promoted non-lipid biomass accumulation. Using this method, a maximum DHA yield of 32.75 g/L was obtained with non-lipid biomass (58.01 g/L) and the ratio of DHA to total fatty acids (52.36%). This study provides an easy strategy for large-scale industrial production of DHA via high-cell-density fermentation of Schizochytrium sp.     

Effect of ammonium-N on malic enzyme and lipid production in Rhodotorula glutinis grown on monosodium glutamate wastewater

Rhodotorula glutinis, an oil producing strain, can utilize monosodium glutamate (MSG) wastewater as a raw material for lipid production. The effects of ammonium-N in the MSG wastewater (ammonium 15,000–25,000?mg/L, COD 30,000–50,000?mg/L) on cell growth, lipid accumulation and malic enzyme activity of R. glutinis have been studied. Four initial ammonium sulfate concentrations in the medium were set, which were 20, 60, 100, and 140?g/L. With an increase in the ammonium sulfate concentration, the uptake of ammonia nitrogen and lipid accumulation increased while the biomass decreased at 72?h. The maximum value of ammonia nitrogen consumption reached 5.77?g/L for an initial ammonium sulfate concentration of 140?g/L at 72?h. In addition, 60?g/L ammonium sulfate concentration may be an appropriate concentration for R. glutinis cultivation. The activity of the malic enzyme was measured and the results showed that there was a linear relationship between the intracellular lipid content and the total malic enzyme activity.     

Dynamic flux balance analysis of biomass and lipid production by Antarctic thraustochytrid Oblongichytrium sp. RT2316-13

Production of biomass and lipids in batch cultures of the Antarctic thraustochytrid Oblongichytrium sp. RT2316-13, is reported. The microorganism proved capable of producing nearly 67% docosahexaenoic acid (DHA) and 15% eicosapentaenoic acid (EPA) in its total lipid fraction. Biomass with a maximum total lipid content of 33.5% (wt/wt) could be produced at 15°C in batch culture using a medium containing glucose (20 g/L), yeast extract (10.5 g/L), and other minor components. A lower culture temperature (5°C) reduced biomass and lipid productivities compared to culture at 15°C, but enhanced the DHA and EPA content of the lipids by 6.4- and 3.3-fold, respectively. Both a simple minimally structured mathematical model and a more complex genome-scale metabolic model (GEM) allowed the fermentation profiles in batch cultures to be satisfactorily simulated, but the GEM provided much greater insight in the biochemical and physiological phenomena underlying the observed behavior. Unlike the simpler model, the GEM could be interrogated for the possible effects of various external factors such as oxygen supply, on the expected outcomes. In silico predictions of oxygen effects were consistent with literature observations for DHA producing thraustochytrids.     

Microbial production of <Emphasis Type="Italic">cis</Emphasis>-1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylate by genetically modified <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid (DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after 24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC production rate was 0.356 g L⁻¹ h⁻¹. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by nuclear magnetic resonance.