Assessment of <Emphasis Type="Italic">Nannochloropsis gaditana</Emphasis> growth and lipid accumulation with increased inorganic carbon delivery

Algal biomass refineries for sustainable transportation fuels, in particular biodiesel, will benefit from algal strain enhancements to improve biomass and lipid productivity. Specifically, the supply of inorganic carbon to microalgal cultures represents an area of great interest due to the potential for improved growth of microalgae and the possibility for incorporation with CO₂ mitigation processes. Combinations of bicarbonate (HCO₃^?) salt addition and application of CO₂ to control pH have shown compelling increases in growth rate and lipid productivity of fresh water algae. Here, focus was placed on the marine organism, Nannochloropsis gaditana, to investigate growth and lipid accumulation under various strategies of enhanced inorganic carbon supply. Three gas application strategies were investigated: continuous sparging of atmospheric air, continuous sparging of 5% CO₂ during light hours until nitrogen depletion, and continuous sparging of atmospheric air supplemented with 5% CO₂ for pH control between 8.0 and 8.3. These gas sparging schemes were combined with addition of low concentrations (5 mM) of sodium bicarbonate at inoculation and high concentration (50 mM) of sodium bicarbonate amendments just prior to nitrogen depletion. The optimum scenario observed for growth of N. gaditana under these inorganic carbon conditions was controlling pH with 5% CO₂ on demand, which increased both growth rate and lipid accumulation. Fatty acid methyl esters were primarily comprised of C16:0 (palmitic) and C16:1 (palmitoleic) aliphatic chains. Additionally, the use of high concentration (50 mM) of bicarbonate amendments further improved lipid content (up to 48.6%) under nitrogen deplete conditions when paired with pH-controlled strategies.     

Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum

The marine diatom Phaeodactylum tricornutum is attracting considerable interest as a candidate for biofuel production due to its fast growth and high lipid content. Nitrogen deficiency can increase the lipid content in certain microalgae species, including P. tricornutum. However, the molecular basis of such changes remains unclear without analyzing metabolism at the proteomic level. We attempted to systematically analyze protein expression level changes of P. tricornutum upon N deprivation. We observed translational level changes that could overall redirect the metabolic network from carbon flux towards lipid accumulation. N deprivation led to an increase in the expression of genes involved in nitrogen assimilation and fatty acid biosynthesis and a concomitant decrease in photosynthesis and lipid catabolism enzymes. These molecular level changes are consistent with the observed physiological changes, e.g., in photosynthesis rate and saturated lipid content. Our results provide information at the proteomic level of the key enzymes involved in carbon flux towards lipid accumulation in P. tricornutum and suggest candidates for genetic manipulation in microalgae breeding for biodiesel production.     

Metabolic flux analysis of hepatocyte function in hormone- and amino acid-supplemented plasma

Understanding the metabolic and regulatory pathways of hepatocytes is important for biotechnological applications involving liver cells. Previous attempts to culture hepatocytes in plasma yielded poor functional results. Recently we reported that hormone (insulin and hydrocortisone) and amino acid supplementation reduces intracellular lipid accumulation and restores liver-specific function in hepatocytes exposed to heparinized human plasma. In the current study, we performed metabolic flux analysis (MFA) using a simplified metabolic network model of cultured hepatocytes to quantitively estimate the changes in lipid metabolism and relevant intracellular pathways in response to hormone and amino acid supplementation. The model accounts for the majority of central carbon and nitrogen metabolism, and assumes pseudo-steady-state with no metabolic futile cycles. We found that beta-oxidation and tricarboxylic acid (TCA) cycle fluxes were upregulated by both hormone and amino acid supplementation, thus enhancing the rate of lipid oxidation. Concomitantly, hormone and amino acid supplementation increased gluconeogenic fluxes. This, together with an increased rate of glucose clearance, caused an increase in predicted glycogen synthesis. Urea synthesis was primarily derived from ammonia and aspartate generated through transamination reactions, while exogenous ammonia removal accounted for only 3-6% of the urea nitrogen. Amino acid supplementation increased the endogenous synthesis of oxaloacetate, and in turn that of aspartate, a necessary substrate for the urea cycle. These findings from MFA provide cues as to which genes/pathways relevant to fatty acid oxidation, urea production, and gluconeogenesis may be upregulated by plasma supplementation, and are consistent with current knowledge of hepatic amino acid metabolism, which provides further credence to this approach for evaluating the metabolic state of hepatocytes under various environmental conditions.     

Comparison of CO2 and bicarbonate as inorganic carbon sources for triacylglycerol and starch accumulation in Chlamydomonas reinhardtii

Microalgae are capable of accumulating high levels of lipids and starch as carbon storage compounds. Investigation into the metabolic activities involved in the synthesis of these compounds has escalated since these compounds can be used as precursors for food and fuel. Here, we detail the results of a comprehensive analysis of Chlamydomonas reinhardtii using high or low inorganic carbon concentrations and speciation between carbon dioxide and bicarbonate, and the effects these have on inducing lipid and starch accumulation during nitrogen depletion. High concentrations of CO₂ (5%; v/v) produced the highest amount of biofuel precursors, transesterified to fatty acid methyl esters, but exhibited rapid accumulation and degradation characteristics. Low CO₂ (0.04%; v/v) caused carbon limitation and minimized triacylglycerol (TAG) and starch accumulation. High bicarbonate caused a cessation of cell cycling and accumulation of both TAG and starch that was more stable than the other experimental conditions. Starch accumulated prior to TAG and then degraded as maximum TAG was reached. This suggests carbon reallocation from starch‐based to TAG‐based carbon storage. Biotechnol. Bioeng. 2013; 110: 87–96. © 2012 Wiley Periodicals, Inc.     

Metabolomic Profiling of 13 Diatom Cultures and Their Adaptation to Nitrate-Limited Growth Conditions

Diatoms are very efficient in their use of available nutrients. Changes in nutrient availability influence the metabolism and the composition of the cell constituents. Since diatoms are valuable candidates to search for oil producing algae, measurements of diatom-produced compounds can be very useful for biotechnology. In order to explore the diversity of lipophilic compounds produced by diatoms, we describe the results from an analysis of 13 diatom strains. With the help of a lipidomics platform, which combines an UPLC separation with a high resolution/high mass accuracy mass spectrometer, we were able to measure and annotate 142 lipid species. Out of these, 32 were present in all 13 cultures. The annotated lipid features belong to six classes of glycerolipids. The data obtained from the measurements were used to create lipidomic profiles. The metabolomic overview of analysed cultures is amended by the measurement of 96 polar compounds. To further increase the lipid diversity and gain insight into metabolomic adaptation to nitrogen limitation, diatoms were cultured in media with high and low concentrations of nitrate. The growth in nitrogen-deplete or nitrogen-replete conditions affects metabolite accumulation but has no major influence on the species-specific metabolomic profile. Thus, the genetic component is stronger in determining metabolic patterns than nitrogen levels. Therefore, lipid profiling is powerful enough to be used as a molecular fingerprint for diatom cultures. Furthermore, an increase of triacylglycerol (TAG) accumulation was observed in low nitrogen samples, although this trend was not consistent across all 13 diatom strains. Overall, our results expand the current understanding of metabolomics diversity in diatoms and confirm their potential value for producing lipids for either bioenergy or as feed stock.     

Effects of carbon and nitrogen sources on fatty acid contents and composition in the green microalga, Chlorella sp. 227

In order to investigate and generalize the effects of carbon and nitrogen sources on the growth of and lipid production in Chlorella sp. 227, several nutritional combinations consisting of different carbon and nitrogen sources and concentrations were given to the media for cultivation of Chlorella sp. 227, respectively. The growth rate and lipid content were affected largely by concentration rather than by sources. The maximum specific growth was negatively affected by low concentrations of carbon and nitrogen. There is a maximum allowable inorganic carbon concentration (less than 500~1,000 mM bicarbonate) in autotrophic culture, but the maximum lipid content per gram dry cell weight (g DCW) was little affected by the concentration of inorganic carbon within the concentration. The lipid content per g DCW was increased when the microalga was cultured with the addition of glucose and bicarbonate (mixotrophic) at a fixed nitrogen concentration and with the lowest nitrogen concentration (0.2 mM), relatively. Considering that lipid contents per g DCW increased in those conditions, it suggests that a high ratio of carbon to nitrogen in culture media promotes lipid accumulation in the cells. Interestingly, a significant increase of the oleic acid amount to total fatty acids was observed in those conditions. These results showed the possibility to induce lipid production of high quality and content per g DCW by modifying the cultivation conditions.     

Metabolic Analysis of Adaptation to Short-Term Changes in Culture Conditions of the Marine Diatom Thalassiosira pseudonana

This report describes the metabolic and lipidomic profiling of 97 low-molecular weight compounds from the primary metabolism and 124 lipid compounds of the diatom Thalassiosira pseudonana. The metabolic profiles were created for diatoms perturbed for 24 hours with four different treatments: (I) removal of nitrogen, (II) lower iron concentration, (III) addition of sea salt, (IV) addition of carbonate to their growth media. Our results show that as early as 24 hours after nitrogen depletion significant qualitative and quantitative change in lipid composition as well as in the primary metabolism of Thalassiosira pseudonana occurs. So we can observe the accumulation of several storage lipids, namely triacylglycerides, and TCA cycle intermediates, of which citric acid increases more than 10-fold. These changes are positively correlated with expression of TCA enzymes genes. Next to the TCA cycle intermediates and storage lipid changes, we have observed decrease in N-containing lipids and primary metabolites such as amino acids. As a measure of counteracting nitrogen starvation, we have observed elevated expression levels of nitrogen uptake and amino acid biosynthetic genes. This indicates that diatoms can fast and efficiently adapt to changing environment by altering the metabolic fluxes and metabolite abundances. Especially, the accumulation of proline and the decrease of dimethylsulfoniopropionate suggest that the proline is the main osmoprotectant for the diatom in nitrogen rich conditions.     

Carbon accumulation in <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> induced by nitrogen limitation

Background

Oleaginous microorganisms, such as bacterium, yeast and algal species, can represent an alternative oil source for biodiesel production. The composition of their accumulated lipid is similar to the lipid of an oleaginous plant with a predominance of unsaturated fatty acid. Moreover this alternative to conventional biodiesel production does not create competition for land use between food and oleo-chemical industry supplies. Despite this promising potential, development of microbial production processes are at an early stage. Nutritional limited conditions, such as nitrogen limitation, with an excess of carbon substrate is commonly used to induce lipid accumulation metabolism. Nitrogen limitation implies modification of the carbon-to-nitrogen ratio in culture medium, which impacts on carbon flow distribution in the metabolic network.

Results

The goal of the present study is to improve our knowledge of carbon flow distribution in oleaginous yeast metabolism by focusing carbon distribution between carbohydrate and lipid pools in order to optimize microbial lipid production. The dynamic effects of limiting nitrogen consumption flux according to carbon flow were studied to trigger lipid accumulation in the oleaginous yeast Rhodotorula glutinis. With a decrease of the specific nitrogen consumption rate from 0.052 Nmol.Cmol_X^?1.h^?1 to 0.003 Nmol.Cmol_X^?1.h^?1, a short and transitory intracellular carbohydrate accumulation occurred before the lipid accumulation phase. This phenomenon was studied in fed-batch culture under optimal operating conditions, with a mineral medium and using glucose as carbon source. Two different strategies of decreasing nitrogen flow on carbohydrate accumulation were investigated: an instantaneous decrease and a progressive decrease of nitrogen flow.

Conclusions

Lipid production performance in these fed-batch culture strategies with R. glutinis were higher than those reported in the previous literature; the catalytic specific lipid production rate was 0.07 Cmol_lip.Cmol_X*^?1.h^?1. Experimental results suggested that carbohydrate accumulation was an intrinsic phenomenon connected to the limitation of growth by nitrogen when the nitrogen-to-carbon ratio in the feed flow was lower than 0.045 Nmol.Cmol^?1. Carbohydrate accumulation corresponded to a 440% increase of carbohydrate content. These results suggest that microbial lipid production can be optimized by culture strategy and that carbohydrate accumulation must be taken account for process design.

     

Triacylglycerol accumulation of Phaeodactylum tricornutum with different supply of inorganic carbon

The diatom Phaeodactylum tricornutum produces large quantities of lipids, especially triacylglycerols (TAGs) under nitrogen or phosphorus limitation. In this study, production of lipids and TAGs during this process was compared under conditions with different inputs of inorganic carbon. With an abundant supply of inorganic carbon, considerable accumulation of biomass, lipids, and TAGs was identified after a nitrogen/phosphorus-limiting “induction incubation.” TAGs were still synthesized and accumulated even under inorganic carbon limitation with a cessation in the production of biomass and cellular lipids. This part of accumulated TAGs could be synthesized through recycling and transformation of other lipids such as glycolipids and phospholipids. Additionally, some alterations in the fatty acid profile following TAG accumulation were found. The content of the C16:0 fatty acid increased with decreases in C16:3 and C20:5, which could have been caused by enzymatic selectivity for these fatty acids during the process of TAG synthesis. It was concluded that nitrogen and phosphorus metabolism regulates the synthesis of TAG, while carbon metabolism promotes it by providing sufficient substrates.     

Essential role of the plasmid hik31 operon in regulating central metabolism in the dark in Synechocystis sp. PCC 6803

The plasmid hik31 operon (P3, slr6039‐slr6041) is located on the pSYSX plasmid in Synechocystis sp. PCC 6803. A P3 mutant (ΔP3) had a growth defect in the dark and a pigment defect that was worsened by the addition of glucose. The glucose defect was from incomplete metabolism of the substrate, was pH dependent, and completely overcome by the addition of bicarbonate. Addition of organic carbon and nitrogen sources partly alleviated the defects of the mutant in the dark. Electron micrographs of the mutant revealed larger cells with division defects, glycogen limitation, lack of carboxysomes, deteriorated thylakoids and accumulation of polyhydroxybutyrate and cyanophycin. A microarray experiment over two days of growth in light‐dark plus glucose revealed downregulation of several photosynthesis, amino acid biosynthesis, energy metabolism genes; and an upregulation of cell envelope and transport and binding genes in the mutant. ΔP3 had an imbalance in carbon and nitrogen levels and many sugar catabolic and cell division genes were negatively affected after the first dark period. The mutant suffered from oxidative and osmotic stress, macronutrient limitation, and an energy deficit. Therefore, the P3 operon is an important regulator of central metabolism and cell division in the dark.     

Use of sodium bicarbonate to stimulate triacylglycerol accumulation in the chlorophyte Scenedesmus sp. and the diatom Phaeodactylum tricornutum

There is potential for algal-derived biofuel to help alleviate part of the world’s dependency on petroleum based fuels. However, research must still be done on strain selection, induction of triacylglycerol (TAG) accumulation, and fundamental algal metabolic studies, along with large-scale culturing techniques, harvesting, and biofuel/biomass processing. Here, we have advanced the knowledge on Scenedesmus sp. strain WC-1 by monitoring growth, pH, and TAG accumulation on a 14:10 light–dark cycle with atmospheric air or 5% CO₂ in air (v/v) aeration. Under ambient aeration, there was a loss of pH-induced TAG accumulation, presumably due to TAG consumption during the lower culture pH observed during dark hours (pH 9.4). Under 5% CO₂ aeration, the growth rate nearly doubled from 0.78 to 1.53 d^?1, but the pH was circumneutral (pH 6.9) and TAG accumulation was minimal. Experiments were also performed with 5% CO₂ during the exponential growth phase, which was then switched to aeration with atmospheric air when nitrate was close to depletion. These tests were run with and without the addition of 50 mM sodium bicarbonate. Cultures without added bicarbonate showed decreased growth rates with the aeration change, but there was no immediate TAG accumulation. The cultures with bicarbonate added immediately ceased cellular replication and rapid TAG accumulation was observed, as monitored by Nile Red fluorescence which has previously been correlated by gas chromatography to cellular TAG levels. Sodium bicarbonate addition (25 mM final concentration) was also tested with the marine diatom Phaeodactylum tricornutum strain Pt-1 and this organism also accumulated TAG.     

Accumulation of poly-beta-hydroxybutyrate in Nostoc muscorum: regulation by pH, light-dark cycles, N and P status and carbon sources

Accumulation of poly-beta-hydroxybutyrate (PHB) in Nostoc muscorum was studied. Cells harvested at stationary phase of growth depicted maximum accumulation i.e. 8.6% (w/w) of dry cells as compared to lag (4.1%) or logarithmic (6.1%) phases of cultures. In contrast to alkaline pH, acidic pH, continuous illumination and cells grown in presence of combined nitrogen sources, such as NH(4)Cl and KNO(3), were found to affect PHB accumulation negatively. However, P-deficiency and addition of exogenous carbon sources (acetate, glucose, maltose, fructose and ethanol) were found stimulatory for PHB accumulation. In this report PHB accumulation in N. muscorum was boosted up to 35% (w/w) of dry cells when cells supplemented with 0.2% acetate were subjected to dark incubation for 7 days. Further studies are needed at metabolic engineering level or to apply genetic engineering techniques to improve the expression level of PHB photoproduction in cyanobacteria.     

Compositional profiles of Rhodosporidium toruloides cells under nutrient limitation

Lipid production by the red yeast Rhodosporidium toruloides was explored under nutrient limitation. To determine the compositional profiles of R. toruloides cells, samples were prepared using a continuous cultivation process under nutrient limitation and analyzed via several methods, including Fourier transform infrared spectroscopy and elemental analysis. Under nitrogen limitation, as the dilution rate increased, the cellular lipid content decreased but the carbohydrate and protein contents increased. Under carbon limitation, the cellular lipid, protein, and carbohydrate contents remained relatively constant at the different dilution rates. Moreover, the cellular elemental composition was essentially identical under nitrogen and carbon limitation at a high dilution rate of 0.20 h⁻¹. We also analyzed the consumed carbon to nitrogen (C/N) under different nutrition conditions. The results indicated that the consumed C/N had a major influence on cell metabolism and product formation, which contributed to our understanding of the physiological characteristics of R. toruloides.

     

Acetate accumulation and regulation by process parameters control in Chinese hamster ovary cell culture

Chinese hamster ovary (CHO) cells represent a group of predominantly used mammalian hosts for producing recombinant therapeutic proteins. Known for their rapid proliferation rates, CHO cells undergo aerobic glycolysis that is characterized by fast glucose consumption, that ultimately gives rise to a group of small-molecule organic acids. However, only the function of lactate has been extensively studied in CHO cell culture. In this study, we observed the accumulation of acetate from the late exponential phase to harvest day, potentially contributing to the pH decline in late culture stage regardless of lactate consumption. In addition, we evaluated the acidification of the fresh media and the cell culture suspension, and the data revealed that acetate presented a lower acidification capacity compared to lactate and exhibited limited inhibitory effect on cells with less than 20 mM supplemented in the media. This study also explored the ways to control acetate accumulation in CHO cell culture by manipulating the process parameters such as temperature, glucose, and pH control. The positive correlation between the specific glucose consumption rate and acetate generation rate provides evidence of the endogenous acetate generation from overflow metabolism. Reducing these parameters (temperature, glucose consumption) and HCl-controlled low pH ultimately suppress acetate build-up. In addition, the specific acetate generation rate and relevant glucose consumption rate are found to be a metabolic trait associated with specific cell lines. Taken together, the results presented in these experiments provide a means to advance industrial CHO cell culture process control and development.     

Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the chlorophyta

Algal-derived biodiesel is of particular interest because of several factors including: the potential for a near-carbon-neutral life cycle, the prospective ability for algae to capture carbon dioxide generated from coal, and algae’s high per acre yield potential. Our group and others have shown that in nitrogen limitation, and for a single species of Chlorella, a rise in culture medium pH yields triacylglycerol (TAG) accumulation. To solidify and expand on these triggers, the influence and interaction of pH and nitrogen concentration on lipid production was further investigated on Chlorophyceae Scenedesmus sp. and Coelastrella sp. Growth was monitored optically and TAG accumulation was monitored by Nile red fluorescence and confirmed by gas chromatography. Both organisms grew in all treatments and TAG accumulation was observed by two distinct conditions: high pH and nitrogen limitation. The Scenedesmus sp. was shown to grow and produce lipids to a larger degree in alkaliphilic conditions (pH >9) and was used to further investigate the interplay between TAG accumulation from high pH and/or nitrate depletion. Results given here indicate that TAG accumulation per cell, monitored by Nile red fluorescence, correlates with pH at the time of nitrate depletion.     

土壤有机碳和微生物群落结构对多年不同耕作方式与秸秆还田的响应

土壤有机碳对维持陆地生态系统功能和缓解土壤退化问题至关重要,土壤微生物参与土壤有机碳的循环过程,而耕作方式与秸秆还田影响土壤有机碳和微生物群落.本试验采用裂区试验设计,耕作方式为主区,分别设深松(ST)和旋耕(RT)处理,副区为秸秆还田量,分别设秸秆全还田(F)和秸秆不还田(0)处理,采用Illumina测序技术测定土壤微生物群落结构和固碳功能基因,并测定了2012—2017试验年间土壤有机碳含量.结果表明: 1)深松耕作和秸秆还田均显著提高了pH、微生物生物量碳、总氮、粉粒含量、黏粒含量,而显著降低了砂粒含量; 2)试验年间各处理的土壤有机碳(SOC)含量均呈增加趋势,但与旋耕耕作和秸秆不还田处理相比,深松耕作和秸秆还田处理的平均有机碳增量分别显著提高30.6%和33.2%; 3)土壤中最丰富的细菌类型为变形菌门,其次为酸杆菌门和芽单胞菌门; 4)深松秸秆还田处理(STF)具有较高的微生物多样性; 5)除砂粒含量外,土壤pH、微生物生物量碳、总氮、粉粒和黏粒含量均促使深松秸秆全还田处理下的微生物群落结构向着有利于有机碳积累的方向发生变异; 6)除二糖和寡糖代谢途径外,CO₂固定、发酵、主要碳水化合物代谢、一碳代谢、糖醇、有机酸、糖苷水解酶类的代谢功能基因丰度均表现为深松耕作显著高于旋耕,且均与土壤有机碳呈正相关关系.因此,深松结合秸秆还田能够改善土壤基本性质与土壤微生物群落结构,有利于增加土壤固碳能力和解决土壤退化问题.     

Organic Osmolytes in Aerobic Bacteria from Mono Lake, an Alkaline, Moderately Hypersaline Environment

The identity and concentrations of intracellular organic solutes were determined by nuclear magnetic resonance spectroscopy for two strains of aerobic, gram-negative bacteria isolated from Mono Lake, Calif., an alkaline, moderately hypersaline lake. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was the major endogenous solute in both organisms. Concentrations of ectoine varied with external NaCl levels in strain ML-D but not in strain ML-G, where the level was high but invariant from 1.5 to 3.0 M NaCl. Hydroxyectoine also occurred in strain ML-D, especially at elevated NaCl concentrations (2.5 and 3.0 M), but at levels lower than those of ectoine. Exogenous organic solutes that might occur in Mono Lake were examined for their effects on the de novo synthesis of ectoine. Dimethylsulfoniopropionate (DMSP) (0.1 or 1 mM) did not significantly lower ectoine levels in either isolate, and only strain ML-G showed any capacity for DMSP accumulation. With nitrogen limitation, however, DMSP (0.1 mM) substituted for ectoine in strain ML-G and became the main organic solute. Glycine betaine (GB) was more effective than DMSP in affecting ectoine levels, principally in strain ML-D. Strain ML-D accumulated GB to 50 or 67% of its organic solute pool at 2.5 M NaCl, at an external level of 0.1 or 1 mM GB, respectively. Strain ML-D also accumulated arsenobetaine. The methylated zwitterionic compounds, probably metabolic products of phytoplankton (DMSP and GB) or brine shrimps (arsenobetaine) in Mono Lake, may function as osmolytes for indigenous bacteria when present at high concentrations or under conditions of nitrogen limitation or salt stress.