Monitoring hybridoma metabolism in continuous suspension culture at the intracellular level

A model mouse hybridoma cell line was grown in continuous culture experiments in a serum-free low-protein lipid-free medium. The steady-state responses of cell numbers, extra- and intracellular metabolite concentrations, substrate and (by) product consumption/production rates, and yield coefficients were investigated as a function of step changes in the glutamine concentration of the feed medium. In addition to the commonly performed analysis of metabolites in culture supernatants, we prepared perchloric acid extracts of cells and determined the amount and the composition of intracellular amino acids and organic acids. Significant differences were found with respect to intracellular metabolite pools for cells growing at nearly identical specific growth rates. To our knowledge this is the first time that data on the intracellular concentrations (pools) of amino acids and Krebs cycle intermediates are reported in the literature that were obtained under carefully defined culture conditions such as those attained in continuous culture experiments.     

Changes in Free Amino Acid Production and Intracellular Amino Acid Pools of Bacillus licheniformis as a Function of Culture Age and Growth Media

Changes in the endogenous intracellular amino acid pool and total free amino acid production in Bacillus licheniformis grown in minimal media were investigated. The total intracellular pool increased during exponential growth and then decreased rapidly after the end of growth. Most of the amino acids were present at low concentrations, but glutamate and alanine comprised 60 to 90% of the total intracellular free amino acid at most times during the growth cycle. It was concluded that, in addition to providing monomers for protein synthesis, the intracellular amino acid pool may be maintained for the storage of energy-providing metabolic intermediates and possibly as a balance to the ionic strength of the medium. The total free amino acid production by the cell was found to be dependent upon the composition of the salts medium as well as the culture age under conditions in which the carbon and nitrogen sources were the same. A 10-fold increase in extracellular amino acid was observed as the cells changed from vegetative to sporulation metabolism, mostly due to the extrusion of intracellular amino acid. The impact of this increase upon amino acid uptake and pulse-labeling studies using unwashed cells is discussed.     

Diatom cultivation and biotechnologically relevant products. Part II: Current and putative products

While diatoms are widely present in terms of diversity and abundance in nature, few species are currently used for biotechnologically applications. Most studies have focussed on intracellularly synthesised eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA) used for pharmaceutical applications. Applications for other intracellular molecules, such as total lipids for biodiesel, amino acids for cosmetic, antibiotics and antiproliferative agents, are at the early stage of development. In addition, the active principle component must be identified amongst the many compounds of biotechnological interest. Biomass from diatom culture may be applied to: (1). aquaculture diets, due to the lipid- and amino-acid-rich cell contents of these microorganisms, and (2). the treatment of water contaminated by phosphorus and nitrogen in aquaculture effluent, or heavy metal (bioremediation). The most original application of microalgal biomass, and specifically diatoms, is the use of silicon derived from frustules in nanotechnology. The competitiveness of biotechnologically relevant products from diatoms will depend on their cost of production. Apart from EPA, which is less expensive when obtained from Phaeodactylum tricornutum than from cod liver, comparative economic studies of other diatom-derived products as well as optimisation of culture conditions are needed. Extraction of intracellular metabolites should be also optimised to reduce production costs, as has already been shown for EPA. Using cell immobilisation techniques, benthic diatoms can be cultivated more efficiently allowing new, biotechnologically relevant products to be investigated.     

Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae

Concerns regarding the depletion of the world's reserves of oil and global climate change have promoted an intensification of research and development toward the production of biofuels and other alternative sources of energy during the last years. There is currently much interest in developing the technology for third-generation biofuels from microalgal biomass mainly because of its potential for high yields and reduced land use changes in comparison with biofuels derived from plant feedstocks. Regardless of the nature of the feedstock, the use of fertilizers, especially nitrogen, entails a potential economic and environmental drawback for the sustainability of biofuel production. In this work, we have studied the possibility of nitrogen biofertilization by diazotrophic bacteria applied to cultured microalgae as a promising feedstock for next-generation biofuels. We have obtained an Azotobacter vinelandii mutant strain that accumulates several times more ammonium in culture medium than wild-type cells. The ammonium excreted by the mutant cells is bioavailable to promote the growth of nondiazotrophic microalgae. Moreover, this synthetic symbiosis was able to produce an oil-rich microalgal biomass using both carbon and nitrogen from the air. This work provides a proof of concept that artificial symbiosis may be considered an alternative strategy for the low-N-intensive cultivation of microalgae for the sustainable production of next-generation biofuels and other bioproducts.     

Recovery of microalgal biomass and metabolites: process options and economics

Commercial production of intracellular microalgal metabolites requires the following: (1) large-scale monoseptic production of the appropriate microalgal biomass; (2) recovery of the biomass from a relatively dilute broth; (3) extraction of the metabolite from the biomass; and (4) purification of the crude extract. This review examines the options available for recovery of the biomass and the intracellular metabolites from the biomass. Economics of monoseptic production of microalgae in photobioreactors and the downstream recovery of metabolites are discussed using eicosapentaenoic acid (EPA) recovery as a representative case study.     

Microalgae-mediated chemicals production and wastes removal

Biotechnology of microalgae has gained importance in recent years due to the development of new production and environmental technologies. Because their growth requires unexpensive substrates such as solar light and CO₂, microalgae can be used as cheap and effective biocatalysts to obtain high added-value compounds, from simple metabolites to complex molecules, i.e., chemicals, vitamins, carotenoids, pigments, or polysaccharides. During productive processes, the algal biomass formed may be used as a food source like proteins. On the other hand, microalgae can also be employed in contaminant bioelimination processes especially for nitrogen, phosphorus, or sulfur compounds. Particularly relevant is their use for heavy metal removal from wastewaters; upon enriching the biomass in the metal, they can be recovered, thereby providing economic advantages.

The use of immobilized microalgae in these processes is very adequate and offers significant advantages in bioreactors.     

Les principales voies enzymatiques de l'assimilation de l'azote chez les microalgues eucaryotes

Nitrogen utilization by eukaryotic microalgae involves 1) the uptake of the nutrient nitrogen, 2) the intracellular processing of the nitrogen to convert it to ammonia if necessary, 3) the assimilation of the ammonia into small organic molecules (amino acids) and 4) the synthesis of larger organic macromolecules. The ammonia is assimilated into the intracellular amino acid pool of eukaryotic microalgae primarily by either the GS/GOGAT pathway or the reaction of GDH. Here we review our current knowledge on the characteristics of these enzymes and on their putative functions in ammonia assimilation. It seems to be generally accepted that the GS/GOGAT pathway is the main pathway for the ammonia assimilation in eukaryotic microalgae. There is, however, evidence for the role of GDH in ammonia assimilation in some species. In other species the role of this enzyme is thought to be in catalysing the conversion of glutamate to ammonia. The distribution of the isoforms of glutamate dehydrogenase that use NADH or 97 NADPH as reductant make any generalisation difficult.     

Augmentation of monocyte intracellular ascorbate in vitro protects cells from oxidative damage and inflammatory responses

Ascorbic acid is present as a primary antioxidant in plasma and within cells, protecting both cytosolic and membrane components of cells from oxidative damage. The effects of intracellular ascorbic acid on F(2)-isoprostanes (biomarkers of oxidative stress) and monocyte chemoattractant protein-1 (marker of inflammatory responses) production in monocytic THP-1 cells were investigated under conditions of 2,2'-Azobis(2-methylpropionamidine)dihydrochloride (AAPH) induced oxidative stress. Cells cultured under normal conditions have extremely low ascorbate levels and the intracellular ascorbate can be augmented significantly by adding ascorbate to the culture medium. While AAPH treatment reduced cell viability, increased F(2)-isoprostanes and MCP-1 production, the presence of intracellular ascorbic acid maintained high cell viability and attenuated both F(2)-isoprostanes and MCP-1 production. Measurement of intracellular ascorbic acid and its oxidised products showed that intracellular ASC was oxidised to a significantly greater extent during AAPH treatment and may be utilised to protect the cells under conditions of oxidative stress. This study demonstrates the importance of intracellular ascorbate, which may be lacking under normal cell culture conditions, under conditions of increased oxidative stress.     

The influence of different cultivation conditions on the metabolome of Fusarium oxysporum

The two most widespread pentose sugars found in the biosphere are d-xylose and l-arabinose. They are both potential substrates for ethanol production. The purpose of this study was to better understand the redox constraints imposed to Fusarium oxysporum during utilization of pentoses. In order to increase ethanol yield and decrease by-product formation, nitrate was used as nitrogen source. The use of NADH, the cofactor in denitrification process when using nitrate as a nitrogen source, improved the ethanol yield on xylose to 0.89 mol mol(-1) compared to the ethanol yield achieved using ammonium as nitrogen source 0.44 mol mol(-1). The improved ethanol yield was followed by a 28% decrease in yield of the by-product xylitol. In order to investigate the metabolic pathway of arabinose and the metabolic limitations for the efficient ethanol production from this sugar, the extracellular and intracellular metabolite profiles were determined under aerobic and anaerobic cultivation conditions. The results of this study clearly show difficulties in channelling of glucose-1-P (G1P) to pentose phosphate pathway (PPP) and reduced NADPH regeneration, suggesting that NADPH becomes a limiting factor for arabinose conversion, resulting in excessive acetate production. Variations of the fungus intracellular amino and non-amino acid pool, under different culture conditions, were evaluated using principal component analysis (PCA). PCA projection of the metabolome data collected from F. oxysporum subjected to environmental perturbations succeeded to visualize different physiological states and the conclusions of this study were that the metabolite profile is unique according to: (1) the carbon source and (2) the oxygen supply, and to a lesser extent to the cultivation phase.     

Penicillin biosynthesis and amino acid metabolism in Penicillium chrysogenum in experiments with washed mycelium]

The study of the amino acid metabolism in Penicillium chrysogenum with the use of washed mycelium showed that the amount of the free intracellular amino acids significantly decreased during the process of penicillin production. Still, such a decrease did not cover the nitrogen requirements of the culture for the antibiotic synthesis and mobilization of the protein nitrogen took place. By the end of the process the amount of the protein nitrogen markedly decreased. At the same time alpha-amino nitrogen was absent in the fermentation broth filtrate. About 14 amino acids (including cysteine and valine) which participate in constriuction of the penicillin molecule nucleus were found in the amino acid poll. However, the amounts of cysteine and valine were not high and probably other free intracellular amino acids participated in their synthesis. It was shown that one of the limiting factors in the process of penicillin biosynthesis was synthesis of cysteine, a sulphur-containing amino acid which is one of the precursors of the antibiotic molecule nucleus.     

荒漠微藻的碳氧转换与调控

为了提高微藻的生物燃料生产效率及其在密闭环境中的碳氧转换效率,以两株荒漠微藻BG18-3、BE6-2和一株淡水蓝藻7924为研究对象,对其进行逆境条件培养,发现荒漠微藻BG18-3在各种逆境中表现最佳。在静态培养中,荒漠微藻BG1-3也具有明显的优势,其生物量干重达到0.26 g/L,硝态氮和磷酸盐去除率分别为36%和99%。在荒漠微藻BG18-3的通气培养中,生物干重量最高（3% CO₂通气培养16天）达到2.63 g/L,生物量产率为164.0 mg/L·d,出口CO₂浓度最低降到0.04%,O₂净含量增加0.68%,这表明荒漠微藻BG18-3具有较高的碳氧转化效率,具有生产生物燃料的潜质。最后根据18s rDNA分析结果将荒漠微藻BG18-3鉴定为栅列藻Scenedesmus littoralis。     

Microalgae for high-value compounds and biofuels production: A review with focus on cultivation under stress conditions

Microalgal biomass as feedstock for biofuel production is an attracting alternative to terrestrial plant utilization for biofuels production. However, today the microalgal cultivation systems for energy production purposes seem not yet to be economically feasible. Microalgae, though cultivated under stress conditions, such as nutrient starvation, high salinity, high temperature etc. accumulate considerable amounts (up to 60–65% of dry weight) of lipids or carbohydrates along with several secondary metabolites. Especially some of the latter are valuable compounds with an enormous range of industrial applications. The simultaneous production of lipids or carbohydrates for biofuel production and of secondary metabolites in a biorefinery concept might allow the microalgal production to be economically feasible. This paper aims to provide a review on the available literature about the cultivation of microalgae for the accumulation of high-value compounds along with lipids or carbohydrates focusing on stress cultivation conditions.     

Cell fragility — The key problem of microalgae mass production in closed photobioreactors

The gap between the theoretical biological potential of microalgae and the biomass productivity obtained with algal culture in tubular biophotoreactors is due to a reduced growth rate related to hydrodynamic stress of pumping. High levels of mixing are necessary to reach a turbulent flow of the culture, in order to optimize the light regime. The optimal conditions of pumping to produce this significant liquid mixing may produce some cell damage. Factors affecting this hydrodynamic stress (geometry of the bioreactor involved, type of pump utilized, morphology of algal cells, physiological conditions of microalgae, etc.) are discussed.     

The Metabolic Disturbances of Motoneurons Exposed to Glutamate

Glutamate-induced excitotoxicity is considered as one of the major pathophysiological factors of motoneuron death in amyotrophic lateral sclerosis and other motoneuron diseases. In order to expand our knowledge on mechanisms of glutamate-induced excitotoxicity, the present study proposes to determine the metabolic consequences of glutamate and astrocytes in primary enriched motoneuron culture. Using liquid chromatography coupled to high-resolution mass spectrometry (LC-HRMS), we showed that the presence of astrocytes and glutamate profoundly modified the metabolic profile of motoneurons. Our study highlights for the first time that crosstalk between astrocytes and enriched motoneuron culture induced alterations in phenylalanine, tryptophan, purine, arginine, proline, aspartate, and glutamate metabolism in motoneurons. We observed that astrocytes modulate the sensitivity of motoneurons to glutamate, since metabolites altered by glutamate in motoneurons cultured alone were different (except 5-hydroxylysine) from those altered in co-cultured motoneurons. Our findings provide new insight into the metabolic alterations associated to astrocytes and glutamate in motoneurons and provide opportunities to identify novel therapeutic targets.     

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.     

微藻细胞油脂含量的快速检测方法

以斜生栅藻(Scenedesmus obliquus JNU20)为实验材料,采用尼罗红(NR)荧光光谱法和傅里叶变换红外光谱法(FTIR)测定该藻细胞中的油脂含量。研究结果表明NR的最佳染色条件为:染色前微波处理40 s,二甲基亚砜体积分数1%, NR最终质量浓度1.5 μg/ml,染色时间5 min,染色温度40℃。比较了NR荧光光谱法、FTIR与传统重量法测定的该藻在不同时相的油脂积累情况,利用激光共聚焦显微镜观察了细胞中油体形成的动态过程。实验结果表明NR荧光光谱法、FTIR与传统重量法测定的结果显著相关(R²=0.9258, R²=0.9844),但NR荧光光谱法和FTIR更简便快速,研究结果为规模化筛选高含油量藻株及跟踪产油微藻油脂积累过程奠定了基础。     

Perspectives on engineering strategies for improving biofuel production from microalgae — A critical review

Although the potential for biofuel production from microalgae via photosynthesis has been intensively investigated, information on the selection of a suitable operation strategy for microalgae-based biofuel production is lacking. Many published reports describe competitive strains and optimal culture conditions for use in biofuel production; however, the major impediment to further improvements is the absence of effective engineering strategies for microalgae cultivation and biofuel production. This comprehensive review discusses recent advances in understanding the effects of major environmental stresses and the characteristics of various engineering operation strategies on the production of biofuels (mainly biodiesel and bioethanol) using microalgae. The performances of microalgae-based biofuel-producing systems under various environmental stresses (i.e., irradiance, temperature, pH, nitrogen depletion, and salinity) and cultivation strategies (i.e., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) are compared. The reasons for variations in performance and the underlying theories of the various production strategies are also critically discussed. The aim of this review is to provide useful information to facilitate development of innovative and feasible operation technologies for effectively increasing the commercial viability of microalgae-based biofuel production.     

Nutritional stress effects under different nitrogen sources on the genes in microalga Isochrysis zhangjiangensis and the assistance of Alteromonas macleodii in releasing the stress of amino acid deficiency

The expressions of nine nitrogen assimilation‐associated genes, NRT2, NAR1, NIA2, NIR, GLN2, GLSF, GSN1, GDH, and AAT2, in the microalga Isochrysis zhangjiangensis were investigated to unveil the effects of limitations of various nitrogen sources (NaNO₃, NH₄Cl, NaNO₂, and an amino acid mixture) on the microalgae. The results demonstrated that the NRT2, NAR1, GLN2, GSN1, and AAT2 genes were highly expressed in lipid‐rich microalgae under inorganic nitrogen‐deficient conditions and they decreased after nitrogen resupply. Significant increases in the expressions of NAR1, GLN2, and GLSF were found in nitrate‐depleted microalgae, whereas significant increases in the expressions of NRT2, NAR1, GLN2, and GSN1 were found in nitrite‐depleted microalgae. Significant increases in the expressions of only NRT2 and GSN1 were found in ammonium‐depleted microalgae (P < 0.05). Except for the NRT2, other genes were expressed at lower levels under amino acid‐deficient conditions compared with amino acid‐sufficient controls. The expression of the NIA2 gene decreased in nitrogen‐depleted microalgae regardless of the initial nitrogen source. However, the results of fatty acid analyses showed that the features of fatty acid profiles followed a similar mode, in which the percentage compositions of C16:0 and C18:1Δ⁹ increased in nitrogen‐depleted cells and that of C16:1Δ⁹, C18:3Δ^9,12,15, C18:4Δ^6,9,12,15, and C18:5Δ^3,6,9,12,15 decreased, regardless of the type of nitrogen source applied. It was also found that the epiphytic bacterium Alteromonas macleodii played a particularly important role in releasing microalgae from the stress of amino acid deficiency. These findings also provide a foundation for regulating microalgal lipid production through manipulation of the nitrogen assimilation‐associated genes.     

Evaluating (13)C enrichment data of free amino acids for precise metabolic flux analysis

Metabolic flux analysis using (13)C enrichment data of intracellular free amino acids (FAAs) can improve the time resolution of flux estimation compared to analysis of proteinogenic amino acid data owing to the faster turnover times of FAAs. The nature of the (13)C enrichment dynamics of FAAs remains obscure, however, especially with regard to its dependence on culture conditions, even though an understanding of dynamic behavior is important for precise metabolic flux estimation. In this study, we analyzed the (13)C enrichment dynamics of free and proteinogenic amino acids in a series of continuous culture experiments with Escherichia coli. The results indicated that the effect of protein degradation on the (13)C enrichment of FAAs was negligible under cellular growth conditions. Furthermore, they showed that the time scale necessary for (13)C enrichment dynamics of FAAs to reach a steady state depends on culture conditions such as oxygen uptake rate, which was likely due to different pool sizes of intracellular metabolites. The results demonstrate the importance of analyzing (13)C enrichment dynamics for the precise estimation of metabolic fluxes using FAA data.     

A mathematical model of growth and alkaloid production in the submerged culture of Claviceps purpurea

A mathematical model of the batch cultivation of Claviceps purpurea was formulated. The main attention was devoted to the effect of exocellular and intracellular phosphate on the growth of the mycelium and production of clavine alkaloid under experimental conditions without limitation by carbon and nitrogen sources. The method of nonlinear regression was used ot predict the optional technological regime of the phosphate addition in the batch culture at different time intervals of additions.