Total Fatty Acid Accumulation Research Articles

Regulatory mechanisms of autophagy on DHA and carotenoid accumulation in Crypthecodinium sp. SUN

BackgroundAutophagy is a crucial process of cellular self-destruction and component reutilization that can affect the accumulation of total fatty acids (TFAs) and carotenoids in microalgae. The regulatory effects of autophagy process in a docosahexaenoic acid (DHA) and carotenoids simultaneously producing microalga, Crypthecodinium sp. SUN, has not been studied. Thus, the autophagy inhibitor (3-methyladenine (MA)) and activator (rapamycin) were used to regulate autophagy in Crypthecodinium sp. SUN.ResultsThe inhibition of autophagy by 3-MA was verified by transmission electron microscopy, with fewer autophagy vacuoles observed. Besides, 3-MA reduced the glucose absorption and intracellular acetyl-CoA level, which resulting in the decrease of TFA and DHA levels by 15.83 and 26.73% respectively; Surprisingly, 3-MA increased intracellular reactive oxygen species level but decreased the carotenoids level. Comparative transcriptome analysis showed that the downregulation of the glycolysis, pentose phosphate pathway and tricarboxylic acid cycle may underlie the decrease of acetyl-CoA, NADPH and ATP supply for fatty acid biosynthesis; the downregulation of PSY and HMGCR may underlie the decreased carotenoids level. In addition, the class I PI3K-AKT signaling pathway may be crucial for the regulation of carbon and energy metabolism. At last, rapamycin was used to activate autophagy, which significantly enhanced the cell growth and TFA level and eventually resulted in 1.70-fold increase in DHA content.ConclusionsOur findings indicate the mechanisms of autophagy in Crypthecodinium sp. SUN and highlight a way to manipulate cell metabolism by regulating autophagy. Overall, this study provides valuable insights to guide further research on autophagy-regulated TFA and carotenoids accumulation in Crypthecodinium sp. SUN.

Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues

BackgroundMetabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG.ResultsIn this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic “push pull and protect” factors correlated with biomass reduction.ConclusionsIntron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.Graphical abstract

Homologous Overexpression of Acyl-CoA Thioesterase 8 Enhanced Free Fatty Acid Accumulation in Oleaginous Fungus Mucor circinelloides WJ11

Thioesterases play an essential role in the metabolism of fatty acids since they are considered one of the key enzymes to change the total amount and composition of fatty acid in an organism’s cells. Acyl-coenzyme A thioesterase 8 (ACOT8) exhibits substrate specificity mainly for short- to long-chain acyl-CoA. To identify and characterize the ACOT8 enzyme’s superfamily in Mucor circinelloides, three genes were characterized and homologously expressed in M. circinelloides WJ11 which has been used as a model organism to investigate the mechanism of lipid accumulation. Multiple sequence alignment showed that McACOT8s had significant conserved motifs in the ACOT8 family. Experimental data indicated that the biomass of the three recombinant strains (McACOT8a, McACOT8b and McACOT8c) was slightly lower than that of the control strain Mc2075, but the total fatty acid (TFA) content was significantly increased by 30.3, 21.5 and 23.9%, respectively, and the free fatty acid production increased from 12.9% (control strain) to 19.2, 25.2 and 26.2% (recombinant types), respectively, with a maximum increase of 103.1% in McACOT8c. The three strains did not show any significant improvement in fatty acid composition compared to the control strain. However, in terms of fatty acid composition of free fatty acid, McACOT8b and McACOT8c contained γ-linolenic acid (C18:3), which was not detected in the control strain or in McACOT8a, indicating that ACOT8b and ACOT8c had substrate specificity for C18:3. These results displayed that ACOT8 can increase TFA accumulation and that it may be an important target of genetic manipulation for microbial oil production.

A scalable model for EPA and fatty acid production by Phaeodactylum tricornutum.

Large-scale photoautotrophic production of microalgae has the potential to provide a sustainable supply of omega-3 fatty acids (eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) for human and animal nutrition. This study presents a kinetic model for the EPA-producing microalga Phaeodactylum tricornutum in photoautotrophic conditions, with light and nitrogen being the growth limiting factors. The model was developed using a dataset obtained from bench-scale (5 L) cultures and was successfully validated against pilot-scale (50 L) cultures. This model is the first to predict the biomass and total fatty acid accumulation along with the EPA concentrations in the biomass and total fatty acid fraction for microalgae. The model was used to develop an optimized repeated-batch strategy; implementation of this led to increases in the biomass and EPA productivities of 50 and 20% respectively. This clearly indicates the potential of the model to be used as a tool in the design, optimization and scale-up of microalgal systems for EPA production.

Effect of sulphur on selenium accumulation and speciation in Nannochloropsis oceanica

• Sulphur deplete and limiting conditions result in more Se accumulation in N. oceanica . • The dominant Se species in N. oceanica is selenomethionine (SeMet) (>97 %). • Se speciation in N. oceanica is SeMet > SeCys 2 > SeMeSeCys > Se. • Selenium enrichment in N. oceanica does not affect total fatty acids accumulation. Sulphur (S) and selenium (Se) are chemically similar. Once Se is taken up, it substitutes S in S-containing amino acids. This study investigated the effect of S on selenite accumulation in the microalga Nannochloropsis oceanica . [0–28 mM] S concentrations and selenite concentrations of 0 and 30 μM were tested. S concentrations of ≤ 3 mM led to decreased cell growth whereas cultures with ≥ 4 mM were not growth limited. Se accumulation increased up to 8-fold when using S ≤ 2 mM and decreased with S 28 mM. The average relative abundance of organic Se species was selenomethionine (SeMet) 98.2 %, selenocystine (SeCys 2 ) 1.4 % and selenomethyl selenocysteine (SeMeSeCys) 0.4 %. Total fatty acids were not affected by S limitation or Se presence. This is the first study on the effect of S on selenite accumulation, organic Se speciation of N. oceanica and its potential as an organic Se-enriched food/feed ingredient.

Mechanisms of Sodium-Acetate-Induced DHA Accumulation in a DHA-Producing Microalga, Crypthecodinium sp. SUN.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (PUFA) that is critical for the intelligence and visual development of infants. Crypthecodinium is the first microalga approved by the Food and Drug Administration for DHA production, but its relatively high intracellular starch content restricts fatty acid accumulation. In this study, different carbon sources, including glucose (G), sodium acetate (S) and mixed carbon (M), were used to investigate the regulatory mechanisms of intracellular organic carbon distribution in Crypthecodinium sp. SUN. Results show that glucose favored cell growth and starch accumulation. Sodium acetate limited glucose utilization and starch accumulation but caused a significant increase in total fatty acid (TFA) accumulation and the DHA percentage. Thus, the DHA content in the S group was highest among three groups and reached a maximum (10.65% of DW) at 96 h that was 2.92-fold and 2.24-fold of that in the G and M groups, respectively. Comparative transcriptome analysis showed that rather than the expression of key genes in fatty acids biosynthesis, increased intracellular acetyl-CoA content appeared to be the key regulatory factor for TFA accumulation. Additionally, metabolome analysis showed that the accumulated DHA-rich metabolites of lipid biosynthesis might be the reason for the higher TFA content and DHA percentage of the S group. The present study provides valuable insights to guide further research in DHA production.

Nutritional programming by maternal diet alters offspring lipid metabolism in a marine teleost

Nutritional programming - the association between the early nutritional environment and long-term consequences for an animal - is an emerging area of research in fish biology. Previous studies reported correlations between maternal provisioning of essential fatty acids to eggs and the whole-body fatty acid composition of larvae reared under uniform conditions for red drum, Sciaenops ocellatus. This study aimed to further investigate the nutritional stimulus and the consequences of nutritional programming by feeding adult red drum several distinct diets and rearing larvae under uniform conditions until 21days post-hatching when larval lipid and fatty acid compositions were assessed. Different maternal diets produced eggs with distinctive lipid and fatty acid compositions, and despite receiving the same larval diet for almost 3weeks, larvae showed differences in total fatty acid accumulation and in retention of highly unsaturated fatty acids (HUFA). Specifically, larvae reared from a maternal diet of shrimp generally showed elevated levels of fatty acids in the initial steps of the n-3 and n-6 HUFA biosynthetic pathways and reduced levels of fatty acid products of the same pathways, especially in triglyceride. Furthermore, the variations in larval fatty acid accumulation induced by maternal diet varied among females. Lipid metabolism altered by parental diet may have consequences for larval physiological processes and behavioral performance, which may ultimately influence larval survival.

Reactive oxygen species derived from NADPH oxidase regulate astaxanthin and total fatty acid accumulation in Chromochloris zofingiensis

Many abiotic stresses can promote astaxanthin and total fatty acid (TFA) accumulation in microalgae, but their signal molecules remain undetermined. The reactive oxygen species (ROS) derived from NADPH oxidase has been reported to act as signals and regulate many cellular responses in cell responses to biotic and abiotic stresses in higher plants. The present study explored the regulatory effects of NADPH oxidase derived ROS on astaxanthin and TFA accumulation in the green alga Chromochloris zofingiensis under nitrogen starvation. Diphenylene iodonium (DPI) inhibition of NADPH oxidase significantly decreased the cellular ROS level under nitrogen starvation. This was accompanied with the decreasing of astaxanthin and secondary carotenoids content, and the increasing of primary carotenoids content. Molecular studies showed that DPI depressed the expression of PSY, LCYb, and BKT, but elevated that of LCYe, which were coordinated with carotenoids content. Meanwhile, the TFA and neutral lipid contents, and the expression of their key biosynthesis genes, ACCase and PDAT, were also reduced in the DPI-treated cells. Additionally, the addition of ROS scavenger, N-acetylcysteine (NAC), decreased the astaxanthin and TFA contents. Taken together, our results revealed that NADPH oxidase-derived ROS was the key regulator in astaxanthin and fatty acid accumulation under nitrogen starvation.

Improving scallop (Pecten maximus and Placopecten magellanicus) spat production by initial larvae size and hydrodynamic cues used in nursery system

There are several factors affecting scallops during the metamorphosis process that could explain the relatively low post-larvae yield observed in hatcheries. Competent bivalve larvae respond to different settlement cues to undergo metamorphosis and without adequate cues, larvae delay their metamorphosis. The objective of this study is to improve the settlement ratio of the two scallop species, Placopecten magellanicus and Pecten maximus by physical cues associated with hydrodynamic conditions, stocking density in settlement units and larval size at time of transfer to settling units. For each treatment, physiological condition was determined by fatty acid analysis to determine the energetic reserves and structural lipids. We observed similar results for the two important commercial pectinid species and validate the hypothesis on the positive effect of increased flow rate and larval size after transfer to settlement systems on settlement success. Increasing flow rate also affects positively the physiological condition of settled post-larvae by a higher accumulation of total fatty acids in neutral lipid fractions. Furthermore, no effect of larval stocking density until 90 larvae cm−2 in the downwelling sieves was observed. To our knowledge this study is the first to characterize the effect of seawater flow rate on settlement success of different pectinid species cultured under similar conditions. The experiments were performed in a close to commercial scale and thus are relevant to industry situations.

Effects of light intensity, light quality, and illumination period on cell growth, TFA accumulation, and DHA production in Crypthecodinium sp. SUN

Crypthecodinium strains are ideal candidates for DHA production. In a previous study, light was found to be efficient in inducing total fatty acid accumulation in Crypthecodinium sp. SUN. In order to further analyze the light-inducing behavior of this microalga, experiments were conducted to elucidate the influence of different light intensities, light qualities, and illumination periods on DHA production. The results showed that an irradiance of 30 μmol photons m−2 s−1 was most suitable for DHA production. Compared to red light and blue light, green light was more efficient in elevating the total fatty acid content in the cells. It was also found that illumination at the first 24 h promoted cell growth, whereas it favored total fatty acid accumulation only during 48–96 h. This is the first systematic investigation of the influence of light on total fatty acid accumulation and DHA production in Crypthecodinium sp. SUN, providing a solid foundation for further research on DHA production.

Kinetic study of growth, lipid and carotenoid formation in β-carotene producing Rhodotorula glutinis

The oleaginous and red yeast Rhodotorula glutinis CCY 20-2-26 is known for its high-storage lipids accumulation in cells as well as for β-carotene biosynthesis. The work is focused on the study of lipogenesis and carotenoid pigments formation by the yeast grown in media with four different C/N ratios (20:1, 50:1, 70:1 and 100:1). Total fatty acids accumulation in cells reached the maximal value of 48% and yielded up to 8.9 g fatty acid/L media under C/N 70:1. On contrary, while the highest pigment accumulation in the yeast (1268 μg/g of cells) was found at the C/N ratio of 20:1, the maximal carotenoid yield (mainly β-carotene) of 12.7 mg/L was obtained under 50:1 ratio. Calculation of kinetic parameters of metabolites production revealed the metabolic correlations among glucose consumption, lipogenesis and carotenoid biosynthesis. It was demonstrated that glucose exhaustion resulted in reduction of growth and lipid accumulation in cells. Then, storage fatty acids, especially palmitic and oleic acids, begun to be degraded by β-oxidations and formed acetyl-CoA which was especially used for biosynthesis of β-carotene. This is the first study providing the new hypothesis about the metabolic connection between fatty acids and β-carotene metabolism in the red yeasts.

Light enhanced the accumulation of total fatty acids (TFA) and docosahexaenoic acid (DHA) in a newly isolated heterotrophic microalga Crypthecodinium sp. SUN

In the present study, light illumination was found to be efficient in elevating the total fatty acid content in a newly isolated heterotrophic microalga, Crypthecodinium sp. SUN. Under light illumination, the highest total fatty acid and DHA contents were achieved at 96h as 24.9% of dry weight and 82.8mgg−1 dry weight, respectively, which were equivalent to 1.46-fold and 1.68-fold of those under the dark conditions. The elevation of total fatty acid content was mainly contributed by an increase of neutral lipids at the expense of starches. Moreover, light was found to alter the cell metabolism and led to a higher specific growth rate, higher glucose consumption rate and lower non-motile cell percentage. This is the first report that light can promote the total fatty acids accumulation in Crypthecodinium without growth inhibition.

Engineering the filamentous fungus Neurospora crassa for lipid production from lignocellulosic biomass

Microbially produced triacylglycerol (TAG) is a potential feedstock for the production of biodiesel, but its commercialization will require high yields from low-cost renewable feedstocks such as lignocellulose. The present study employs a multi-gene approach to increasing TAG biosynthesis in the filamentous fungus Neurospora crassa. We demonstrate the redirection of carbon flux from glycogen biosynthesis towards fatty acid biosynthesis in a glycogen synthase deletion strain (Δgsy-1). Furthermore, combining Δgsy-1 with an enhanced TAG biosynthetic strain (acyl-Coenzyme A synthase; Δacs-3) of N. crassa yielded a twofold increase in total fatty acid accumulation over the control strain. The cellulose degrading potential of this double deletion strain was improved by deleting of the carbon catabolite regulation transcription factor (Δcre-1) to create the triple deletion strain Δacs-3 Δcre-1; Δgsy-1. This strain exhibited early and increased cellulase expression, as well as fourfold increased total fatty acid accumulation over the control on inhibitor-free model cellulose medium. The Δcre-1 mutation, however, was not beneficial for total fatty acid accumulation from pretreated lignocellulose. Conversion of dilute-acid pretreated Miscanthus to TAG was maximum in the constructed strain Δacs-3; Δgsy-1, which accumulated 2.3-fold more total fatty acid than the wild-type control strain, corresponding to a total fatty acid yield of 37.9 mg/g dry untreated Miscanthus.

Probing the effects of high-light stress on pigment and lipid metabolism in nitrogen-starving microalgae by measuring chlorophyll fluorescence transients: Studies with a Δ5 desaturase mutant of Parietochloris incisa (Chlorophyta, Trebouxiophyceae)

We investigated effects of irradiance on the relationships between chlorophyll fluorescence transients (OJIP), carotenoid-to-chlorophyll ratio, and fatty acids in a nitrogen-deprived Parietochloris incisa (Chlorophyta, Trebouxiophyceae) Δ5 desaturase mutant accumulating valuable LC-PUFA dihomo-γ-linolenic acid (DGLA). High light (270μE·m−2·s−1 PAR) and nitrogen starvation brought about a decrease in maximum quantum yield of photosystem II (ΦP0) and electron transport (ΦE0) but enhanced the quantum yield of thermal dissipation (ΦD0) and induced non-photochemical quenching (NPQ) in an irradiance-dependent manner. Under high irradiance a decline in the rate of total fatty acid accumulation and DGLA percentage in comparison with the cultures grown under 130μE·m−2·s−1 PAR was recorded. Increasing irradiance from 130 to 270μE·m−2·s−1 enhanced total fatty acid accumulation only within the first week of nitrogen starvation and negatively affected DGLA production. Regardless of irradiance, ΦP0, ΦE0, and ΦD0 exhibited tight (r2=0.8–0.9) relationships with the stress-induced changes of total fatty acid and DGLA content and the carotenoid-to-chlorophyll ratio. The applicability and limitations of OJIP and its derived parameters for on-line monitoring of physiological condition and accumulation of value-added products in microalgal cultures grown in photobioreactors are discussed.

Algal swimming velocities signal fatty acid accumulation

The use of microalgae for biofuel production will be beneficial to society if we can produce biofuels at large scales with minimal mechanical energy input in the production process. Understanding micro-algal physiological responses under variable environmental conditions in bioreactors is essential for the optimization of biofuel production. We demonstrate that measuring micro-algal swimming speed provides information on culture health and total fatty acid accumulation. Three strains of Chlamydomonas reinhardtii were grown heterotrophically on acetate and subjected to various levels of nitrogen starvation. Other nutrient levels were explored to determine their effect on micro-algal kinetics. Swimming velocities were measured with two-dimensional micro-particle tracking velocimetry. The results show an inverse linear relationship between normalized total fatty acid mass versus swimming speed of micro-algal cells. Analysis of RNA sequencing data confirms these results by demonstrating that the biological processes of cell motion and the generation of energy precursors are significantly down-regulated. Experiments demonstrate that changes in nutrient concentration in the surrounding media also affect swimming speed. The findings have the potential for the in situ and indirect assessment of lipid content by measuring micro-algal swimming kinetics.

Stearoyl-acyl carrier protein desaturase gene from the oleaginous microalga Chlorella zofingiensis: cloning, characterization and transcriptional analysis

The green alga Chlorella zofingiensis can accumulate high level of oleic acid (OA, C18:1△(9)) rich oils in response to stress conditions. To understand the regulation of biosynthesis of fatty acid in particular OA at the molecular level, we cloned and characterized the stearoyl acyl carrier protein (ACP) desaturase (SAD) responsible for OA formation through desaturation of stearic acid (C18:0) from C. zofingiensis. Southern blot indicated that the C. zofingiensis genome contained a single copy of SAD, from which the deduced amino acid sequence shared high identity to the corresponding homologs from other microalgae and higher plants. The desaturation activity of SAD was demonstrated in vitro using C18:0-ACP as a substrate. Stress conditions such as high light (HL), nitrogen deficiency (N(-)), or combination of HL and N(-) (HL+N(-)) drastically up-regulated the transcripts of biotin carboxylase (BC, a subunit of ACCase) and SAD, and therefore induced considerably the cellular accumulation of total fatty acids including OA. Glucose (50mM) gave rise to the similar up-regulation of the two genes and induction of fatty acid accumulation. The accumulation of intracellular reactive oxygen species was found to be associated with the up-regulation of genes. This is the first report of characterization of Chlorella-derived SAD and the results may contribute to understanding of the mechanisms involved in fatty acid/lipid biosynthesis in microalgae.

Carotenoid and fatty acid metabolism in nitrogen-starved Dunaliella salina, a unicellular green microalga

Nitrogen availability and light intensity affect β-carotene overproduction in the green alga Dunaliella salina. Following a previous study on high-light stress, we here report on the effect of nitrogen depletion on the growth characteristics and β-carotene as well as fatty acid metabolism of D. salina under a constant light regime in a turbidostat. Upon nitrogen depletion, the biomass yield on absorbed light approximately doubled, due to a transient increase in cell division rate, swelling of the cells and a linear increase of the density of the cells. Simultaneously, β-carotene started to accumulate up to a final intracellular concentration of 14mgLCV−1 (i.e. 2.7% of AFDW). This β-carotene production accounted for 6% of the increased density of the cells, indicating that other biochemical constituents accumulated as well. Since D. salina accumulates β-carotene in lipid globules, we also determined the fatty acid content and composition of D. salina. The intracellular concentration of the total fatty acid pool did not change significantly during nitrogen starvation, indicating that β-carotene and total fatty acid accumulation were unrelated, similar to what was found previously for high-light treated cells. However, for both high-light and nitrogen stress, β-carotene accumulation negatively correlated with the degree of unsaturation of the total fatty acid pool and, within the individual fatty acids, correlated positively with oleic acid biosynthesis, suggesting that oleic acid may be a key component of the lipid-globule-localized triacylglycerols and thereby in β-carotene accumulation.

Stress-Induced Changes in Optical Properties, Pigment and Fatty Acid Content of Nannochloropsis sp.: Implications for Non-destructive Assay of Total Fatty Acids

In order to develop a practical approach for fast and non-destructive assay of total fatty acid (TFA) and pigments in the biomass of the marine microalga Nannochloropsis sp. changes in TFA, chlorophyll, and carotenoid contents were monitored in parallel with the cell suspension absorbance. The experiments were conducted with the cultures grown under normal (complete nutrient f/2 medium at 75μmol PAR photons/(m(2) s)) or stressful (nitrogen-lacking media at 350 μmol PAR photons/(m(2) s)) conditions. The reliable measurement of the cell suspension absorbance using a spectrophotometer without integrating sphere was achieved by deposition of cells on glass-fiber filters in the chlorophyll content range of 3-13mg/L. Under stressful conditions, a 30-50% decline in biomass and chlorophyll, retention of carotenoids and a build-up of TFA (15-45 % of dry weight) were recorded. Spectral regions sensitive to widely ranging changes in carotenoid-to-chlorophyll ratio and correlated changes of TFA content were revealed. Employing the tight inter-correlation of stress-induced changes in lipid metabolism and rearrangement of the pigment apparatus, the spectral indices were constructed for non-destructive assessment of carotenoid-to-chlorophyll ratio (range 0.3-0.6; root mean square error (RMSE) = 0.03; r (2) = 0.93) as well as TFA content of Nannochloropsis sp. biomass (range 5.0-45%; RMSE = 3.23 %; r (2) = 0.89) in the broad band 400-550nm normalized to that in chlorophyll absorption band (centered at 678nm). The findings are discussed in the context of real-time monitoring of the TFA accumulation by Nannochloropsis cultures under stressful conditions.

Carotenoid-to-chlorophyll ratio as a proxy for assay of total fatty acids and arachidonic acid content in the green microalga Parietochloris incisa

The relationships between pigment (carotenoid and chlorophyll) content with accumulation of total fatty acids (TFA) and arachidonic acid (AA) were studied in the green microalga Parietochloris incisa (Trebouxiophyceae, Chlorophyta) grown under different PFDs (35, 200, and 400 μmol photons m �2 s �1 ) and nitrogen availabilities. The growthofP. incisa under higher light and nitrogen deficiency was accompanied by accumulation of FA, an increase in carotenoid and a decline in chlorophyll content. It was found that the carotenoid-to-chlorophyll ratio (but not the individ- ual pigment content) correlates closely with the volumetric content of both TFA and AA. Analysis of scattering- compensated absorption spectra of P. incisa suspensions revealed their tight relationship in the blue-green range of the spectrum with the carotenoid-to-chlorophyll ratio, TFA, and AA content. These findings allowed the development of algorithms for the non-destructive assay of TFA and AA in cell suspensions in the ranges of 0.09-3.04 and 0.04-1.7 μg mL �1 , with accuracy of 0.06 and 0.01 μ gm L �1 ,r espectively, via analytically measured carotenoid-to-chlorophyll ratio and using the ratio of absorption coefficients at 510 and 678 nm, with accuracy of 0.07 and 0.02 μ gm L �1 , respectively. The feasibility of obtaining essential spectral information con- cerning the physiological condition of P. incisa using a standard spectrophotometer is also shown.

Effect of pH on growth and fatty acid composition ofLactobacillus büchneri andLactobacillus fermentum

Growth and fatty acid composition of two lactobacilli,Lactobacillus buchneri andLactobacillus fermentum, were studied at different pHs of the media in a small-scale fermenter with particular interest in lactobacillic acid production of the cultures. Generally, the total fatty acid content of the bacterial cells increased with increasing culture age. The production of lactobacillic acid was affected in the two lactobacilli by both culture age and pH of the media, but in a very different manner. In L.buchneri cultures, the relative proportion of lactobacillic acid was highest when the pH was lowest (pH 4.5), whereas inL. fermentum cultures, the proportion of lactobacillic acid was highest at pH 7.0. The pH of the medium affected not only the relative proportion of lactobacillic acid, but also biomass production and total fatty acid accumulation of the cultures. Thus, by controlling the pH of the cultures, the volumetric yield of lactobacillic acid could be improved considerably compared to cultures without pH control.