Optimization of <Emphasis Type="Italic">Prochlorothrix hollandica</Emphasis> cyanobacteria culturing for obtaining myristoleic acid

Plankton filament cyanobacteria Prochlorothrix hollandica is characterized by a very high content of C₁₄ and C₁₆ fatty acids (FA) in the lipid membranes. Depending on culturing conditions of the cyanobacteria, total concentrations of myristic and myristoleic acids can reach 35% and those of palmitic and palmitoleic acids can reach 60% of all esterified FA cells. In P. hollandica, a variety of monounsaturated FA is represented by myristoleic and palmitic acids, and by hexadecenoic (C_16:1) acid with olefin bond of cis-configuration, located in the Δ4 position. The process of intensive culturing for P. hollandica cells to yield a maximal biomass in order to isolate the pure drug of myristoleic acid derivative has been optimized. The use of a threestage purification gives 30 mg of chromatographically pure myristoleic acid methyl ester from 17 g of P. hollandica raw biomass (dry mass is 3 g), which is 1% of dry cell mass.     

Modification of fatty acid selectivity of <Emphasis Type="Italic">Candida antarctica</Emphasis> lipase A by error-prone PCR

Objective

To generate Candida antarctica lipase A (CAL-A) mutants with modified fatty acid selectivities and improved lipolytic activities using error-prone PCR (epPCR).

Results

A Candida antarctica lipase A mutant was obtained in three rounds of epPCR. This mutant showed a 14 times higher ability to hydrolyze triacylglycerols containing conjugated linoleic acids, and was 12 and 14 times more selective towards cis-9, trans-11 and trans-10, cis-12 isomers respectively, compared to native lipase. Lipolytic activities towards fatty acid esters were markedly improved, in particular towards butyric, lauric, stearic and palmitic esters.

Conclusion

Directed molecular evolution is an efficient method to generate lipases with desirable selectivity towards CLA isomers and improved lipolytic activities towards esters of fatty acids.

     

Changes in growth and composition of the marine microalgae <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> and <Emphasis Type="Italic">Nannochloropsis salina</Emphasis> in response to changing sodium bicarbonate concentrations

Oils, carbohydrates, and fats generated by microalgae are being refined in an effort to produce biofuels. The research presented here examines two marine microalgae, Nannochloropsis salina (green alga) and Phaeodactylum tricornutum (diatom), when grown with 0 (no addition), 0.5, 1.0, 2.0, and 5.0 g L^?1 NaHCO₃ added to an f/2 medium during the growth phase (GP) and a nutrient induced (nitrate limitation) lipid formation phase (LP). We hypothesize that the addition of NaHCO₃ is a sustainable and practical strategy to increase cellular density and concentrations of lipids in microalgae as well as the rate of lipid accumulation. In N. salina, final cell densities were significantly (p?<?0.05) higher in the NaHCO₃-treated cells than the control while in P. tricornutum the cell densities were higher with >[NaHCO₃] during the GP. During the LP, cell densities were generally higher in the NaHCO₃-treated cells compared with controls. F _V/F _M (efficiency of photosystem II) patterns paralleled those for cell density with generally higher values with higher concentrations of NaHCO₃ and significantly different values between controls and 5.0 g L^?1 NaHCO₃ at the end of the GP (p?<?0.05). F _V/F _M was variable between treatments in P. tricornutum (0.3–0.65) but less so in N. salina for (0.5–0.7) regardless of [NaHCO₃]. The lipid index (measured with Nile red), used as a proxy for triacylglycerides (TAGs), was 10.2?±?6.5 and 4.4?±?2.9 (fluorescence units/OD cells ×1000) for N. salina and P. tricornutum, respectively, at the end of the GP. At the end of the LP, the lipid index was eight and four times higher than during the GP in the corresponding 5.0 g L^?1 NaHCO₃ treatments, revealing that N. salina was accumulating more lipid than P. tricornutum. Dry weights essentially doubled during LP compared with GP for N. salina; this was not the case for P. tricornutum. In general, the percentage of ash in dry weights was significantly higher in the LP relative to the corresponding GP treatments for P. tricornutum; this was not the case for N. salina. During the LP, there was also less soluble protein in N. salina compared to GP; differences were not significant in cells growing with 2.0 or 5.0 g L^?1 NaHCO₃. In P. tricornutum, faster growing cells had more soluble protein during the GP and LP; differences between treatments were significant. P. tricornutum generally accumulated significantly more crude protein than N. salina at higher [NaHCO₃]; there was three times more crude protein in the highest NaHCO₃ (5.0 g L^?1) treatment compared with the controls. C:N ratios (mol:mol) were similar across treatments during GP: 7.03?±?0.12 and 10.16?±?0.41 for N. salina and P. tricornutum, respectively. Further, C:N ratios increased with increasing [NaHCO₃] during LP. Species-specific fatty acid methyl ester (FAMEs) profiles were observed. While C16:0 was lower in P. tricornutum compared to N. salina, the diatom produced more C16:1 and C14 but not C18:3. Monounsaturated fatty acids (MUFA) significantly increased in N. salina in the LP compared to GP and in response to increasing [NaHCO₃] (t tests; p?<?0.05). Saturated fatty acids (SFA) responded similarly but to a lesser degree. There were more polyunsaturated fatty acids (PUFA) in N. salina than MUFAs or SFAs. In P. tricornutum, there were generally more SFAs, MUFAs and PUFAs in P. tricornutum during LP than GP in the corresponding NaHCO₃ treatments. These findings reveal the importance of considering NaHCO₃ as a supplemental carbon source in the culturing marine phytoplankton in large-scale production for biofuels.     

Extractive compounds of birch buds (<Emphasis Type="Italic">Betula pendula</Emphasis> Roth.): I. Composition of fatty acids,hydrocarbons, and esters

This is the first report devoted to study of the hydrocarbon composition of the extract of buds of European birch Betula pendula (family Betulacea). We have identified saturated (C₁₆ to C₂₈, even number of carbon atoms) and unsaturated (linoleic and linolenic) fatty acids, β-caryophyllene, α-humulene, and the components of epicuticular waxes of cover scales, such as n-alkanes (C₂₁ to C₂₆), esters of fatty acids (C₁₆ to C₂₈, even number of carbon atoms), and fatty alcohols (C₁₈ to C₃₀, even number of carbon atoms). The gas chromatographic retention indices of all identified compounds have been determined.     

Biotic Interactions between Two Species of Microalgae in Mixed Culture

Biotic interactions in a mixed culture of two microalgae species—Scenedesmus quadricauda (Turp.) Breb. and Monoraphidium arcuatum (Korsch.) Hind.—used in bioassay in monocultures as test objects were studied. The toxic effect of cell-free filtrates from different “age” monoculture (2, 7, 10, 15, 21, and 28 days) of S. quadricauda on the growth of the “young” test culture of M. arcuatum and, conversely, the toxic effect of cell-free filtrates from the different “age” (2, 7, 10, 15, 21, and 28 days) monoculture of M. arcuatum on the growth of the “young” test culture of S. quadricauda was evaluated. Simultaneously, the toxicity of their own filtrates of different “ages” was monitored by a test culture of each species. The interactions of the species in the mixed culture can be regarded as negative, as an antagonistic one, when both populations inhibit the growth of each other through metabolites and food resource competition, while the effect of S. quadricauda on M. arcuatum is much stronger. The main factor constraining the growth of monoculture S. quadricauda is the rapid depletion of the food resource from the medium and not the inhibition of growth by its own metabolites. The depletion of the food resources from the medium in monoculture of M. arcuatum occurs much later than in monoculture of S. quadricauda. Metabolites of S. quadricauda cause a strong inhibitory effect on the growth of M. arcuatum, and the metabolites of M. arcuatum cause a weak inhibitory effect on the growth of S. quadricauda. The filtrates of the “old” culture of S. quadricauda (21–28 days) cause the greatest inhibitory effect on cell division of M. arcuatum. The filtrates of the “old” culture of S. quadricauda (21–28 days) cause the greatest inhibitory effect on cell division of M. arcuatum. Comparative analysis of the cell number dynamics of two species, S. quadricauda and M. arcuatum, in mono- and two-species algal cultures, as well as experiments with filtrates of these monocultures, showed that the interaction of species can be explained by the food resource competition and allelopathic interaction (exometabolite effect).     

Proteomic profiling of liver tissue from the <Emphasis Type="Italic">mdx</Emphasis>-<Emphasis Type="Italic">4cv</Emphasis> mouse model of Duchenne muscular dystrophy

Background

Duchenne muscular dystrophy is a highly complex multi-system disease caused by primary abnormalities in the membrane cytoskeletal protein dystrophin. Besides progressive skeletal muscle degeneration, this neuromuscular disorder is also associated with pathophysiological perturbations in many other organs including the liver. To determine potential proteome-wide alterations in liver tissue, we have used a comparative and mass spectrometry-based approach to study the dystrophic mdx-4cv mouse model of dystrophinopathy.

Methods

The comparative proteomic profiling of mdx-4cv versus wild type liver extracts was carried out with an Orbitrap Fusion Tribrid mass spectrometer. The distribution of identified liver proteins within protein families and potential protein interaction patterns were analysed by systems bioinformatics. Key findings on fatty acid binding proteins were confirmed by immunoblot analysis and immunofluorescence microscopy.

Results

The proteomic analysis revealed changes in a variety of protein families, affecting especially fatty acid, carbohydrate and amino acid metabolism, biotransformation, the cellular stress response and ion handling in the mdx-4cv liver. Drastically increased protein species were identified as fatty acid binding protein FABP5, ferritin and calumenin. Decreased liver proteins included phosphoglycerate kinase, apolipoprotein and perilipin. The drastic change in FABP5 was independently verified by immunoblotting and immunofluorescence microscopy.

Conclusions

The proteomic results presented here indicate that the intricate and multifaceted pathogenesis of the mdx-4cv model of dystrophinopathy is associated with secondary alterations in the liver affecting especially fatty acid transportation. Since FABP5 levels were also shown to be elevated in serum from dystrophic mice, this protein might be a useful indicator for monitoring liver changes in X-linked muscular dystrophy.

     

pH and CO<Subscript>2</Subscript> effects on <Emphasis Type="Italic">Coelastrella</Emphasis> (<Emphasis Type="Italic">Scotiellopsis</Emphasis>) <Emphasis Type="Italic">rubescens</Emphasis> growth and metabolism

We studied effects of рН and СО₂ enrichment on the physiological condition and biochemical composition of a carotenogenic microalga Coelastrella (Scotiellopsis Vinatzer) rubescens Kaufnerová et Eliás (Scenedesmaceae, Sphaeropleales, Chlorophyceae), a promising source of natural astaxanthin. The microalga was grown at a constant pH (5, 6, 7 or 8) maintained by direct СО₂ injection. The air-sparged culture served as the control. Cell division rate and size, dry biomass productivity, the rates of nitrogen and phosphorus uptake as well as photosynthetic pigment and total lipid content and fatty acid composition were followed. С. rubescens possessed a narrow-range рН tolerance (the optimum рН 6–7). Under these conditions, the highest values of the maximum (1.0–1.1 1/day) and average (0.3–0.35 1/day) specific growth rate, chlorophyll а (4.8–4.9%) and total carotenoid dry weight percentages (1.7–1.8%) were recorded. Cell lipid fatty acid unsaturation index (1.851) and polyunsaturated fatty acid percentage (36–39%) and С18:3 ω3/С18:1 ω9 ratio (3.8–4.5) were also the highest under these conditions. A decline of рН to 5 brought about severe stress manifesting itself as a cell division cessation, photosynthetic apparatus reduction, two-fold increase in cell volume, accumulation of dry weight and lipids and a considerable decline in fatty acid unsaturation. Cultivation of С. rubescens without СО₂ enrichment resulted in a rapid alkalization of the medium to рН 9.5–10.5 impairing the physiological condition of the cells. Reasons of the deteriorative effects of suboptimal pH values on the physiological condition of C. rubescens are discussed.     

The combined effects of blue light and dilution rate on lipid class and fatty acid composition of <Emphasis Type="Italic">Tisochrysis lutea</Emphasis>

The lipid class and the fatty acid compositions of microalgae highly influence bivalve larval and post-larval development. Light is an essential environmental factor for microalgal culture, and quantity and quality of light may induce changes in the biochemical composition of the algae. The objective of this study was to investigate the effect of light spectrum (blue vs. white light) on lipid class and fatty acid compositions of Tisochrysis lutea cultured in a chemostat. Two different dilution rates (D) were assayed for each light spectrum: 0.2 and 0.7 day^?1. Triacylglycerol (TAG), sterol, and hydrocarbon (HC) content increased sharply at low D. The proportion of alkenones was significantly reduced under blue light. Polyunsaturated fatty acids (PUFA), and particularly n-3 PUFA, content in phospholipids (PL) increased under blue light compared to white light at low D. Thus, blue light raised 22:6(n-3) levels in total lipids of T. lutea at low D. The cultivation of T. lutea in a chemostat at low D under blue light may improve nutritional value as feed for bivalve larvae by modifying the PUFA profile, especially increasing 22:6(n-3).     

Lipid composition of the three co-existing <Emphasis Type="Italic">Calanus</Emphasis> species in the Arctic: impact of season,location and environment

Arctic species of Calanus are critical to energy transfer between higher and lower trophic levels and their relative abundance, and lipid content is influenced by the alternation of cold and warm years. All three species of Calanus were collected during different periods in Kongsfjorden (Svalbard, 79°N) and adjacent shelf during the abnormally warm year of 2006. Lipid composition and fatty acid structure of individual lipid classes were examined in relation with population structure. Wax esters dominated the neutral lipid fraction. Phosphatidylcholine (PC) dominated the structural lipids followed by phosphatidylethanolamine (PE). PC/PE ratios of 3–6 suggested an increase in PC proportions compared to earlier studies. Depending on the time scale, fatty acids of wax esters illustrated either trophic differences between fjord and offshore conditions for C. hyperboreus and C. finmarchicus or trophic differences related to seasonality for C. glacialis. Similarly, seasonality and trophic conditions controlled the changes in fatty acids of triglycerides, but de novo synthesis of long-chain monoenes suggested energy optimization to cope with immediate metabolic needs. Polar lipids fatty acid composition was species specific and on the long-term (comparison with data from the past decade) composition appears related to changes in trophic environment. Fatty acid composition of PC and PE indicated relative dominance of 20:5n-3 in PC and 22:6n-3 in PE for all three species. The combination of PE and PC acyl chain and phospholipid head group restructuring indicates an inter-annual variability and suggests that membrane lipids are the most likely candidate to evaluate adaptive changes in Arctic copepods to hydrothermal regime.     

Feruloyl esterase from <Emphasis Type="Italic">Alternaria tenuissima</Emphasis> that hydrolyses lignocellulosic material to release hydroxycinnamic acids

An extracellular feruloyl esterase from the culture filtrates of the isolated fungus Alternaria tenuissima was successfully purified to apparent homogeneity by anion-exchange and size-exclusion chromatography. Peptide fragments of purified enzyme (designated as AltFAE; molecular weight of 30.3 kDa determined by SDS-PAGE) were identified by mass spectrometry using a NanoLC-ESI-MS/MS system. Michaelis-Menten constants (K_M) and catalytic efficiencies (k_cat/K_M) were determined for typical substrates of feruloyl esterase, and the lowest K_M of 50.6 μM (i.e., the highest affinity) and the highest k_cat/K_M (3.1 × 10⁵ s^—1 M^–1) were observed for methyl ^p-coumarate and methyl ferulate, respectively. Not least, AltFAE catalyzed conversion of lignocellulosic material (e.g. wood meal) to release hydroxycinnamic products, i.e. ferulic- and ^p-coumaric acids.     

Ammonium,nitrate, and urea play different roles for lipid accumulation in the nervonic acid—producing microalgae <Emphasis Type="Italic">Mychonastes afer</Emphasis> HSO-3-1

Producing valuable coproducts from oleaginous microalgae is an option to reduce the total cost of biofuel production. Here, the influence of nitrogen sources on biomass yield and lipid accumulation of a newly identified oleaginous green microalgal strain, Mychonastes afer HSO-3-1, was evaluated. Carbon assimilation and the following lipid biosynthesis of M. afer were inhibited to some extent under weak acidic conditions (6 < pH < 7) and any of the tested nitrogen source. The highest lipid productivity of 50.7 mg L^?1 day^?1 was achieved with a 17.6 mM nitrogen supplement in the form of urea. The cell polar lipid content was significantly higher than triacylglycerol (TAG), and saturated palmitic acid (C16:0) occupied a dominant position in the fatty acid profiles while culturing M. afer in acidic medium with NH₄ ⁺ as the nitrogen source. Under neutral conditions, the lipid productivities of M. afer cultivated in media containing 17.6 mM of NaNO₃, NH₄Cl, and NH₄NO₃ were 76.2, 77.5, and 79.0 mg L^?1 day^?1, respectively. The greatest TAG content (58.56%) of total lipids was obtained when NaNO₃ was used as the nitrogen source. There was no significant difference in the fatty acid composition of M. afer cells when they were cultivated in neutral media supplemented with NaNO₃, urea, NH₄Cl, and NH₄NO₃. Therefore, NH₄ ⁺ was not a suitable nitrogen source for M. afer cultivation due to the additional labor, working procedures, and alkali required to adjust the medium pH. Considering that using urea as nitrogen source could reduce the cost of nutrient salts substantially and urea can be taken up and utilized by most microalgae, it is a preferred nitrogen source. The major properties of biodiesel derived from M. afer HSO-3-1 met biodiesel quality, and nervonic acid concentrations remained at approximately 3.0% of total fatty acids.     

Lipid production by microalgae <Emphasis Type="Italic">Chlorella protothecoides</Emphasis> with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment

Volatile fatty acids (VFAs) that can be derived from food wastes were used for microbial lipid production by Chlorella protothecoides in heterotrophic cultures. The usage of VFAs as carbon sources for lipid accumulation was investigated in batch cultures. Culture medium, culture temperature, and nitrogen sources were explored for lipid production in the heterotrophic cultivation. The concentration and the ratio of VFAs exhibited significant influence on cell growth and lipid accumulation. The highest lipid yield coefficient and lipid content of C. protothecoides grown on VFAs were 0.187 g/g and 48.7 %, respectively. The lipid content and fatty acids produced using VFAs as carbon sources were similar to those seen on growth and production using glucose. The techno-economic analysis indicates that the biodiesel derived from the lipids produced by heterotrophic C. protothecoides with VFAs as carbon sources is very promising and competitive with other biofuels and fossil fuels.     

Microalgae from the Selenastraceae as emerging candidates for biodiesel production: a mini review

Over the years, microalgae have been identified to be a potential source of commercially important products such as pigments, polysaccharides, polyunsaturated fatty acids and in particular, biofuels. Current demands for sustainable fuel sources and bioproducts has led to an extensive search for promising strains of microalgae for large scale cultivation. Prospective strains identified for these purposes were among others, mainly from the genera Hematococcus, Dunaliella, Botryococcus, Chlorella, Scenedesmus and Nannochloropsis. Recently, microalgae from the Selenastraceae emerged as potential candidates for biodiesel production. Strains from the Selenastraceae such as Monoraphidium sp. FXY-10, M. contortum SAG 47.80, Ankistrodesmus sp. SP2-15 and M. minutum were high biomass and lipid producers when cultivated under optimal conditions. A number of Selenastraceae strains were also reported to be suitable for cultivation in wastewater. This review highlights recent reports on potential strains from the Selenastraceae for biodiesel production and contrasts their biomass productivity, lipid productivity as well as fatty acid profile. Cultivation strategies employed to enhance their biomass and lipid productivity as well as to reduce feedstock cost are also discussed in this paper.     

Chemical Composition of Lipids from Peat-Forming Leafy Mosses of Eutrophic Swamps of Western Siberia and Altai

The molecular composition of lipids in three samples of leafy mosses (Aulacomnium palustre, Warnstorfia fluitans, and Calliergon giganteum) has been determined. The revealed acyclic compounds included normal and isoprenoid alkanes, isoprenoid alkenes, normal and isoprenoid ketones, carboxylic acids and their esters, alcohols, and aldehydes. Among cyclic compounds, bi-, tri- and tetracyclic polycycloaromatic hydrocarbons (PAHs), bicyclic and pentacyclic terpenoids, steroids and tocopherols have been observed. The identified organic compounds consisted mainly of carbocyclic acids and n-alkanes with the prevalence of C₂₇ homologues. A. palustre is characterized by a reduced content of isoprenoid compounds, alcohols, and ketones, while the content of unsaturated acids, pentacyclic terpenoids, and aldehydes is rather heightened. A. palustre differs from W. fluitans and C. giganteum in the steroid composition and contains eremophylene, a sesquiterpenoid, which is absent in the mosses of the family Amblystegiaceae. Compared to C. giganteum, W. fluitans has a higher content of lycopadiene, carboxylic acids, n-alkanes, phyt-2-ene, aldehydes, esters, squalene, diploptene, α-tocopherol, and triphenyl phosphates.     

Microalgal Biomass and Lipid Production on Dairy Effluent Using a Novel Microalga, <Emphasis Type="Italic">Chlorella</Emphasis> sp. Isolated from Dairy Wastewater

The present study focused on cost-effective production of microalgal biomass and lipid production on dairy effluent. The novel microalga, Chlorella sp. isolated from the dairy effluent showed high growth and lipid production on the undiluted and two-fold diluted dairy effluent which were four to five times higher than those of Chlorella vulgaris (control). The high growth of Chlorella sp. was thought to be possibly due to its heterotrophic growth capacity, high turbidity, COD, nutrients and trace elements. In contrast, C. vulgaris showed poor heterotrophic and photoautotrophic growth under the highly turbid conditions of dairy effluent. Both Chlorella sp. and C. vulgaris showed similar total FAME (mg FAME/g algal cells). The fatty acid composition analysis revealed that both Chlorella sp. and C. vulgaris possessed major C18 and C20 fatty acids which will be used for biodiesel production. Overall, the novel microalga, Chlorella sp. isolated from the dairy effluent showed high potential for cost-effective algal cultivation and lipid production on dairy effluent without any modification of process.     

Characterization of cell structural change,growth, lipid accumulation,and pigment profile of a novel oleaginous microalga, <Emphasis Type="Italic">Vischeria stellata</Emphasis> (Eustigmatophyceae), cultured with different initial nitrate supplies

The appropriate microalgal species and the optimal nitrogen supply in culture medium are vital factors in maximizing biomass and metabolite productivities in microalgae. Vischeria stellata is an edaphic unicellular eustigmatophycean microalga. Cytological and ultrastructural characteristics and the effects of different initial nitrate-nitrogen concentrations on growth, lipid accumulation, fatty acid profile, and pigment composition were investigated in the present study. The cell structures of V. stellata changed with the degree of nutrient utilization and growth phase. The initial nitrate concentration for the optimal growth of V. stellata ranged from 6.0 to 9.0 mM. The maximum total lipid (TLs), neutral lipid (NLs), and total fatty acid (TFAs) contents were 55.9, 51.9, and 44.7 % of dry biomass, respectively. The highest volumetric productivity of TLs, NLs, and TFAs reached 0.28, 0.25, and 0.21 g L^?1 day^?1, respectively. V. stellata had a suitable fatty acid profile for biodiesel production, as well as containing eicosapentaenoic acid (EPA) for nutraceutical applications. In addition, the content β-carotene, increased gradually as culture time was prolonged, resulting in its exclusive production at the end of cultivation. V. stellata is a promising microalgal strain for the production of biofuels and bioproducts.