Productivity and biochemical composition of <Emphasis Type="Italic">Tetradesmus obliquus</Emphasis> and <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis>: effects of different cultivation approaches

The present work evaluated biomass productivity, carbon dioxide fixation rate, and biochemical composition of two microalgal species, Phaeodactylum tricornutum (Bacillariophyta) and Tetradesmus obliquus (Chlorophyta), cultivated indoors in high-technology photobioreactors (HT-PBR) and outdoors both in pilot ponds and low-technology photobioreactors in a greenhouse in southern Italy. Microalgae were grown in standard media, under nitrogen starvation, and in two liquid digestates obtained from anaerobic digestion of agro-zootechnical and vegetable biomass. P. tricornutum, cultivated in semi-continuous mode in indoor HT-PBRs with standard medium, showed a biomass productivity of 21.0?±?2.3 g m^?2 d^?1. Applying nitrogen starvation, the lipid productivity increased from 2.3 up to 4.5?±?0.5 g m^?2 d^?1, with a 24 % decrease of biomass productivity. For T. obliquus, a biomass productivity of 9.1?±?0.9 g m^?2 d^?1 in indoor HT-PBR was obtained using standard medium. Applying liquid digestates as fertilizers in open ponds, T. obliquus gave a biomass productivity (10.8?±?2.0 g m^?2 d^?1) not statistically different from complete medium such as P. tricornutum (6.5?±?2.2 g m^?2 d^?1). The biochemical data showed that the fatty acid composition of the microalgal biomass was affected by the different cultivation conditions for both microalgae. In conclusion, it was found that the microalgal productivity in standard medium was about doubled in HT-PBR compared to open ponds for P. tricornutum and was about 20 % higher for T. obliquus.     

Magnetophoretic harvesting of freshwater microalgae using polypyrrole/Fe<Subscript>3</Subscript>O<Subscript>4</Subscript> nanocomposite and its reusability

A magnetophoretic harvesting agent, a polypyrrole/Fe₃O₄ magnetic nanocomposite, is proposed as a cost and energy efficient alternative to recover biomass of the microalgae Botryococcus braunii, Chlorella protothecoides, and Chlorella vulgaris from their culture media. The maximal recovery efficiency reached almost 99 % for B. braunii, 92.4 % for C. protothecoides, and 90.8 % for C. vulgaris. The maximum adsorption capacity (Q ₀) of the magnetic nanocomposite for B. braunii (63.49 mg dry biomass mg^?1 PPy/Fe₃O₄) was higher than that for C. protothecoides (43.91 mg dry biomass mg^?1 PPy/Fe₃O₄) and C. vulgaris (39.98 mg dry biomass mg^?1 PPy/Fe₃O₄). The highest harvesting efficiency for all the studied microalgae were at pH 10.0, and measurement of zeta-potential confirmed that the flocculation was induced by charge neutralization. This study showed that polypyrrole/Fe₃O₄ can be a promising flocculant due to its high efficacy, low dose requirements, short settling time, its integrity with cells, and with great potential for saving energy because of its recyclability.     

Potential Outdoor Cultivation of Green Microalgae Based on Response to Changing Temperatures and by Combining with Air Temperature Occurrence

In this study, our working hypothesis was to examine whether temperature alters biomass and metabolite production by microalgae according to strain. We also addressed whether it is possible to choose a strain suitable for growing in each season of a given region. A factorial experiment revealed a significant interaction between chlorophylls a and b (Chl a and Chl b), carotenoid/Chl (a?+?b) ratio, biomass and total lipid productivity of six green microalgae (four Chlorella spp., Chlorella sorokiniana and Neochloris oleoabundans) after 15 days at four temperatures. At 39/35 °C, two Chlorella sp. strains (IPR7115 and IPR7117) showed higher total carotenoids/Chl (a?+?b) (0.578 and 0.830), respectively. N. oleoabundans had the highest Chl a (8210 μg L^?1) and Chl b (1909 μg L^?1) at 19/15 °C and highest maximum dry biomass (2900 mg L^?1), specific growth rate (0.538 day^?1) and total lipids (1003 mg L^?1) at 15/8 °C. We applied a method to infer the growth of these six green microalgae in outdoor ponds, as based on their response to changing temperatures and by combining with historical data on day/night air temperature occurrence for a given region. We conclude that the use of regionalized maps based on air temperature is a good strategy for predicting microalgal cultivation in outdoor ponds based on their features and tolerance to changing temperature.     

Mixotrophic cultivation of <Emphasis Type="Italic">Chlorococcum</Emphasis> sp. under non-controlled conditions using a digestate from pig manure within a biorefinery

A number of business opportunities may arise from microalgae and wastewater treatment becoming an integrated system, as biofuels and high-added value products could be obtained simultaneously. This study, performed under controlled and non-controlled conditions, aimed at cultivating Chlorococcum sp. using a digestate from pig manure as culture medium and assessing its growth and biochemical composition for further applications. Under controlled conditions, cultures of Chlorococcum sp. were established testing various digestate dilutions (v/v). It was found that all tested dilutions (up to 8% v/v) promoted a higher biomass density, compared to the control culture in modified Bold’s Basal Medium (modified BBM). Under non-controlled conditions, it was found that the biomass productivity using the digestate diluted 5.6% v/v (23.4 mg L^?1 day^?1) was statistically similar to the one obtained using modified BBM (26.4 mg L^?1 day^?1). The volatile fatty acids contained in the digestate might have allowed mixotrophic growth for Chlorococcum sp. The intracellular lipid content in Chlorococcum sp. remained constant throughout the experiments in both, treatment and control cultures, while carbohydrates increased from 20 to 45% of the cell dry weight in the treatment and from 20 to 42% in the control one. It was concluded that conditions of nitrogen starvation and fluctuating irradiance and temperature benefit carbohydrate accumulation in this strain, since intracellular carbohydrate content increased nearly two-fold during this period. Additionally, the obtained biomass has the potential to be used as feedstock for bioethanol production. This system can meet the concept of a microalgae-based biorefinery, in which biofuels and high-added value products are produced from microalgae and wastewater.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

Growth and photosynthetic activity of <Emphasis Type="Italic">Botryococcus braunii</Emphasis> biofilms

Botryococcus braunii is a green microalga capable of producing large amounts of external long-chain hydrocarbons suitable as a source of biofuel. There have been several studies indicating that cultures of B. braunii can reduce the energy and water requirement for mass biofuel production, especially if non-destructive extraction methods for milking hydrocarbons are used. Growing microalgae as a raw material for biofuel using conventional liquid-based cultivation (i.e., raceway ponds) has yet to be shown to be economically successful. An alternative solid growth (biofilm) cultivation method can markedly reduce the energy requirements and costs associated with the harvesting and dewatering processes. We evaluated the growth of biofilms of several strains of B. braunii (from races A, B, L and S) and found that three of the four tested races successfully grew to stationary phase in 10 weeks with no contamination. Among all races, B. braunii BOT22 (race B) reached the highest biomass and lipid yields (3.80 mg dry weight cm^?2 day^?1 and 1.11 mg dry weight cm^?2). Irrespective of the race, almost all photosynthetic parameters (F _V /F ₀ , PI_ABS and the OJIP curve) showed that the biofilm cultures were more stressed during lag and stationary phases than in logarithmic phase. We also studied the Botryococcus biofilm profiles using confocal microscopy and found that this method is suitable for estimating the overall biomass yield when compared with gravimetric measurement. In conclusion, the growth characteristics (biomass and lipid) and photosynthetic performance of all races indicated that B. braunii BOT22 is the most promising strain for biofilm cultivation.     

Impacts of enhanced UVB radiation on photosynthetic characteristics of the marine diatom <Emphasis Type="Italic">Phaeodactylum tricornutum</Emphasis> (Bacillariophyceae,Heterokontophyta)

Solar ultraviolet B (UVB) irradiance at the Earth’s surface is increasing due to anthropogenic influences. To evaluate the effects of enhanced UVB radiation on photosynthetic characteristics of the marine diatom Phaeodactylum tricornutum, the species was exposed to four levels of UVB radiation, 0, 0.25, 0.75, and 1.50 KJ m^?2 day^?1 for 7 days. Effects of UVB stress on net photosynthetic rate, net respiration rate, variable chlorophyll (Chl) fluorescence parameters, Chl a and carotenoid contents, and UV-absorbing compounds (UVACs) were investigated. Results showed that there were no significant differences in terms of net respiration rate or maximal photochemical efficiency of photosystem II (F_v/F_m) between the treatments in the short or long term. However, enhanced UVB radiation at an intensity of 0.16 W m^?2 had a negative effect on the net photosynthetic rate, electron transport rate, and on the pathway of excess energy dissipation over the short term (1 to 5 days). Carotenoid and UVACs content increased under UVB radiation. Photosynthetic parameters were unaffected by UVB radiation on the seventh day indicating that P. tricornutum can adapt to UVB radiation in the long term. Results of the present study indicate that there is a dynamic balance between damage and adaptation in microalgae that enables them to adapt to UVB-induced photosystem alterations during both short-term and long-term exposure.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

The effect of gradual increase in salinity on the biomass productivity and biochemical composition of several marine,halotolerant<Emphasis Type="Bold">,</Emphasis> and halophilic microalgae

Open ponds are the preferred cultivation system for large-scale microalgal biomass production. To be more sustainable, commercial scale biomass production should rely on seawater, as freshwater is a limiting resource, especially in places with high irradiance. If seawater is used for both pond fill and evaporative volume makeup, salinity of the growth media will rise over time. It is not possible for any species to achieve optimum growth over the whole saline spectrum (from seawater salinity level up to salt saturation state). In this study, we investigated the effects of gradual salinity increase (between 35 and 233 ppt) on biomass productivity and biochemical composition (lipid and carbohydrate) of six marine, two halotolerant, and a halophilic microalgae. A gradual and slow stepped salinity increase was found to expand the salinity tolerance range of tested species. A gradual reduction in biomass productivity and maximum photochemical efficiency was observed as a consequence of increased salinity in all tested species. Among the marine microalgae, Tetraselmis showed highest biomass productivity (32 mg L^?1 day^?1) with widest salinity tolerance range (35 to 109 ppt). Halotolerant Amphora and Navicula were able to grow from 35 ppt to 129 ppt salinity. Halophilic Dunaliella was the only species capable of growing between 35 and 233 ppt and showed highest lipid content (56.2%) among all tested species. This study showed that it should be possible to maintain high biomass in open outdoor cultivation utilizing seawater by growing Tetraselmis, Amphora, and Dunaliella one after another as salinity increases in the cultivation system.     

Analysis of the effect of plant growth regulators and organic elicitors on antibacterial activity of <Emphasis Type="Italic">Eucomis autumnalis</Emphasis> and <Emphasis Type="Italic">Drimia robusta</Emphasis> ex vitro-grown biomass

The effects of plant growth regulators (PGRs) and organic elicitors (OEs) on in vitro propagation of Eucomis autumnalis was established. Three-year-old ex vitro grown plants from organogenesis of E. autumnalis and somatic embryogenesis (previously reported protocol) of Drimia robusta were investigated for antibacterial activity. In vitro propagation from leaf explants of E. autumnalis was established using different PGRs and OE treatments for mass propagation, biomass production and bioactivity analysis to supplement the use of wild plant material. Prolific shoots (16.0?±?0.94 shoots per explant) were obtained with MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium containing 100 mg l^?1 haemoglobin (HB), 10 µM benzyladenine (BA) and 2 µM naphthaleneacetic acid (NAA). The shoots were rooted effectively with a combination of 2.5 µM indole-3-acetic acid and 5.0 µM indole-3-butyric acid. The plantlets were successfully acclimatized in a vermiculite-soil mixture (1:1 v/v) in the greenhouse. Three-year-old ex vitro-grown E. autumnalis and D. robusta plants derived via organogenesis and somatic embryogenesis respectively exhibited antibacterial activity and varied with PGR and OE treatments, plant parts and bacteria. The leaves of E. autumnalis ex vitro-derived from a combination of HB, BA and NAA followed by the individual treatments of BA and HB gave the best antibacterial activities (<?1 mg ml^?1: minimum inhibitory concentration from 0.098 to 0.78 mg ml^?1) against all tested pathogenic bacteria (Bacillus subtilis, Enterococcus faecalis, Micrococcus luteus, Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa). The bulbs of D. robusta ex vitro-derived from solid culture with 10 µM picloram, 1 µM thidiazuron and 20 µM glutamine exhibited good antibacterial activity against E. faecalis, M. luteus and S. aureus when compared with other treatments and mother plants. The ex vitro-grown E. autumnalis and D. robusta biomass produced with PGRs along with OE treatments confirmed a good potent bioresource and can be used as antibacterial agents. The in vitro plant regeneration of E. autumnalis and D. robusta protocols and ex vitro plants could be used for conservation strategies, bioactivity and traditional medicinal use.     

Accumulation and tolerance characteristics of lead in <Emphasis Type="Italic">Althaea rosea</Emphasis> Cav. and <Emphasis Type="Italic">Malva crispa</Emphasis> L.

Two ornamental plants of Althaea rosea Cav. and Malva crispa L. were exposed to various concentrations of lead (Pb) (0, 50, 100, 200 and 500 mg·kg^?1) for 70 days to evaluate the accumulating potential and the tolerance characteristics. The results showed that both plant species grown normally under Pb stress, and A. rosea had a higher tolerance than M. crispa, while M. crispa had a higher ability in Pb accumulation than A. rosea. Besides, lower Pb concentration (50 mg·kg^?1) stimulated the shoot biomass in both plant species. Pb accumulation in plants was consistent with the increase of Pb levels, and the main accumulation sites were the roots and the older leaves. In addition, the photosynthetic pigments content and chlorophyll fluorescence parameters were influenced by Pb stress. In such case, both of the plants could improve the activities of antioxidant enzymes of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) and ascorbate peroxidase (APX), and the contents of the total soluble sugar and soluble protein, which reached the highest value at Pb 100 mg·kg^?1, as well as the accumulation of the total thiols (T-SH) and non-protein thiols (NP-SH) to adapt to Pb stress. Thus, it provides the theoretical basis and possibility for ornamental plants of A. rosea and M. crispa in phytoremediation of Pb contaminated areas.     

Abscisic Acid-Induced Starch Accumulation in Bioenergy Crop Duckweed <Emphasis Type="Italic">Spirodela polyrrhiza</Emphasis>

Spirodela polyrrhiza, a fast-growing duckweed with high starch and low lignin content, shows promise as a feedstock for bioenergy. Abscisic acid (ABA) is a biological hormone that controls plant growth and stress response. The effects of different ABA concentrations (0, 1.0 × 10^?5, 1.0 × 10^?4, 1.0 × 10^?3, 1.0 × 10^?2, and 1.0 × 10^?1 mg/L) on duckweed biomass growth, carbon dioxide fixation, formation of photosynthetic pigments (Chlorophyll a (Chla), Chlorophyll b (Chlb), and carotenoids), the activities of soluble starch synthase (SSS) and starch branching enzyme (SBE), and the starch content of biomass were investigated in this study. ABA at concentrations lower than 1.0 × 10^?3 mg/L promoted carbon dioxide fixation, whereas it inhibited carbon dioxide fixation at concentrations over 1.0 × 10^?3 mg/L. ABA enhanced SSS and SBE activities at concentrations lower than 1.0 × 10^?2 mg/L. ABA treatment increased the content of Chla, Chlb, and carotenoids and resulted in the enhancement of starch content. Chla content gradually increased with the increasing concentration of ABA (1.0 × 10^?5 to 1.0 × 10^?2 mg/L). After culturing for 10 days, starch content in 1.0 × 10^?2 mg/L ABA medium reached 35.3% of dry weight (DW), which was the highest level in this study. This suggests that there is a great potential to develop a technology to increase starch accumulation in duckweed which can be used as an alternative to corn, sugarcane, or other food crops as a starch source.     

Physiological and Biochemical Mechanisms Preventing Cd Toxicity in the New Hyperaccumulator <Emphasis Type="Italic">Abelmoschus manihot</Emphasis>

Abelmoschus manihot, an ornamental plant, was examined for phytoremediation purposes in accordance with the ability to accumulate cadmium and physiological mechanisms of cadmium tolerance. A net photosynthetic rate (A _N) glasshouse experiment for 60 days was conducted to investigate the influence of different cadmium amounts (0–100 mg kg^?1) on the growth, biomass, photosynthetic performance, reactive oxygen species (ROS) production, antioxidative enzyme activities, Cd uptake and accumulation of A. manihot. Exposure to cadmium enhanced plant growth even at 100 mg kg^?1, without showing symptoms of visible damage. The cadmium concentration of shoots (stems or leaves) and roots was more than the critical value of 100 mg kg^?1 and reached 126.17, 185.26 and 210.24 mg kg^?1, respectively. BCF values of A. manihot plants exceeded the reference value 1.0 for all the Cd treatments, and TF values were greater than 1 at 15–60 mg kg^?1 Cd treatment. The results also showed that cadmium concentrations of 60 mg kg^?1 or less induced a significant enhancement in plant net photosynthetic rate (A _N), stomatal conductance (G _s), transpiration rate (T _r), photosynthetic pigments and F _v/F _m. These parameters were slightly decreased at the higher concentration (100 mg kg^?1). The ROS production (O₂ ^?, H₂O₂) and antioxidative response including SOD, CAT and POD were significantly enhanced by increasing cadmium. These results suggest that A. manihot can be considered as a Cd-hyperaccumulator and the hormetic effects may be taken into consideration in remediation of Cd contamination soil.     

Evaluation of growth and phycobiliprotein composition of cyanobacteria isolates cultivated in different nitrogen sources

Phycobiliproteins, light-harvesting pigments found in cyanobacteria and in some eukaryotic algae, have numerous commercial applications in food, cosmetic, and pharmaceutical industries. Colorant production from cyanobacteria offers advantages over their production from higher plants, as cyanobacteria have fast growth rate and high photosynthetic efficiency and require less space. In this study, three cyanobacteria strains were studied for phycobiliprotein production and the influence of sodium nitrate, potassium nitrate and ammonium chloride on the growth and phycobiliprotein composition of the strains were evaluated. In the batch culture period of 12 days, Phormidium sp. and Pseudoscillatoria sp. were able to utilize all tested nitrogen sources; however, ammonium chloride was the best nitrogen source for both strains to achieve maximum growth rate μ?=?0.284?±?0.03 and μ?=?0.274?±?0.13 day^?1, chlorophyll a 16.2?±? 0.5 and 12.2?±? 0.2 mg L^?1, and phycobiliprotein contents 19.38?±?0.09 and 19.99?±?0.14% of dry weight, whereas, for Arthrospira platensis, the highest growth rate of μ?=?0.304?±?0.0 day^?1, chlorophyll a 19.1?±?0.5 mg L^?1, and phycobiliprotein content of 22.27?±?0.21% of dry weight were achieved with sodium nitrate. The phycocyanin from the lyophilized cyanobacterial biomass was extracted using calcium chloride and food grade purity (A₆₂₀/A₂₈₀ ratio >?0.7) was achieved. Furthermore, phycocyanin was purified using two-step chromatographic method and the analytical grade purity (A₆₂₀/A₂₈₀ ratio >?4) was attained. SDS-PAGE demonstrated the purity and presence of two bands corresponding to α- and β-subunits of the C-phycocyanin. The results showed that Phormidium sp. and Pseudoscillatoria sp. could be good candidates for phycocyanin production.     

Improving of lipid productivity of the biodiesel promising green microalga <Emphasis Type="Italic">Chlorella pyrenoidosa</Emphasis> via low-energy ion implantation

High lipid content in microalgae is an essential parameter for adopting of microalgal biomass as a feedstock for biodiesel. Mutation is one approach to obtain desired algal strain with high lipid production. In this study, a mutant strain of Chlorella pyrenoidosa was isolated using 1.5?×?10¹⁵ ions cm^?2 s^?1 of N⁺ ion beam implantation technique, which has been widely used in mutagenesis of agricultural crops. N⁺ implantation slightly improved the growth of the mutant over the corresponding wild strain with significant increase in lipid content (32.4 % higher than the wild strain), which resulted in significant increase in lipid productivity by 35 %. In addition, ion implantation mutagenesis of C. pyrenoidosa resulted in 21.4 % decrease in total saturated fatty acids (SFAs) compared to the wild type, with a noticeable increase in polyunsaturated fatty acids (PUFAs). The increase in PUFAs was due mainly to stimulation of hexadecadienoic acid (C16:2) and octadecadienoic acid (C18:2) production. However, the SFA content of wild and mutant strains was 31.7 and 24.9 % of total fatty acids, respectively, highlighting the oxidative stability of biodiesel produced by both strains according to the European standards. Cultivation of C. pyrenoidosa mutant in selenite enrichment medium for five successive cultivation experiments showed insignificant changes in biomass productivity, lipid content, and lipid productivity alongside the study period, which confirms the genetic stability of the produced mutant. The present study confirmed the feasibility of generation of microalgae mutants with significant high lipid production using ion beam implantation.     

Culture medium influence on growth,fatty acid,and pigment composition of <Emphasis Type="Italic">Choricystis minor</Emphasis> var. <Emphasis Type="Italic">minor</Emphasis>: a suitable microalga for biodiesel production

The purposes of this study were to assess the influence of culture medium on biomass production, fatty acid, and pigment composition of Choricystis minor var. minor and to evaluate the use of this microalga as a source of fatty raw material for biodiesel production. Cultures of C. minor var. minor were grown using WC (Wright’s cryptophyte) and BBM (Bold’s Basal medium) media. BBM medium produced more biomass (984.3 mg L^?1) compared to the WC medium (525.7 mg L^?1). Despite this result, WC medium produced a higher methyl ester yield for biodiesel production than the BBM medium (170.0 and 90.2 mg g^?1 of biomass, respectively). The average percentage of fatty acids obtained using the WC medium (17.0 %) was similar to soybean (18.0 %) and with similar biomass fatty acid profile. However, for pigment production, carotenoids and chlorophyll concentrations were twice as high when using the BBM medium.     

Screening of microalgae for primary metabolites including β-glucans and the influence of nitrate starvation and irradiance on β-glucan production

Βeta-glucans, widespread glucose polymers in mushrooms, yeasts, and bacteria, but rarely found in microalgae, have wide applications and high medicinal and economical potential. Some β-glucans like paramylon from algae-like Euglena gracilis are well investigated, but there is little information about the β-glucan content of microalgae. Therefore, more than 40 species of cultured microalgae have been investigated for composition of their biomass regarding to lipids, carbohydrates including β-glucans and proteins. Most of algae species showed a rather similar biomass composition of about 10 % lipids, 25 % carbohydrates, and 40 % proteins if they have been cultivated in a full medium, rather low light conditions of 50 μmol photons m^?2 s^?1, 12/12 h light/dark cycle under aeration and a temperature of 25?±?2 °C. The content of β-glucans varied between 1.7 and 24.2 % of dry weight, respectively. Two microalgae, Scenedesmus ovalternus SAG 52.80 and Porphyridium purpureum SAG 1380-1d with a yield of more than 20 % of dry weight were identified as the best β-glucan producers under standard cultivation conditions. Culture optimization experiments revealed that enhanced irradiance increased the β-glucan content of Scenedesmus obtusiusculus A 189, a novel green algae isolate, from 6.4 to 19.5 %, but the β-glucan content of the green alga S. ovalternus SAG 52.80 remained unaffected (24.2 vs. 23.3 %). Nitrate starvation enhanced the β-glucan content of S. obtusiusculus A 189 from 16 to 23 % and of S. ovalternus SAG 52.80 from 23.3 to 36.7 %.     

The effect of temperature on growth and lipid and fatty acid composition on marine microalgae used for biodiesel production

Cultivation temperature is one of the major factors affecting the growth and lipid accumulation of microalgae. In this study, the effects of temperature on the growth, lipid content, fatty acid composition and biodiesel properties of the marine microalgae Chaetoceros sp. FIKU035, Tetraselmis suecica FIKU032 and Nannochloropsis sp. FIKU036 were investigated. These species were cultured at different temperatures (25, 30, 35 and 40 °C). The results showed that the specific growth rate, biomass and lipid content of all microalgae decreased with increasing temperature. With regards to fatty acids, the presence of saturated fatty acids (SFAs) in T. suecica FIKU032 and Nannochloropsis sp. FIKU036 decreased with increasing temperature, in contrast with polyunsaturated fatty acids (PUFAs). Moreover, Chaetoceros sp. FIKU035 was the only species that could grow at 40 °C. The highest lipid productivity was observed in Chaetoceros sp. FIKU035 when cultivated at 25 °C (66.73 ± 1.34 mg L^?1 day^?1) and 30 °C (61.35 ± 2.89 mg L^?1 day^?1). Moreover, the biodiesel properties (cetane number, cold filter plugging point, kinematic viscosity and density) of the lipids obtained from this species were in accordance with biodiesel standards. This study indicated that Chaetoceros sp. FIKU035 can be considered as a suitable species for biodiesel production in outdoor cultivation.     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Growth and palmitoleic acid accumulation of filamentous oleaginous microalgae <Emphasis Type="Italic">Tribonema minus</Emphasis> at varying temperatures and light regimes

Palmitoleic acid (C16:1Δ9), contributes greatly to human health, industrial chemicals and biodiesel. The filamentous oleaginous microalgae Tribonema sp. has been identified as a highly efficient producer of palmitoleic acid. Temperature and light regime were adapted to regulate the palmitoleic acid content in this study. Strain T. minus was able to grow well at all the tested temperatures, even at 5 °C. The optimum temperature for palmitoleic acid accumulation (54.25 % of total fatty acid) was 25 °C. Moreover, both light intensity and photoperiod affect the growth, lipid content and fatty acid files of T. minus. The culture exposed to 240 μmol photons m^?2 s^?1 with a photoperiod of 24:0 showed the highest biomass (6.87 g L^?1) and biggest lipid content (61.27 % of dry weight), whereas the most amount of palmitoleic acid (50.47 % of total fatty acid) was detected at 120 μmol photons m^?2 s^?1. These findings make tangible contributions to culture T. minus for commercial production of lipid or palmitoleic acid.