Microalgae as a raw material for biofuels production

Biofuels demand is unquestionable in order to reduce gaseous emissions (fossil CO₂, nitrogen and sulfur oxides) and their purported greenhouse, climatic changes and global warming effects, to face the frequent oil supply crises, as a way to help non-fossil fuel producer countries to reduce energy dependence, contributing to security of supply, promoting environmental sustainability and meeting the EU target of at least of 10% biofuels in the transport sector by 2020. Biodiesel is usually produced from oleaginous crops, such as rapeseed, soybean, sunflower and palm. However, the use of microalgae can be a suitable alternative feedstock for next generation biofuels because certain species contain high amounts of oil, which could be extracted, processed and refined into transportation fuels, using currently available technology; they have fast growth rate, permit the use of non-arable land and non-potable water, use far less water and do not displace food crops cultures; their production is not seasonal and they can be harvested daily. The screening of microalgae (Chlorella vulgaris, Spirulina maxima, Nannochloropsis sp., Neochloris oleabundans, Scenedesmus obliquus and Dunaliella tertiolecta) was done in order to choose the best one(s), in terms of quantity and quality as oil source for biofuel production. Neochloris oleabundans (fresh water microalga) and Nannochloropsis sp. (marine microalga) proved to be suitable as raw materials for biofuel production, due to their high oil content (29.0 and 28.7%, respectively). Both microalgae, when grown under nitrogen shortage, show a great increase (~50%) in oil quantity. If the purpose is to produce biodiesel only from one species, Scenedesmus obliquus presents the most adequate fatty acid profile, namely in terms of linolenic and other polyunsaturated fatty acids. However, the microalgae Neochloris oleabundans, Nannochloropsis sp. and Dunaliella tertiolecta can also be used if associated with other microalgal oils and/or vegetable oils.     

The marine microalgaChlorella stigmatophora as a potential source of single cell protein: Enhancement of the protein content in response to nutrient enrichment

Summary Mass cultures ofChlorella stigmatophora were carried out in order to obtain maximum protein production and to study the chemical variations in function of the nutrient concentration. Cultures reached maximum cellular densities of 2.2·10⁸ cells/ml, with a growth velocity between 0.49 and 0.55 doublings/day. Carbohydrate content in the stationary phase ranged between 2.23 and 2.74 pg/cell, RNA between 0.78 and 1.36 pg/cell and DNA between 0.013 and 0.016 pg/cell. The maximum value for chlorophylla was 0.13 pg/cell. Maximum protein content was obtained with a nutrient concentration of 16 mM of NaNO₃, giving 4.85 pg/cell and a protein concentration of 0.7 g/l. The protein content can be manipulated by changes in the nutrient concentration, showing differences up to a 9.2-fold increase. This characteristic makesChlorella stigmatophora a suitable source of single cell protein.     

Measuring the trophic ecology of ants using stable isotopes

Ants are prominent components of most terrestrial arthropod food webs, yet due to their highly variable diet, the role ants play in arthropod communities can be difficult to resolve. Stable isotope analysis is a promising method for determining the dietary history of an organism, and has the potential to advance our understanding of the food web ecology of social insects. However, some unique characteristics of eusocial organisms can complicate the application of this technique to the study of their trophic ecology. Using stable isotopes of N and C, we investigated levels of intraspecific variation both within and among colonies. We also examined the effect of a common preservation technique on δ¹⁵N and δ¹³C values. We discuss the implications of our results on experimental design and sampling methods for studies using stable isotopes to investigate the trophic ecology of social insects. Received 4 February 2005; revised 23 June 2005; accepted 4 July 2005.     

Cell-wall abnormalities of a Chlamydomonas reinhardtii mutant strain under suboptimal growth conditions

Sporangia were accumulated in autotrophically and mixotrophically growing cultures of the Chlamydomonas reinhardtii mutant strain ls entering the stationary phase. Such an accumulation of sporangia was never observed in stationary-phase cultures of wildtype strains. Sporangia harvested from stationary-phase cultures of the mutant strain ls released their zoospores after being resuspended in fresh culture medium. Liberation of zoospores was also observed during fixation of these sporangia with glutaraldehyde and OsO₄. Release of zoospores during fixation was prevented by pretreatment with 3 mol·l^–1 LiCl. Ultrastructural analyses of these LiCl-pretreated sporangia revealed that they contained abnormal sporangial walls: sporangia containing sporangia and sporangia surrounded by additional multilayered cell walls have been observed. Similar abnormal cell-wall structures were found in sporangia accumulated at the end of the dark period, when the mutant strain ls was grown photoautotrophically under a 12 h light-12 h dark regime with suboptimal aeration. When grown under optimal conditions, this particular mutant did not show any abnormal wall structures.This work has been supported by a grant from the Deutsche Forschungsgemeinschaft. The authors thank Mrs. C. Adami for the photographic work.     

Microalgae aquaculture feeds

Microalgae feeds are currently used in relatively small amounts in aquaculture, mainly for the production of larvae and juvenile shell- and finfish, as well as for raising the zooplankton required for feeding of juvenile animals. The blue-green algaSpirulina is used in substantial amounts (over 100 t y^–1) as a fish and shrimp feed, and even larger markets can be projected if production costs could be reduced. Another potential large-scale application of microalgae is the cultivation ofHaematococcus for the production of the carotenoid astaxanthin, which gives salmon flesh its reddish color. In the long-term microalgae biomass high in lipids (omega-3 fatty acids) may be developed as substitutes for fish oil-based aquaculture feeds. In shrimp ponds the indigenous algal blooms supply a part of the dietary requirements of the animals, but it is difficult to maximize algal productivities. A separate algal production system could feed the shrimps and minimize the need for added feed. Bivalves feed essentially exclusively on marine microalgae throughout their life cycle. The development of cultivation technologies for such microalgae would allow the onshore production of these animals, with greatly improved product quality and safety.This paper was presented at the Symposium on Applied Phycology at the Fourth International Phycological Congress, Duke University.     

Intrapopulation variation in carbon and nitrogen stable isotope ratios in the earthworm Aporrectodea longa

The natural abundance variations in carbon and nitrogen stable isotope ratios in a population of the earthworm Aporrectodea longa, a species known to feed on both soil and plant litter, is reported in this paper. Worms were collected from a small land area of an old white clover field and body tissue and mucus were analyzed separately. The range of isotopic values was small, but patterns of variation were not random. Tissue carbon and nitrogen isotope ratios were significantly higher in adult than in juvenile A. longa and tissue nitrogen isotope ratios tended to increase with increasing biomass of individuals. Further, carbon and nitrogen isotope ratios were positively correlated in both tissue and mucus. Possible causes of the observed patterns, including physiological effects, body composition and assimilation of C and N from different plant, soil and microbial sources are discussed. It is concluded that the causes of natural variability in isotopic composition must be understood and validated experimentally before natural abundance stable isotope methods can be used for the analysis of trophic relations among detritivorous soil invertebrates.     

The bacterial phleomycin resistance geneble as a dominant selectable marker inChlamydomonas

A chimeric gene composed of the coding sequence of theble gene fromStreptoalloteichus hindustanus fused to the 5 and 3 untranslated regions of theChlamydomonas reinhardtii nuclear geneRBCS2 has been constructed. Introduction of this chimeric gene into the nuclear genome ofC. reinhardtii by co-transformation with theARG7 marker yields Arg⁺ transformants of which approximately 80% possess theble gene. Of these co-transformants, approximately 3% display a phleomycin-resistant (Pm^R) phenotype. Western blot analysis using antibodies against theble gene product confirms the presence of the protein in the Pm^R transformants and genetic analysis demonstrates the co-segregation of theble gene with the phenotype in progeny arising from the mating of a Pm^R transformant to wild-type strains. Direct selection of Pm^R transformants was achieved by allowing an 18-h period for recovery and growth of transformed cells prior to selection. This work represents the first demonstration of stable expression and inheritance of a foreign gene in the nuclear genome ofC. reinhardtii and provides a useful dominant marker for nuclear transformation.     

Comparing the response of Antarctic,tropical and temperate microalgae to ultraviolet radiation (UVR) stress

The response of Antarctic, tropical and temperate microalgae of similar taxonomic grouping to ultraviolet radiation (UVR) stress was compared based on their growth and fatty acid profiles. Microalgae of similar taxa from the Antarctic (Chlamydomonas UMACC 229, Chlorella UMACC 237 and Navicula UMACC 231), tropical (Chlamydomonas augustae UMACC 246, Chlorella vulgaris UMACC 001 and Amphiprora UMACC 259) and temperate (Chlamydomonas augustae UMACC 247, Chlorella vulgaris UMACC 248 and Navicula incerta UMACC 249) regions were exposed to different UVR conditions. The cultures were exposed to the following conditions: PAR (42 μmol photons m⁻² s⁻¹), PAR + UVA (854 μW cm⁻²) and PAR + UVA + UVB (117 μW cm⁻²). The cultures were subjected to UVA doses of 46.1, 92.2 and 184.4 J cm⁻² and UVB doses of 6.3, 12.6 and 25.2 J cm⁻² by varying the duration of their exposure (1.5, 3 and 6 h) to UVR during the light period (12:12 h light-dark cycle). UVA did not affect the growth of the microalgae, even at the highest dose. In contrast, growth was adversely affected by UVB, especially at the highest dose. The dose that caused 50% inhibition (ID₅₀) in growth was used to assess the sensitivity of the microalgae to UVB. Sensitivity of the microalgae to UVB was species-dependent and also dependent on their biogeographic origin. Of the nine microalgae, the Antarctic Chlorella was most tolerant to UVB stress (ID₅₀ = 21.0 J cm⁻²). Except for this Chlorella, the percentage of polyunsaturated fatty acids of the microalgae decreased in response to high doses of UVB. Fatty acid profile is a useful biomarker for UVB stress for some microalgae. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Utilization of organic compounds as the sole source of nitrogen by Thiobacillus thiooxidans

Thiobacillus thiooxidans DSM 504 was shown to grow with adenine, hypoxanthine, xanthine and uric acid as sole sources of nitrogen. Growth with these compounds was observed after lag periods of varying lengths, unless the cells had been previously grown with the same purine base. The disappearance of adenine was accompanied by a temporary accumulation of hypoxanthine in the medium. The utilization of purines was inhibited by ammonia (1 mM). Guanine, pyrimidines and some other organic compounds were not utilized.Non-standard abbreviation U-¹⁴C uniformly labeled by ¹⁴C     

Phytoavailability of Cd and Zn in soil estimated by stable isotope exchange and chemical extraction

The distribution of labile Cd and Zn in two contrasting soils was investigated using isotopic exchange techniques and chemical extraction procedures. A sewage sludge amended soil from Great Billings (Northampton, UK) and an unamended soil of the Countesswells Association obtained locally (Aberdeen, UK) were used. ¹¹⁴Cd and ⁶⁷Zn isotopes were added to a water suspension of each soil and the labile metal pool (E-value) determined from the isotope dilution. Samples were obtained at 13 time points from 1h to 50 days. For the sewage sludge amended soil, 29 g Cd g^–1 (86% of total) and 806 g Zn g^–1 (65% of total) were labile and for the Countesswells soil the value was 8.6 g Zn g^–1 (13% of total); limits of detection prevented a Cd E-value from being measured in this soil. The size of the labile metal pool was also measured by growing plants for 90 days and determining the isotopic content of the plant tissue (L-value). Thlaspi caerulescensJ. & C. Presl (alpine penny cress), a hyperaccumulator of Zn and Cd, Taraxacum officinale Weber (dandelion) and Hordeum vulgare L. (spring barley) were used. L-values were similar across species and lower than the E-values. On average the L-values were 23±0.8 g Cd g^–1 and 725±14 g Zn g^–1 for the Great Billings soil and 0.29±0.16 g Cd g^–1 and 7.3±0.3 g Zn g^–1 for the Countesswells soil. The extractable metal content of the soils was also quantified by extraction using 0.1 M NaNO₃, 0.01 M CaCl₂, 0.5 M NaOH, 0.43 M CH₃COOH and 0.05 M EDTA at pH 7.0. Between 1.3 and 68% of the total Cd and between 1 and 50% of the total Zn in the Great Billings soil was extracted by these chemicals. For the Countesswells soil, between 6 and 83% of the total Cd and between 0.1 and 7% of the total Zn was extracted. 0.05 M EDTA and 0.43 M CH₃COOH yielded the greatest concentrations for both soils but these were less than the isotopic estimates. On the whole, E-values were numerically closer to the L-values than the chemical extraction values. The use of isotopic exchange provides an alternative estimate of the labile metal pool within soils compared to existing chemical extraction procedures. No evidence was obtained that T. caerulescens is able to access metal within the soil not freely available to the other plants species. This has implications for long term remediation strategies using hyperaccumulating plant species, which are unlikely to have any impact on non-labile Cd and Zn in contaminated soil.     

Incorporation of a stable isotopically labeled amino acid into multiple human apolipoproteins

Procedures are presented for the separation and determination of the isotopic enrichment of multiple human apolipoproteins labeled in vivo with a stable isotope amino acid. The isotopic enrichments of plasma lysine and plasma apolipoproteins were monitored for 16 days after a single intravenous dose of [4,4,5,5-2H4]lysine (5 mg/kg body weight). The use of a multiply deuterated amino acid enabled the measurement of isotopic enrichments above background over the entire 16-day time course in all proteins. Individual apolipoproteins were separated on a specially designed gradient sodium dodecyl sulfate polyacrylamide gel electrophoresis system cast in a conventional slab gel apparatus which resolved apoB-100, apoE, apoA-I, apoA-II, apoC-I, apoC-II, apoC-III-1, and apoC-III-2 on a single gel. After staining with Coomassie blue, proteins bands (containing 5 to 30 micrograms of individual apolipoprotein) were excised from the gel. Amino acids were recovered from hydrolyzed gel slices, derivatized, and analyzed by gas chromatography-mass spectrometry for determination of lysine isotopic enrichments. The utility of the method is demonstrated using examples of apolipoproteins B-100, A-I, A-II, C-I, C-II, and C-III from either total plasma d less than 1.21 g/ml lipoproteins or selected lipoprotein subfractions. Lysine isotopic enrichments of proteins were generally determined with a precision of better than 5%. The isotopic enrichment profiles were consistent with literature reports of apolipoprotein metabolic kinetics based on the use of radioiodinated apolipoproteins. The procedures outlined can be used to separate and measure the isotopic enrichment of virtually any apolipoprotein from any chosen lipoprotein fraction. Thus, these procedures should find wide application in the study of apolipoprotein metabolic kinetics.     

Green microalgae can use naphthalenesulfonic acids as sources of sulfur

1-Naphthalenesulfonate (1-NS) was utilized by axenic cultures of Scenedesmus obliquus and by 5 other green microalgae as the sole source of sulfur. For all algae under study, 2-naphthalenesulfonate was definitely inferior to 1-NS as a source of sulfur. The rate of disapperance of 1-NS from the medium was measured by HPLC and, indirectly, by relating growth to sulfur supply. The physiological availability of 1-NS sulfur for Scenedesmus obliquus amounted to about 14% of sulfate sulfur. 1-Naphthol appeared as the major metabolite of 1-NS. Hence, it was concluded that 1-NS underwent a desulfonation which also took place in the presence of moderate concentrations of sulfate.Abbreviations HPLC high performance liquid chromatography - 1-NS 1-naphthalenesulfonic acid - 2-NS 2-naphthalenesulfonic acid - OD optical density - OD₀ optical density at time 0 of the light-and-dark cycleDedicated to Prof. Dr. W. Habsguth on the occasion of his 75th birthday     

Species-specific ability ofChlorella strains (Chlorophyceae) to form stable symbioses withHydra viridis

46 strains ofChlorella, identified by physiological and biochemical characters, were examined for their ability to form stable symbioses with aposymbioticHydra viridis. It was found to be a species-specific characteristic. Among the 15 taxa studied, onlyC. saccharophila var.ellipsoidea, C. saccharophila var.saccharophila, C. fusca var.vacuolata, C. kessleri, C. luteoviridis, andC. protothecoides formed stable symbioses withHydra viridis. Among the 11 known physiological and biochemical characters of theseChlorella species, only acid tolerance seems to be correlated with symbiosis: All symbiotic species are capable of growing at or below pH 4.0.     

Identification of 4-O-methyl-D-xylose as a constituent of the cell wall of Chlorella vulgaris

From the cell wall of a strain of Chlorella vulgaris a sugar was isolated after acid hydrolysis and was identified as 4-O-methyl-D-xylose by the following criteria: (i) mass spectroscopy of its alditol acetate revealed characteristic primary fragments with m/e 117 and m/e 261, and, when one deuterium atom was substituted at C-1, with m/e 262 instead of m/e 261; (ii) after demethylation with BCl₃, xylose was identified as its parent sugar by chromatographic methods; (iii) L-iditol: NAD 5-oxidoreductase (sorbitol dehydrogenase) catalyzed the oxidation of its alditol, but not of 4-O-methyl-L-xylitol. 4-O-Methyl-D-xylose amounted to approx. 10% of the cell walls' dry weight or 1.6% of the cells' dry weight.     

Plant cell factories as a source for anti-cancer lignans

The review places podophyllotoxin, a powerful anti-cancer material used in clinical treatment of small cell cancers, in focus. The economical synthesis of podophyllotoxin is not feasible and demand for this material outstrips supply. At present, Podophyllum hexandrum (Indian May apple) is the commercial source but it grows in an inhospitable region (the Himalayas) where it is collected from wild stands. Furthermore, the plant is now an endangered species. Alternative sources of podophyllotoxin are considered, e.g., the supply of podophyllotoxin and related lignans by establishing plant cell cultures that can be grown in fermentation vessels. Increase of product yields, by variation of medium and culture conditions or by varying the channelling of precursors into side-branches of the biosynthetic pathway by molecular approaches, are discussed.     

Conidia of Trichoderma virens as a phosphorus source for mycorrhizal Pinus sylvestris seedlings

In this study, the mobilization and further translocation of phosphorus from conidia of saprotrophic fungus Trichoderma virens into Pinus sylvestris seedlings by nondestructive measuring of ³²P was assessed. The radioactive phosphorus flux from the conidia to the Scots pine seedlings forming mycorrhiza with Laccaria laccata and Suillus bovinus amounted up to 27.82% and 7.42%, respectively, on the 28th day of the experiment, while at the same time in nonmycorrhizal pine seedlings, the detected radioactivity reached only 0.56%. Our studies revealed that both ectomycorrhizal fungi: L. laccata and S. bovinus, mobilized the phosphorus from radioactive conidia of T. virens. On this basis, we conclude that activities of the mycosymbionts may facilitate absorption and further translocation of phosphorus from organic matter into the host plants.     

The cell wall of Chlamydomonas reinhardtii gametes: Composition,structure and autolysin-mediated shedding and dissolution

The cell walls of Chlamydomonas gametes are multilayered structures supported on frameworks of polypeptides extending from the plasma membrane. The wall-polypeptide catalogue reported by Monk et al. (1983, Planta 158, 517–533) and extended by U.W. Goodenough et al. (1986, J. Cell Biol. 103, 405–417) was re-evaluated by comparative analysis of mechanically isolated cell walls purified from several strains. The extracellular locus of wall polypeptides was verified by in vivo iodogen-catalysed iodination and by autolysin-mediated elimination of the bulk of these polypeptides from the cell surface. Three (w15, w16, w17) and possibly four (w14) polypeptides were located to the most exterior aspect of the wall because of their susceptibility to Enzymobeadcatalysed iodination and their retention by a cell-wall-less mutant. The composition of shed walls stabilised with ethylenediaminetetraacetic acid during natural mating and kinetic analysis of the dissolution of walls purified from a bald-2 mutant demonstrated the rapid and specific destruction of polypeptide w3. Differential solubilisation of wall polypeptides occurred after loss of w3. Wall dissolution, characterised by the generation of fishbone structures from the W2 layer, gave as many as four additional polypeptides. Charged detergents and sodium perchlorate extracted a comparable range of polypeptides at room temperature from mechanically isolated walls, i.e. components of the W4–W6 layers, hot sodium dodecyl sulphate solubilised framework polypeptides, while reducing agent was required to solubilise the W2 layer. A model of wall structure is presented.Abbreviations DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

Biosynthesis of ferredoxin in Chlamydomonas reinhardii

The selective action of the antibiotics chloramphenicol and cycloheximide on the synthesis of ferredoxin in liquid cultures of Chlamydomonas reinhardii was studied. Highly specific antibodies raised against Chlamydomonas ferredoxin were used to determine the in vivo synthesis of apoferredoxin and conversion into native protein. The results indicate that 80S ribosomes are involved in the synthesis. Chlamydomonas cells growing in the absence of iron did not synthesize immunologically detectable amounts of ferredoxin. We suggest that this is based upon feed-back inhibition of apoferredoxin synthesis at the translational level.Abbreviations CAP chloramphenicol - CHI cycloheximide - IgG Immunoglobulin G - PBS 140.4 mM NaCl. 9 mM Na₂HPO₄, 1.3 mM NaH₂PO₄ (pH 74) - SDS sodium dodecvl sulphate - Fd Ferredoxin - apoFd Apoferredoxin - CM-Fd Scarboxymethylated Fd - TCA-Fd Fd treated with trichloroacetic acid