An improved colony PCR procedure for genetic screening of <Emphasis Type="Italic">Chlorella</Emphasis> and related microalgae

A colony PCR technique was applied for both genomic and chloroplast DNA in the green microalgae Chlorella. Of five different lysis buffers, Chelex-100 was superior for DNA extraction, PCR and DNA storage. It also was insensitive to variations in cell density. The conditions established for an improved PCR formulation are applicable for screening of genetically-engineered transformants as well as bioprospecting of natural microalgal isolates. Besides multiple Chlorella species, we also demonstrate the efficacy of Chelex-100 for colony PCR with a number of other microalgal strains, including Chlamydomonas reinhardtii, Dunaliella salina, Nannochloropsis sp., Coccomyxa sp., and Thalassiosira pseudonana.     

A Simple Novel Agar Diffusion Method for Isolation of Indigenous Microalgae Chlamydomonas sp. CRP7 and Chlorella sp. CB4 from Operational Swampy Top Soil

A simple agar diffusion method is developed where pure colony of Chlamydomonas sp. CRP7 was isolated from Chlorella sp. CB4 mixtures by passing through agar migration with a light exposure of 6,000 lux for 7 h. The main concept behind it is that Chlamydomonas has flagella and the rhodopsin pigment is attracted towards light. Thus the above two microalgae species can be separated from the mixtures as eye spot serves as a navigator and flagella serves as a propeller for Chlamydomonas spp. Further the genomic DNA was isolated and purified from the above mentioned two species after the separation from the mixtures. PCR amplification was carried out for ITS1, 5.8S and ITS2 regions. The amplified products were sequenced and the sequence analysis confirmed that they belong to Chlamydomonas sp. and Chlorella sp. This is an important augmentation for isolation and separation of microalgae.     

Multiple microalgal partners in symbiosis with the acantharian Acanthochiasma sp. (Radiolaria)

Acantharia (Radiolaria) are widespread and abundant heterotrophic marine protists, some of which can host endosymbiotic eukaryotic microalgae. Although this photosymbiotic association was first described at the end of the 19th century, the diversity of the symbiotic microalgae remains poorly characterized. Here, we examined the identity of the microalgae associated with the acantharian species Acanthochiasma sp. by sequencing partial 18S and internal transcribed spacer (ITS) ribosomal DNA genes from cultured symbionts and directly from isolated holobiont specimens. Single Acanthochiasma cells contained multiple symbiotic partners, including distantly related dinoflagellates (Heterocapsa sp., Pelagodinium sp., Azadinium sp. and Scrippsiella sp.) as well as a haptophyte (Chrysochromulina sp.). This original association of multiple symbiotic microalgae within a single host cell raises questions about the specificity and functioning of the relationship. These microalgae exhibit the common ecological feature of being abundant and widely distributed in coastal and oceanic waters, some occasionally forming extensive blooms. Some of the microalgal genera found in association with Acanthochiasma (i.e. Pelagodinium and Chrysochromulina) are known to occur in symbiosis with other heterotrophic protists such as Foraminifera and other Radiolaria, whereas Heterocapsa, Scrippsiella and Azadinium have never previously been reported to be involved in putative symbiotic relationships. The unusual association unveiled in this study contributes to our understanding of the ecological and evolutionary significance of photosymbiosis in Acantharia and also provides new insights into the nature of such partnerships in the planktonic realm.     

Phylogeny,morphology, and physiology of Micractinium strains isolated from shallow ephemeral freshwater in Antarctica

Cryotolerant eukaryotic microalgae were isolated from meltwater streams on Ardley Island and King George Island in Antarctica, and their morphological, molecular, and physiological characteristics were investigated. Owing to their simple morphology, distinctive characters were not observed with neither light microscopy nor transmission electron microscopy. However, molecular phylogenetic inferences drawn from the concatenated small subunit rRNA and internal transcribed spacer sequence data indicated that these microalgal strains belonged to the genus Micractinium. All the Micractinium strains showed cryotolerant properties, while their optimum growth temperature was around 20°C. Similar to other cryotolerant organisms, these Antarctic microalgae also contained a higher ratio of polyunsaturated fatty acids to saturated fatty acids. In this study, new Antarctic Micractinium spp. were discovered and added to the culture collection. These cryotolerant strains may serve as a promising source of nutritionally important linoleic (C_18:2 ω6) and α‐linolenic (C_18:3 ω3) acids.     

Comparative evaluation of inorganic and organic amendments for their flocculation efficiency of selected microalgae

Cost-efficient harvesting of microalgae is a major challenge due to their small size and often low concentration in the culture medium. The flocculation efficacy of different inorganic and organic amendments was evaluated on various microalgae genera—one strain each belonging to Chlamydomonas, Chlorococcum, two of Botryococcus, and of Chlorella. An improvised medium comprising of commercial grade urea, single super phosphate, and muriate of potash was used to grow the microalgae for flocculation experiments. High pH induced increased flocculation efficiency (72–76 %) in selected microalgal strains. Ferric chloride was found to be the most efficient for most of the microalgal strains, while maize starch and rice starch proved superior for Chlorella sp. MCC6 and Botryococcus sp. MCC32. Although the highest flocculation efficiency was obtained with inorganic flocculant, i.e., ferric chloride (87.3 %) with Botryococcus MCC31, this was comparable with rice starch (86.8 %) for Botryococcus MCC32. This study showed that widely available cheaper biopolymers such as rice starch, maize, and potato starch can be promising flocculants due to their better harvesting efficiency (>80 %) and low price, thereby contributing to economical production of biodiesel from algae.     

Nutritional Evaluation of Australian Microalgae as Potential Human Health Supplements

This study investigated the biochemical suitability of Australian native microalgal species Scenedesmus sp., Nannochloropsis sp., Dunaliella sp., and a chlorophytic polyculture as nutritional supplements for human health. The four microalgal cultures were harvested during exponential growth, lyophilized, and analysed for proximate composition (moisture, ash, lipid, carbohydrates, and protein), pigments, and amino acid and fatty acid profiles. The resulting nutritional value, based on biochemical composition, was compared to commercial Spirulina and Chlorella products. The Australian native microalgae exhibited similar, and in several cases superior, organic nutritional properties relative to the assessed commercial products, with biochemical profiles rich in high-quality protein, nutritious polyunsaturated fats (such as α-linolenic acid, arachidonic acid, and eicosapentaenoic acid), and antioxidant pigments. These findings indicate that the microalgae assessed have great potential as multi-nutrient human health supplements.     

Sequence Variation in the Ribosomal DNA Internal Transcribed Spacer of Tridacna crocea

DNA-based genetic markers are needed to augment existing allozyme markers in the assessment of genetic diversity of wild giant clam populations. The dearth of polymorphic mitochondrial DNA regions amplified from known universal polymerase chain reaction (PCR) primers has led us to search other regions of the genome for viable sources of DNA polymorphism. We have designed tridacnid-specific PCR primers for the amplification of internal transcribed spacer regions. Sequences of the first internal transcribed spacer segment (ITS-1) revealed very high polymorphism, showing 29% variation arising from base substitutions alone. Preliminary restriction analysis of the ITS regions using 8 restriction enzymes revealed cryptic changes in the DNA sequence. These mutations are promising as marker tools for differentiating geographically separated populations. Such variation in the ITS region can possibly be used for population genetic analysis. Received February 1, 2000; accepted May 8, 2000.     

Molecular genetic analysis of new Anabaena strains isolated from a plant-cyanobacterial community

Ecosystems of rice paddies are good sources of new strains of heterocyst-forming cyanobacteria that can be used in biotechnological systems for production of photohydrogen. The morphological and physiological properties of two novel epiphytic strains of cyanobacteria, Anabaena sp. 182 and Anabaena sp. 281, were studied. DNA typing of these strains based on PCR amplification of hydrogenase-encoding genes and DNA analysis using RAPD and Rep primers was carried out. The properties of the genome of strain Anabaena sp. 281 differed considerably from those of two reference strains (Anabaena variabilis ATCC 29413 and Nostoc sp. PCC 7120) with sequenced genomes, whereas strain Anabaena sp. 182 was found to be a close relative of A. variabilis ATCC 29413. Due to a number of physiological and biochemical advantages, Anabaena sp. 182 may be considered a new promising model for molecular and genetic engineering studies aimed at the development of H₂ producers.     

Screening of the culture media with different concentrations of nutrients for cultivation of the microalgae associated with the invertebrates of the White Sea

The growth and biomass accumulation of three microalgal strains of Desmodesmus (Scenedesmaceae, Chlorophyceae), 1Рm66В, 2Cl66E, and 3Dp86Е-1, isolated from the White Sea benthic invertebrates were studied under conditions of batch culture in different standard media (BG-11, Prat, Goldberg, Gromov, Tamiya, artificial seawater) and modified media. The culture condition, biomass accumulation, and uptake of nitrate and phosphate were recorded. A significant alkalization of the culture medium up to pH 10 has been observed during a vigorous growth of the microalgae. The most significant biomass accumulation has been recorded in BG-11 (in complete or modified medium with addition of artificial seawater), Tamiya, and Prat media. Addition of seawater did not affect the growth of Desmodesmus sp. in the nitrate-containing media, although that maintained growth of the microalgae in the nitrogen-lacking media without cell aggregation. The BG-11 medium appears suitable for isolation and cultivation of both symbiotic and free-living microalgae by all the tested features. The Prat medium is the best for maintaining the microalgal strains in living collection.     

Molecular Characterization of Brevibacillus laterosporus and Its Potential Use in Biological Control

Thirty-three strains of Brevibacillus laterosporus, including three novel strains isolated from Brazilian soil samples, were examined for genetic variability by the use of different PCR-based methods. Molecular markers that could characterize bacterial strains with regards to their pathogenic potential were investigated. In addition, toxicity was assessed by the use of insects belonging to the orders Lepidoptera and Coleoptera and the mollusk Biomphalaria glabrata. Among the targets tested, Biomphalaria glabrata demonstrated the highest degree of sensitivity to B. laterosporus, with some strains inducing 90 to 100% mortality in snails aged 3 and 12 days posteclosion. Larvae of the coleopteron Anthonomus grandis were also susceptible, presenting mortality levels of between 33 and 63%. Toxicity was also noted towards the lepidopteron Anticarsia gemmatalis. In contrast, no mortality was recorded among test populations of Tenebrio molitor or Spodoptera frugiperda. The application of intergenic transcribed spacer PCR and BOX-PCR generated 15 and 17 different genotypes, respectively. None of the molecular techniques allowed the identification of a convenient marker that was associated with any entomopathogenic phenotype. However, a 1,078-bp amplicon was detected for all strains of B. laterosporus when a primer for amplification of the BOXA1R region was used. Similarly, a 900-bp amplicon was generated from all isolates by use of the primer OPA-11 for randomly amplified polymorphic DNA analysis. These amplicons were not detected for other phenotypically related Brevibacillus species, indicating that they represent markers that are specific for B. laterosporus, which may prove useful for the isolation and identification of new strains of this species.     

Rapid Characterization of Microalgae and Microalgae Mixtures Using Matrix-Assisted Laser Desorption Ionization Time-Of-Flight Mass Spectrometry (MALDI-TOF MS)

Current molecular methods to characterize microalgae are time-intensive and expensive. Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) may represent a rapid and economical alternative approach. The objectives of this study were to determine whether MALDI-TOF MS can be used to: 1) differentiate microalgae at the species and strain levels and 2) characterize simple microalgal mixtures. A common protein extraction sample preparation method was used to facilitate rapid mass spectrometry-based analysis of 31 microalgae. Each yielded spectra containing between 6 and 56 peaks in the m/z 2,000 to 20,000 range. The taxonomic resolution of this approach appeared higher than that of 18S rDNA sequence analysis. For example, two strains of Scenedesmus acutus differed only by two 18S rDNA nucleotides, but yielded distinct MALDI-TOF mass spectra. Mixtures of two and three microalgae yielded relatively complex spectra that contained peaks associated with members of each mixture. Interestingly, though, mixture-specific peaks were observed at m/z 11,048 and 11,230. Our results suggest that MALDI-TOF MS affords rapid characterization of individual microalgae and simple microalgal mixtures.     

Local bioprospecting for high-lipid producing microalgal strains to be grown on concentrated municipal wastewater for biofuel production

Mass cultivation of microalgae for biofuel production depends heavily on the performance of the microalgae strains used. In this study, 60 algae-like microorganisms collected from different sampling sites in Minnesota were examined using multi-step screening and acclimation procedures to select high-lipid producing facultative heterotrophic microalgae strains capable of growing on concentrated municipal wastewater (CMW) for simultaneous energy crop production and wastewater treatment. Twenty-seven facultative heterotrophic microalgae strains were found, among which 17 strains were proved to be tolerant to CMW. These 17 top-performing strains were identified through morphological observation and DNA sequencing as Chlorella sp., Heynigia sp., Hindakia sp., Micractinium sp., and Scenedesmus sp. Five strains were chosen for other studies because of their ability to adapt to CMW, high growth rates (0.455-0.498 d⁻¹) and higher lipid productivities (74.5-77.8 mg L⁻¹ d⁻¹). These strains are considered highly promising compared with other strains reported in the literature.     

Characteristics of Fusobacterium ulcerans,a New and Unusual Species Compared with Fusobacterium varium and Fusobacterium mortiferum

Fusobacterium ulcerans is a newly described obligately anaerobic Gram-negative, non-spore-forming rod [1] that has been isolated from tropical ulcers. Two morphotypes were described: one resemblingFusobacterium varium and the other Fusobacterium mortiferum[1]. Because of the weak or negative fermentation reactions of most fusobacteria, the standard carbohydrate tests used for identification of anaerobe organisms are of little use for identification, and other rapid and simple methods are needed. We characterized eight F. ulcerans strains using conventional biochemical testing. We further analysed these strains by PCR employing a single non-specific primer AP3 and by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell proteins. PCR using a self-designed pair of primers for the amplification of the spacer (intergenic) region between the 16S and 23S rRNA genes, led to the development of genetic markers for species identification. All F. ulcerans clinical isolates appeared very similar to each other in all the test parameters, but were distinctly different from the type strains of the two phenotypically similar species, F. mortiferum and F. varium. High similarity in PCR- and protein-profiles also raise the possibility that all these F. ulcerans strains came from one clone. We noted significant differences among the strains of F. mortiferum and F. varium.     

Integration of biological waste conversion and wastewater treatment plants by microalgae cultivation

In this study, growth performance and lipid content of two microalgae species Neochloris oleoabundans and Chlorella vulgaris are monitored by using three different types of sludge waste feedstocks obtained from the water treatment plants located in Bedonia, Borgotaro and Fornovo (Montagna2000 Spa, Province of Parma, Italy). The sludge waste is optimized in order to achieve microalgal growth media and dispose of the sewage sludge produced at the wastewater treatment facilities. Both photoautotrophic and heterotrophic growth conditions are applied to the microalgal cultivations. The growth parameters of microalgae strains such as cell concentration, growth rate, optical density, cell biovolume, photosynthetic pigments and lipid contents are monitored. The amounts of total dried lipid biomass, obtained by the biological conversion of the wet sludge waste, are determined. Lipid production of microalgal cells grown in the medium optimized from sludge waste from the Fornovo site provides the highest amount of microalgal lipid content for N. oleoabundans and C. vulgaris photoautotrophic cultivations, while sludge waste from the Bedonia site provides for N. oleoabundans heterotrophic cultivation.     

Single-tube colony PCR for DNA amplification and transformant screening of oleaginous microalgae

Recently, several colony PCR methods have been developed to simplify DNA isolation procedure and facilitate PCR-based colony screening efforts in microalgae. A main drawback of current protocols is that cell collection, disruption, and genomic DNA extraction are required preceding the PCR step, making the colony PCR process laborious and costly. In the present study, we have developed a novel procedure that eliminates any steps of DNA extraction and allows the colony screening to be performed in a single PCR tube: algal cells (as low as 5,000) from agar plates or liquid cultures were directly transferred into a PCR tube containing 2× PCR buffer and boiled for 5–10 min depending on different algal strains, followed by addition of other PCR components (dNTPs, primers, and polymerase) and then subjected to conventional PCR reaction. The procedure documented here worked well not only for the model alga Chlamydomonas reinhardtii, but also for the thick-walled oleaginous strains such as Chlorella, Haematococcus, Nannochloropsis, and Scenedesmus with its efficacy independent on amplicon sizes and primer pairs. In addition, screening of Chlorella zofingiensis transformants was achieved using this method. Collectively, our single-tube colony PCR is a much simpler and more cost-effective procedure as compared to those previously reported and has broad applications including gene cloning, strain determination, and high-throughput screening of algae colonies and transformants for biomass and biofuel production.     

Molecular characterization of Hysterothylacium fabri (Nematoda: Anisakidae) from Zeus faber (Pisces: Zeidae) caught off the Mediterranean coasts of Turkey based on nuclear ribosomal and mitochondrial DNA sequences

In the present study, Hysterothylacium fabri was found in the coasts of the Mediterranean Sea, Turkey and characterized by sequencing of nuclear (internal transcribed spacer, ITS) and mitochondrial (cytochrome c oxidase subunit 2, cox2) markers. Pairwise comparison between the entire ITS fragment including ITS-1, 5.8S, ITS-2 sequences of the H. fabri isolates from the Mediterranean Sea (Turkey, KC852206) and other H. fabri isolates from the South China Sea (JQ520158), the South Korea waters (JX974558) showed differences ranged from 0.1 and 1.1%. With the present study, H. fabri from the Mediterranean Sea was characterized for the first time by sequencing of the cox2 gene.     

Template-blocking PCR: An advanced PCR technique for genome walking

This article describes the development of an improved method for the isolation of genomic fragments adjacent to a known DNA sequence based on a cassette ligation-mediated polymerase chain reaction (PCR) technique. To reduce the nonspecific amplification of PCR-based genome walking, the 3′ ends of the restriction enzyme-digested genomic DNA fragments were blocked with dideoxynucleoside triphosphate (ddNTP) and ligated with properly designed cassettes. The modified genomic DNA fragments flanked with cassettes were used as a template for the amplification of a target gene with a gene-specific primer (GSP) and a cassette primer (CP). The ddNTP blocking of the genomic DNA ends significantly reduced the nonspecific amplification and resulted in a simple and rapid walking along the genome. The efficiency of the template-blocking PCR method was confirmed by a carefully designed control experiment. The method was successfully applied for the cloning of the PGK1 promoter from Pichia ciferrii and two novel cellulase genes from Penicillium sp.     

Fatty acids as biomarkers of microalgae

Microalgae are primary producers of the food chain and hold prominence towards pharmaceutical and nutraceutical applications. Fatty acids (FAs) are one of the primary metabolites of microalgae, which enrich their utility both in the form of food and fuels. Additionally, the vast structural diversity coupled with taxonomic specificity makes these FAs as potential biomarkers. The determination of lipid and fatty acid profiling of 12 different strains of microalgae has been accomplished in this study and further discussed in respect to their chemotaxonomic perspective in microalgae. Palmitic acid (C16:0) and oleic acid (C18:1n9c) were found to be dominant among the members of Cyanophyceae whereas members of Chlorophyceae were rich in palmitic acid (C16:0), oleic acid (C18:1n9c) and linoleic acid (C18:2n6). The application of principal component analysis (PCA) and algorithmic hierarchical clustering (AHC) resulted in the segregation of the studied microalgal strains into 8 different orders belonging to 2 distinct phyla according to their phylogenetic classification. Nutritionally important FAs like eicosapentaenoic acid (EPA, C20:5n3) and docosahexaenoic acid (DHA, C22:6n3) were detected only in Chlorella sp. belonging to Chlorophyceaen family. Differential segregation of microalgae with respect to their fatty acid profile indicated the potential utility of FAs as biomarkers.     

Amplicon restriction patterns associated with nitrogenase activity of root nodules for selection of superior Myrica seedlings

Trees of Myrica sp. grow abundantly in the forests of Meghalaya, India. These trees are actinorhizal and harbour nitrogen-fixing Frankia in their root nodules and contribute positively towards the enhancement of nitrogen status of forest areas. They can be used in rejuvenation of mine spoils and nitrogen-depleted fallow lands generated due to slash and burn agriculture practiced in the area. We have studied the association of amplicon restriction patterns (ARPs) of Myrica ribosomal RNA gene and internal transcribed spacer (ITS) region and nitrogenase activity of its root nodules. We found that ARPs thus obtained could be used as markers for early screening of seedlings that could support strains of Frankia that fix atmospheric nitrogen more efficiently.