Direct colony PCR for rapid identification of varied microalgae from freshwater environment

Molecular identification of microalgal species is vital and involves sequencing of specific markers present in the genome, which are unique to a genus. PCR is a vital tool for identification of microalgae; the preparation of template DNA for PCR by traditional methods requires large amount of algal cells and a time-consuming process. One simple way to reduce these complications is to use the microalgal colonies directly for amplification of required DNA fragments from the genome. In this study, a simple cell-disrupting method, using autoclaved glass powder, has been used for direct colony PCR of microalgae. Four morphologically different microalgal strains were chosen from freshwater samples, and the PCR amplification reaction was evaluated with three different molecular markers (rbcL, internal transcribed spacer 2, and 18S rDNA). PCR amplification was optimized with less number of cells (0.04?×?10⁵), minimal quantity of glass powder (0.5 mg), and in the presence of Milli-Q water for internal transcribed spacer marker. The isolated strains were identified as Desmodesmus sp. JQ782747, Coelastrum proboscideum JQ898144, Chlorella sorokiniana JQ898145, and Scenedesmus sp. JQ782746 based on sequence similarity. This direct microalgal colony PCR proves to be a simple and rapid method for detection of varied microalgal species.     

Novel approach for the development of axenic microalgal cultures from environmental samples

We demonstrated a comprehensive approach for development of axenic cultures of microalgae from environmental samples. A combination of ultrasonication, fluorescence‐activated cell sorting (FACS), and micropicking was used to isolate axenic cultures of Chlorella vulgaris Beyerinck (Beijerinck) and Chlorella sorokiniana Shihira & R.W. Krauss from swine wastewater, and Scenedesmus sp. YC001 from an open pond. Ultrasonication dispersed microorganisms attached to microalgae and reduced the bacterial population by 70%, and when followed by cell sorting yielded 99.5% pure microalgal strains. The strains were rendered axenic by the novel method of micropicking and were tested for purity in both solid and liquid media under different trophic states. Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene confirmed the absence of unculturable bacteria, whereas fluorescence microscopy and scanning electron microscopy (SEM) further confirmed the axenicity. This is the most comprehensive approach developed to date for obtaining axenic microalgal strains without the use of antibiotics and repetitive subculturing.     

Photoadaptation of two members of the Chlorophyta (Scenedesmus and Chlorella) in laboratory and outdoor cultures: changes in chlorophyll fluorescence quenching and the xanthophyll cycle

The role of the xanthophyll cycle in the adaptation of two chlorococcal algae Scenedesmus quadricauda and Chlorella sorokiniana to high irradiance was studied under laboratory and outdoor conditions. We wished to elucidate whether the xanthophyll cycle plays a key role in dissipating the excesses of absorbed light, as in higher plants, and to characterise the relationship between chlorophyll fluorescence parameters and the content of xanthophyll-cycle pigments. The xanthophyll cycle was found to be operative in both species; however, its contribution to overall non-photochemical quenching (NPQ) could only be distinguished in Scenedesmus (15–20% of total NPQ). The Scenedesmus cultures showed a larger pool of xanthophyll-cycle pigments than Chlorella, and lower sensitivity to photoinhibition as judged from the reduction of maximum quantum yield of photosystem II. In general, both algae had a larger xanthophyll-cycle pool when grown outdoors than in laboratory cultures. Comparing the two species, Scenedesmus exhibited a higher capacity to adapt to high irradiance, due to an effective quenching mechanism and high photosynthetic capacity; in contrast, Chlorella represents a species with a larger antennae system, less-efficient quenching and lower photosynthetic performance. Non-photochemical quenching (NPQ) induced through the xanthophyll cycle can, to a limited extent, represent a regulatory factor in diluted algal cultures grown in outdoor solar photobioreactors, as well as in natural algal phytoplankton populations exposed transiently to high irradiance. However, it does not play an appreciable role in dense, well-mixed microalgal suspensions. Received: 6 August 1998 / Accepted: 12 February 1999     

Flocculation of microalgae using cationic starch

Due to their small size and low concentration in the culture medium, cost-efficient harvesting of microalgae is a major challenge. We evaluated the potential of cationic starch as a flocculant for harvesting microalgae using jar test experiments. Cationic starch was an efficient flocculant for freshwater (Parachlorella, Scenedesmus) but not for marine microalgae (Phaeodactylum, Nannochloropsis). At high cationic starch doses, dispersion restabilization was observed. The required cationic starch dose to induce flocculation increased linearly with the initial algal biomass concentration. Of the two commercial cationic starch flocculants tested, Greenfloc 120 (used in wastewater treatment) was more efficient than Cargill C*Bond HR 35.849 (used in paper manufacturing). For flocculation of Parachlorella using Greenfloc 120, the cationic starch to algal biomass ratio required to flocculate 80% of algal biomass was 0.1. For Scenedesmus, a lower dose was required (ratio 0.03). Flocculation of Parachlorella using Greenfloc 120 was independent of pH in the pH range of 5 to 10. Measurements of the maximum quantum yield of PSII suggest that Greenfloc 120 cationic starch was not toxic to Parachlorella. Cationic starch may be used as an efficient, nontoxic, cost-effective, and widely available flocculant for harvesting microalgal biomass.     

Variations in the microalgal structure in paddy soil in Osaka, Japan: comparison between surface and subsurface soils

Seasonal variations in microalgal communities were compared between surface and subsurface paddy soils in Osaka, Japan. Soil samples were collected from depths of 0–1 (surface), 8–9, and 17–18 cm. Diatom cells were counted directly, and the numbers of other microalgae were estimated using a culture method. The microalgal community as well as the soil properties changed drastically in the surface soil as a consequence of alternate flooding and drainage. In the soil collected at a depth of 0–1 cm, the cell density of diatoms and the viable count of other microalgae markedly increased, and Chlorella spp., Nitzschia spp., and Navicula spp. were predominant during the flooding period, whereas Scenedesmus spp. and Hantzschia spp. were predominant during the drainage period. In contrast, in the soils collected at depths of 8–9 and 17–18 cm, the cell density of diatoms and the viable count of other microalgae remained constant. Despite the unavailability of light, a large number of microalgae were present in these subsurface soils throughout the annual cultivation cycle, and Scenedesmus spp. and Nitzschia spp. were always dominant. Cyanophytes were also present at all the depths but had low relative frequencies. These results suggest that the algae that are predominant in paddy soil can survive not only drastic changes in water content but also complete darkness.     

Light respiration in <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>

Respiration and photosynthesis are two important processes in microalgal growth that occur simultaneously in the light. To know the rates of both processes, at least one of them has to be measured. To be able to measure the rate of light respiration of Chlorella sorokiniana, the measurement of oxygen uptake must be fast, preferably in the order of minutes. We measured the immediate post-illumination respiratory O₂ uptake rate (OUR) in situ, using fiber-optic oxygen microsensors, and a small and simple extension of the cultivation system. This method enables rapid and frequent measurements without disturbing the cultivation and growth of the microalgae. Two batch experiments were performed with C. sorokiniana in a short light-path photobioreactor, and the OUR was measured at different time points. The net oxygen production rate (net OPR) was measured online. Adding the OUR and net OPR gives the gross oxygen production rate (gross OPR), which is a measure for the oxygen evolution by photosynthesis. The gross OPR was 35–40% higher than the net OPR for both experiments. The respiration rate is known to be related to the growth rate, and it is suggested that faster algal growth leads to a higher energy (ATP) requirement, and as such, respiratory activity increases. This hypothesis is supported by our results, as the specific OUR is highest in the beginning of the batch culture when the specific growth rate is highest. In addition, the specific OUR decreases toward the end of the experiments until it reaches a stable value of around 0.3 mmol O₂ h⁻¹ g⁻¹. This value for the specific OUR is equal to the maintenance requirement of C. sorokiniana as determined in an independent study of (Zijffers et al. 2010 (in press)). This suggests that respiration could fulfill the maintenance requirements of the microalgal cells.     

Phycoremediation of Tannery Wastewater Using Microalgae Scenedesmus Species

A number of microalgae species are efficient in removing toxicants from wastewater. Many of these potential species are a promising, eco-friendly, and sustainable option for tertiary wastewater treatment with a possible advantage of improving the economics of microalgae cultivation for biofuel production. The present study deals with the phycoremediation of tannery wastewater (TWW) using Scenedesmus sp. isolated from a local habitat. The test species was grown in TWW under laboratory conditions and harvested on the 12th day. The results revealed that the algal biomass during the growth period not only reduced the pollution load of heavy metals (Cr-81.2–96%, Cu-73.2–98%, Pb-75–98% and Zn-65–98%) but also the nutrients (NO₃ >44.3% and PO₄ >95%). Fourier Transform Infrared (FTIR) spectrums of Scenedesmus sp. biomass revealed the involvement of hydroxyl amino, carboxylic and carbonyl groups. The scanning electron micrograph (SEM) and Energy Dispersive X-ray Spectroscopic analysis (EDS) revealed the surface texture, morphology and element distribution of the biosorbent. Furthermore, the wastewater generated during wet-blue tanning process can support dense population of Scenedesmus sp., making it a potential growth medium for biomass production of the test alga for phycoremediation of toxicants in tannery wastewaters.     

Mixotrophic and photoautotrophic cultivation of 14 microalgae isolates from Saskatchewan, Canada: potential applications for wastewater remediation for biofuel production

Northern regions are generally viewed as unsuitable for microalgal biofuel production due to unfavorable climate and solar insolation levels. However, these conditions can potentially be mitigated by coupling microalgal cultivation to industrial processes such as wastewater treatment. In this study, we have examined the biomass and lipid productivity characteristics of 14 microalgae isolates (Chlorophyta) from the Canadian province of Saskatchewan. Under both photoautotrophic and mixotrophic cultivation, a distinct linear trend was observed between biomass and lipid productivities in the 14 SK isolates. The most productive strain under cultivation in TAP media was Scenedesmus sp.-AMDD which displayed rates of biomass and fatty acid productivities of 80 and 30.7?mg?L^?1?day^?1, respectively. The most productive strain in B3NV media was Chlamydomonas debaryana-AMLs1b which displayed rates of biomass and fatty acid productivities of 51.7 and 5.9?mg?L^?1?day^?1, respectively. In 11 of the isolates tested, secondary municipal wastewater (MCWW) supported rates of biomass productivity between 21 and 33?mg?L^?1?day^?1 with Scenedesmus sp.-AMDD being the most productive. Three strains, Chlamydomonas debaryana-AMB1, Chlorella sorokiniana-RBD8 and Micractinium sp.-RB1b, showed large increases in biomass productivity when cultivated mixotrophically in MCWW supplemented with glycerol. High relative oleic acid content was detected in 10 of the 14 isolates when grown mixotrophically in media supplemented with acetate. There was no detectable effect on the fatty acid profiles in cells cultivated mixotrophically in glycerol-supplemented MCWW. These data indicate that biomass and lipid productivities are boosted by mixotrophic cultivation. Exploiting this response in municipal wastewater is a promising strategy for the production of environmentally sustainable biofuels.     

Bioprospecting and indexing the microalgal diversity of different ecological habitats of India

Our study reports the collection, biodiversity analyses, isolation and identification of microalgae from different habitats of India. Cyanophyceae and Chlorophyceae were the most dominant algal groups recorded, with the highest number being recorded for non-heterocystous cyanobacteria (48), followed by 44 unicellular forms. Sagar Island, Sunderbans recorded the greatest number of algae, and unicellular/colonial green algae were present in all the samples. Shannon’s Diversity Index was highest in Koikhali, Sunderbans, followed by Rushikulya River, Odisha. Selective enrichment, purification through serial dilution followed by plating and regular observations led to the isolation of sixteen strains. Identification was done by using microscopic observations, supported with standard monographs and classified as belonging to seven genera (Chlorella, Chlorococcum, Kirchneria, Scenedesmus, Chlamydomonas, Tetracystis and Ulothrix). 18S rDNA sequencing was undertaken for four strains. The set of sixteen strains were screened under standard cultural conditions for their growth kinetics and Chlorella sorokiniana MIC-G5, followed by Chlorella sp. MIC-G4 exhibited the highest growth rates. The strain Chlorococcum sp. MIC-G2 recorded highest chlorophyll, while MIC-G3 ranked highest for carbohydrates. The study aided in identifying the dominant microalgae in the diverse habitats and characterizing their growth rate and carbohydrate content, providing a valuable germplasm for further utilization in agriculture and industry.     

Anonymous nuclear markers for the eastern fence lizard,Sceloporus undulatus

We present results from a screen for de novo variable nuclear loci using a genomic library approach in Sceloporus undulatus, the eastern fence lizard. We tested amplification success for 77 primer pairs in S. undulatus, Sceloporus occidentalis and Sceloporus grammicus. Many loci amplified in all three species suggesting that our primers will be useful for developing sequencing or single nucleotide polymorphism (SNP) genotyping markers in other sceloporine lizards. We also sequenced 19 loci, containing 158 variable sites, for 91 S. undulatus individuals. We report high levels of nucleotide variation in this species with an average of 38 SNPs per kilobase.     

Improving carbohydrate production of Chlorella sorokiniana NIES‐2168 through semi‐continuous process coupled with mixotrophic cultivation

Biofuels from microalgae is now a hot issue of great potential. However, achieving high starch productivity with photoautotrophic microalgae is still challenging. A feasible approach to enhance the growth and target product of microalgae is to conduct mixotrophic cultivation. The appropriate acetate addition combined with CO₂ supply as dual carbon sources (i.e., mixotrophic cultivation) could enhance the cell growth of some microalgae species, but the effect of acetate‐mediated mixotrophic culture mode on carbohydrate accumulation in microalgae remains unclear. Moreover, there is still lack of the information concerning how to increase the productivity of carbohydrates from microalgae under acetate‐amended mixotrophic cultivation and how to optimize the engineering strategies to achieve the goal. This study was undertaken to develop an optimal acetate‐contained mixotrophic cultivation system coupled with effective operation strategies to markedly improve the carbohydrate productivity of Chlorella sorokiniana NIES‐2168. The optimal carbohydrate productivity of 695 mg/L/d was obtained, which is the highest value ever reported. The monosaccharide in the accumulated carbohydrates is mainly glucose (i.e., 85–90%), which is very suitable for bio‐alcohols fermentation. Hence, by applying the optimal process developed in this study, C. sorokiniana NIES‐2168 has a high potential to serve as a feedstock for subsequent biofuels conversion.     

DNA:ATP RATIOS IN MARINE MICROALGAE AND BACTERIA: IMPLICATIONS FOR GROWTH RATE ESTIMATES BASED ON RATES OF DNA SYNTHESIS1

DNA: ATP and carbon: DNA (C:DNA) ratios were measured in a total of 14 species of marine microalgae and bacteria. Comparison of several DNA assay methods with results obtained with cultures uniformly labeled with ³³P indicated that by far the most accurate results were obtained using diaminobenzoic acid (DABA) or diphen-ylamine, with DABA having the highest precision. Both the Hoechst and DAPI methods seriously underestimated DNA concentrations in algal cultures. Average DNA: ATP ratios in the algal and bacterial cultures were I7 and 34 by weight, respectively, with almost all values lying in the range of 10–40. DNA: ATP ratios in the microalgae showed no correlation with growth conditions but varied by about a factor of 3 among species. C:DNA ratios for individual species of microalgae and bacteria ranged from 21 to 155 by weight and averaged 50 for the microalgae and bacteria taken together. Growth rates of microalgal species grown in cyclostats were estimated to within 8% of dilution rates when calculated from the uptake of ³H-adenine and the DNA: ATP ratio of the species. Use of the ³H-adenine method for estimating microalgal growth rates in the field may thus be a useful tool for investigating the physiology of microalgae in nature.     

Association between algal productivity and phycosphere composition in an outdoor Chlorella sorokiniana reactor based on multiple longitudinal analyses

Microalgae as a biofuel source are of great interest. Bacterial phycosphere inhabitants of algal cultures are hypothesized to contribute to productivity. In this study, the bacterial composition of the Chlorella sorokiniana phycosphere was determined over several production cycles in different growing seasons by 16S rRNA gene sequencing and identification. The diversity of the phycosphere increased with time during each individual reactor run, based on Faith’s phylogenetic diversity metric versus days post-inoculation (R = 0.66, P < 0.001). During summer months, Vampirovibrio chlorellavorus, an obligate predatory bacterium, was prevalent. Bacterial sequences assigned to the Rhizobiales, Betaproteobacteriales and Chitinophagales were positively associated with algal biomass productivity. Applications of the general biocide, benzalkonium chloride, to a subset of experiments intended to abate V. chlorellavorus appeared to temporarily suppress phycosphere bacterial growth, however, there was no relationship between those bacterial taxa suppressed by benzalkonium chloride and their association with algal productivity, based on multinomial model correlations. Algal health was approximated using a model-based metric, or the ‘Health Index’ that indicated a robust, positive relationship between C. sorokiniana fitness and presence of members belonging to the Burholderiaceae and Allorhizobium–Neorhizobium–Pararhizobium–Rhizobium clade. Bacterial community composition was linked to the efficiency of microalgal biomass production and algal health.     

Permanent Genetic Resources added to Molecular Ecology Resources Database 1 August 2012 – 30 September 2012

This article documents the addition of 83 microsatellite marker loci and 96 pairs of single‐nucleotide polymorphism (SNP) sequencing primers to the Molecular Ecology Resources Database. Loci were developed for the following species: Bembidion lampros, Inimicus japonicus, Lymnaea stagnalis, Panopea abbreviata, Pentadesma butyracea, Sycoscapter hirticola and Thanatephorus cucumeris (anamorph: Rhizoctonia solani). These loci were cross‐tested on the following species: Pentadesma grandifolia and Pentadesma reyndersii. This article also documents the addition of 96 sequencing primer pairs and 88 allele‐specific primers or probes for Plutella xylostella.     

Phospholipid Metabolism in an Industry Microalga Chlorella sorokiniana: The Impact of Inoculum Sizes

Chlorella sorokiniana is an important industry microalga potential for biofuel production. Inoculum size is one of the important factors in algal large-scale culture, and has great effects on the growth, lipid accumulation and metabolism of microalgae. As the first barrier of cell contents, membrane plays a vital role in algal inoculum-related metabolism. The knowledge of phospholipids, the main membrane component and high accumulation of phospholipids as the major content of total lipids mass in some microalgae, is necessary to understand the role of membrane in cell growth and metabolism under different inoculum density. Profiling of C. sorokiniana phospholipids with LC-MS led to the identification of 119 phospholipid species. To discover the phospholipid molecules most related to change of inoculum sizes, Partial Least Squares Discriminant Analysis (PLS-DA) was employed and the results revealed that inoculum sizes significantly affected phospholipid profiling. Phosphatidylglycerol (PG), phosphatidyl- ethanolamine (PE) and several phosphatidylcholine (PC) species might play an important role under our experimental conditions. Further analysis of these biomarkers indicated that cell membrane status of C. sorokiniana might play an important role in the adaption to the inoculum sizes. And the culture with inoculum size of 1×10⁶ cells mL⁻¹ presented the best membrane status with the highest content of PC and PG, and the lowest content of PE. We discovered that the inoculum size of 1×10⁶ cells mL⁻¹ might provide the best growth condition for C. sorokiniana. Also we proposed that PG, PE and several PC may play an important role in inoculum-related metabolism in C. sorokiniana, which may work through thylakoid membrane and photosynthetic pathway. Thus this study would provide more potential targets for metabolic engineering to improve biofuel production and productivity in microalgae.     

Production stability and biomass quality in microalgal cultivation – Contribution of community dynamics

The prospect of using constructed communities of microalgae in algal cultivation was confirmed in this study. Three different algal communities, constructed of diatoms (Diatom), green algae (Green), and cyanobacteria (Cyano), each mixed with a natural community of microalgae were cultivated in batch and semi‐continuous mode and fed CO₂ or cement flue gas (12–15% CO₂). Diatom had the highest growth rate but Green had the highest yield. Changes in the community composition occurred throughout the experiment. Green algae were the most competitive group, while filamentous cyanobacteria were outcompeted. Euglenoids, recruited from scarce species in the natural community became a large part of the biomass in semi‐steady state in all communities. High temporal and yield stability were demonstrated in all communities during semi‐steady state. Valuable products (lipids, proteins, and carbohydrates) comprised 61.5 ± 5% of ash‐free biomass and were similar for the three communities with lipids ranging 14–26% of dry mass (DM), proteins (15–28% DM) and carbohydrates (9–23% DM). Our results indicate that culture functions (stability, biomass quality) were maintained while dynamic changes occurred in community composition. We propose that a multispecies community approach can aid sustainability in microalgal cultivation, through complementary use of resources and higher culture stability.     

Recent progress in the use of processed microalgae in aquaculture

Mass-cultured algal biomass has been tested as a food source for a number of aquaculture animals because of its low cost and convenience. This paper reviews the results of nutritional studies on processed microalgae with respect to mollusc, crustacean, rotifer and fish culture. Research using species of Spirulina, Chlorella, Scenedesmus and other mass-produced algae indicates that microalgae can be an effective dietary component provided that processing, diet formulation and presentation requirements are met. Processed microalgae can be used to correct specific dietary deficiencies in artificial diets. Our research found that the growth and pigmentation of marron, Cherax tenuimanus (Decapoda, Crustacea), can be significantly enhanced by the incorporation of Dunaliella salina in its artificial diet. Likewise, rainbow trout, Oncorhynchus mykiss, were pigmented by Haematococcus pluvialis.     

“DNA signaturing” database construction for Tetradesmus species identification and phylogenetic relationships of Scenedesmus-like green microalgae (Scenedesmaceae,Chlorophyta)

Species identification of Scenedesmus-like microalgae, comprising Desmodesmus, Tetradesmus, and Scenedesmus, has been challenging due to their high morphological and genetic similarity. After developing a DNA signaturing tool for Desmodesmus identification, we built a DNA signaturing database for Tetradesmus. The DNA signaturing tool contained species-specific nucleotide sequences of Tetradesmus species or strain groups with high similarity in ITS2 sequences. To construct DNA signaturing, we collected data on ITS2 sequences, aligned the sequences, organized the data by ITS2 sequence homology, and determined signature sequences according to hemi-compensatory base changes (hCBC)/CBC data from previous studies. Four Tetradesmus species and 11 strain groups had DNA signatures. The signature sequence of the genus Tetradesmus, TTA GAG GCT TAA GCA AGG ACCC, recognized 86% (157/183) of the collected Tetradesmus strains. Phylogenetic analysis of Scenedesmus-like species revealed that the Tetradesmus species were monophyletic and closely related to each other based on branch lengths. Desmodesmus was suggested to split into two subgenera due to their genetic and morphological distinction. Scenedesmus must be analyzed along with other genera of the Scenedesmaceae family to determine their genetic relationships. Importantly, DNA signaturing was integrated into a database for identifying Scenedesmus-like species through BLAST.     

二氧化碳减排产柴油微藻培养体系研究进展

污水资源化、二氧化碳减排及微藻生物柴油是当前能源与环境领域的前沿课题。以下围绕污水及烟道气资源化培养产油微藻的培养体系,就藻种、营养条件、培养方式、培养环境及微藻生物反应器等影响产油微藻培养的因素研究进展进行了综述。在综述的基础上提出：由于微藻具有特殊营养方式,通过藻种筛选、微藻营养条件和培养环境的优化以及高效光生物反应器和生产工艺等的创新,可利用污水进行产油微藻生产,以获得生物柴油等高附加值产品,实现微藻生物能源、污水资源化处理和CO2减排三者高度耦合的产油微藻生产体系,从而减少微藻培养费用及污水处理费用,因此,该体系具有重要的环境、社会、经济价值和商业化应用前景。     

Screening microalgae for some potentially useful agricultural and pharmaceutical secondary metabolites

Nearly two hundred microalgal strains (174 Chlorophyta and 23 Cyanobacteria) were screened against some bacteria, filamentous fungi and yeasts using a disc-diffusion type bioassay. From this initial screening, 10 Chlorophyta strains from three genera (Desmococcus, Chlorella and Scenedesmus) were selected because of their high antimicrobial activity. These 10 strains were partially purified and tested using MIC antimicrobial and microtiter IC₅₀ anticancer assays. These preselected algal strains showed a high incidence of antibacterial activity against both Gram-positive (9 out of 10 species) and Gram-negative (7 out of 10 species) bacteria. The extracts were also effective against some tumour cell lines.