Efficient isolation and cultivation of endosymbiotic Chlorella from Paramecium bursaria on agar plates by co-culture with yeast cells.

Paramecium bursaria is a ciliate that harbors Chlorella-like unicellular green algae as endosymbionts. The relationship between the host P. bursaria and the endosymbiotic Chlorella is facultative; therefore, both partners can be cultured independently and re-combined to re-establish symbiosis, making this system suitable for studying algal endosymbiosis. However, despite many previous studies, cultivation of endosymbiotic Chlorella remains difficult, particularly on agar plates. Here we describe a simple agar plate method for efficiently isolating and culturing cells of the endosymbiotic alga Chlorella variabilis from an individual P. bursaria cell, by co-culturing them with yeast Saccharomyces cerevisiae. The co-culture with the yeast significantly improved the colony-forming efficiency of the alga on agar. Growth assays suggest that the main role of the co-cultured yeast cells is not to provide nutrients for the algal cells, but to protect the algal cells from some environmental stresses on the agar surface. Using the algal cells grown on the plates and a set of specially designed primers, direct colony PCR can be performed for screening of multiple endosymbiont clones isolated from a single host ciliate. These methods may provide a useful tool for studying endosymbiotic Chlorella species within P. bursaria and various other protists.

Molecular and Structural Characterizations of Lipases from Chlorella by Functional Genomics.

Microalgae have been poorly investigated for new-lipolytic enzymes of biotechnological interest. In silico study combining analysis of sequences homologies and bioinformatic tools allowed the identification and preliminary characterization of 14 putative lipases expressed by Chlorella vulagaris. These proteins have different molecular weights, subcellular localizations, low instability index range and at least 40% of sequence identity with other microalgal lipases. Sequence comparison indicated that the catalytic triad corresponded to residues Ser, Asp and His, with the nucleophilic residue Ser positioned within the consensus GXSXG pentapeptide. 3D models were generated using different approaches and templates and demonstrated that these putative enzymes share a similar core with common α/ß hydrolases fold belonging to family 3 lipases and class GX. Six lipases were predicted to have a transmembrane domain and a lysosomal acid lipase was identified. A similar mammalian enzyme plays an important role in breaking down cholesteryl esters and triglycerides and its deficiency causes serious digestive problems in human. More structural insight would provide important information on the enzyme characteristics.

High-Throughput In Vitro Screening of Changed Algal Community Structure Using the PhotoBiobox.

In a previous study, the sequential optimization and regulation of environmental parameters using the PhotoBiobox were demonstrated with high-throughput screening tests. In this study, we estimated changes in the biovolume-based composition of a polyculture built in vitro and composed of three algal strains: Chlorella sp., Scenedesmus sp., and Parachlorella sp. We performed this work using the PhotoBiobox under different temperatures (10-36°C) and light intensities (50-700 µmol/m-2/s-1) in air and in 5% CO2. In 5% CO2, Chlorella sp. exhibited better adaptation to high temperatures than in air conditions. Pearson's correlation analysis showed that the composition of Parachlorella sp. was highly related to temperature whereas Chlorella sp. and Scenedesmus sp. showed negative correlations in both air and 5% CO2. Furthermore, light intensity slightly affected the composition of Scenedesmus sp., whereas no significant effect was observed in other species. Based on these results, it is speculated that temperature is an important factor in influencing changes in algal polyculture community structure (PCS). These results further confirm that the PhotoBiobox is a convenient and available tool for performance of lab-scale experiments on PCS changes. The application of the PhotoBiobox in PCS studies will provide new insight into polyculture-based ecology.

Characterization of a novel herbicide and antibiotic-resistant Chlorella sp. with an extensive extracellular matrix.

A herbicide and antibiotic-resistant microalgal strain, isolated from a eutrophic site at Giofyros river (Heraklion, Crete, Greece) was extensively characterized. In the presence of relatively high concentrations of common photosynthesis inhibitors (DCMU and atrazine), as well as various antibiotics (spectinomycin, kanamycin, and chloramphenicol), the green microalga was able to increase its biomass in approximately equal levels compared to the control. Despite the high concentrations of the inhibitors, photosynthetic efficiency and chlorophyll a amount per dry cell biomass were comparable to those of control cultures in almost all cases. 18S rDNA analysis showed that this microalga belongs to the Chlorella genus. Optical and electron microscopy studies revealed the presence of an extensive extracellular matrix (EM) that surrounds the cells and plays an important role in colony formation and cell-cell interactions. Fourier transform infrared spectroscopy provided evidence that the EM consists of a polysaccharide. This matrix could be separated from the cells with a simple centrifugation. Depending on growth conditions, the dry cell biomass of this Chlorella strain was found to contain 35-39% proteins and 27-42% carbohydrates. The results of this study have demonstrated that the EM plays a protective role for cell homeostasis maintenance against the various chemical agents. This green microalga is a suitable candidate for further studies regarding sustainable biomass production in waste waters for a series of applications.

Lipid production from indigenous Greek microalgae: a possible biodiesel source.

OBJECTIVE: Microalgae gained interest for potential use as biodiesel producers, since they synthesize and accumulate significant quantities of lipids. The aim of this work was to isolate indigenous microalgae strains from Greek habitats, study their physicochemical growth conditions and finally select the best ones with respect to overall lipid production and profile. RESULTS: Two sampling sites of marine aquatic ecosystems were selected in Attica prefecture, Greece in order to screen for novel wild type strains with lipid production capacity. Microalgae isolates (59) were obtained from the selected areas and were morphologically and molecularly characterized. Fatty acids were estimated through Flow Cytometry combined with BODIPY staining method. Four isolates were selected for their lipid production properties and were cultivated in 15 L tank cultures. The four isolates were also identified by 18S rDNA gene sequencing. Two of them, Chlorella sp. ΑCΑ9 and ACA17, exhibited both maximum biomass and lipid productivity. Optimization of growth conditions with respect to pH and initial NaNO3 concentration was performed for the two microalgae in 15 L cultures. Finally, 20 L fed batch cultures were set up using the optimum culture conditions. Lipid profiles were stabilized for both strains at dry biomass levels over 1 g L-1 and lipid content of 25% (w/w). CONCLUSIONS: Two Chlorella strains (ACA9 and ACA17) were promising candidates for biodiesel production as they were easily grown in sea water in fed batch systems and produce lipids suitable for biodiesel-especially Chlorella sp. ACA9.

Evaluation of Chlorella sorokiniana isolated from local municipal wastewater for dual application in nutrient removal and biodiesel production.

The isolated microalga Chlorella sorokiniana BENHA721_ABO4 was grown in Bold's basal medium (BBM) as a control, municipal wastewater (WW), and wastewater enriched with BBM elements (WW+). Cultivation in WW+ showed the highest cell number which represented 25.3 and 47.3% over that grown in WW and BBM, respectively. However, rapid growth in WW+ was accompanied by significant reduction in lipid content. Due to lipid accumulation in WW, it showed the maximum significant lipid productivity of 16.2 mg L-1 day-1. Microalgae cultivation in WW for 10 days showed 74.2, 83.3, and 78.0% removal efficiency for NO3-N, NH3-N and TP, respectively. In addition, growth in WW significantly reduced polyunsaturated fatty acids by 36.0% with respect to BBM in favor of monounsaturated fatty acids. The present results confirmed that C. sorokiniana isolate BENHA721_ABO4 grown in secondary effluent municipal wastewater offers real potential for future application in wastewater treatment and biodiesel production.

Subcritical Water Extraction of Chlorella pyrenoidosa: Optimization through Response Surface Methodology.

Subcritical water extraction (SCW) was used to extract oil from Chlorella pyrenoidosa. The operational factors such as reaction temperature, reaction time, and biomass loading influence the oil yield during the extraction process. In this study, response surface methodology was employed to identify the desired extraction conditions for maximum oil yield. Experiments were carried out in batch reactors as per central composite design with three independent factors including reaction temperature (170, 220, 270, 320, and 370°C), reaction time (1, 5, 10, 15, and 20 min), and biomass loading (1, 3, 5, 10, and 15%). A maximum oil yield of 12.89 wt.% was obtained at 320°C and 15 min, with 3% biomass loading. Sequential model tests showed the good fit of experimental data to the second-order quadratic model. This study opens the great potential of SCW to extract algal oil for use in algal biofuel production.

Molecular Investigation of the Ciliate Spirostomum semivirescens, with First Transcriptome and New Geographical Records.

The ciliate Spirostomum semivirescens is a large freshwater protist densely packed with endosymbiotic algae and capable of building a protective coating from surrounding particles. The species has been rarely recorded and it lacks any molecular investigations. We obtained such data from S. semivirescens isolated in the UK and Sweden. Using single-cell RNA sequencing of isolates from both countries, the transcriptome of S. semivirescens was generated. A phylogenetic analysis identified S. semivirescens as a close relative to S. minus. Additionally, rRNA sequence analysis of the green algal endosymbiont revealed that it is closely related to Chlorella vulgaris. Along with the molecular species identification, an analysis of the ciliates' stop codons was carried out, which revealed a relationship where TGA stop codon frequency decreased with increasing gene expression levels. The observed codon bias suggests that S. semivirescens could be in an early stage of reassigning the TGA stop codon. Analysis of the transcriptome indicates that S. semivirescens potentially uses rhodoquinol-dependent fumarate reduction to respire in the oxygen-depleted habitats where it lives. The data also shows that despite large geographical distances (over 1,600km) between the sampling sites investigated, a morphologically-identical species can share an exact molecular signature, suggesting that some ciliate species, even those over 1mm in size, could have a global biogeographical distribution.

Modeling of microalgal shear-induced flocculation and sedimentation using a coupled CFD-population balance approach.

In this study, shear-induced flocculation modeling of Chlorella sp. microalgae was conducted by combination of population balance modeling and CFD. The inhomogeneous Multiple Size Group (MUSIG) and the Euler-Euler two fluid models were coupled via Ansys-CFX-15 software package to achieve both fluid and particle dynamics during the flocculation. For the first time, a detailed model was proposed to calculate the collision frequency and breakage rate during the microalgae flocculation by means of the response surface methodology as a tool for optimization. The particle size distribution resulted from the model was in good agreement with that of the jar test experiment. Furthermore, the subsequent sedimentation step was also examined by removing the shear rate in both simulations and experiments. Consequently, variation in the shear rate and its effects on the flocculation behavior, sedimentation rate and recovery efficiency were evaluated. Results indicate that flocculation of Chlorella sp. microalgae under shear rates of 37, 182, and 387 s-1 is a promising method of pre-concentration which guarantees the cost efficiency of the subsequent harvesting process by recovering more than 90% of the biomass. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:160-174, 2018.

Comparison of different artificial neural network architectures in modeling of Chlorella sp. flocculation.

Biodiesel production from microalgae feedstock should be performed after growth and harvesting of the cells, and the most feasible method for harvesting and dewatering of microalgae is flocculation. Flocculation modeling can be used for evaluation and prediction of its performance under different affective parameters. However, the modeling of flocculation in microalgae is not simple and has not performed yet, under all experimental conditions, mostly due to different behaviors of microalgae cells during the process under different flocculation conditions. In the current study, the modeling of microalgae flocculation is studied with different neural network architectures. Microalgae species, Chlorella sp., was flocculated with ferric chloride under different conditions and then the experimental data modeled using artificial neural network. Neural network architectures of multilayer perceptron (MLP) and radial basis function architectures, failed to predict the targets successfully, though, modeling was effective with ensemble architecture of MLP networks. Comparison between the performances of the ensemble and each individual network explains the ability of the ensemble architecture in microalgae flocculation modeling.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium "Candidatus Holospora parva".

Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of "green" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name "Candidatus Holospora parva" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.

Uptake of inorganic and organic nutrient species during cultivation of a Chlorella isolate in anaerobically digested dairy waste.

A natural assemblage of microalgae from a facultative lagoon system treating municipal wastewater was enriched for growth in the effluents of an anaerobic digester processing dairy waste. A green microalga with close resemblance to Chlorella sp. was found to be dominant after multiple cycles of sub-culturing. Subsequently, the strain (designated as LLAI) was isolated and cultivated in 20× diluted digester effluents under various incident light intensities (255-1,100 µmoles m-2 s-1 ) to systematically assess growth and nutrient utilization. Our results showed that LLAI production increased with increasing incident light and a maximum productivity of 0.34 g L-1 d-1 was attained when the incident irradiance was 1,100 µmoles m-2 s-1 . Lack of growth in the absence of light indicated that the cultures did not grow heterotrophically on the organic compounds present in the medium. However, the cultures were able to uptake organic N and P under phototrophic conditions and our calculations suggest that the carbon associated with these organic nutrients contributed significantly to the production of biomass. Overall, under high light conditions, LLAI cultures utilized half of the soluble organic nitrogen and >90% of the ammonium, orthophosphate, and dissolved organic phosphorus present in the diluted waste. Strain LLAI was also found to accumulate triacylglycerides (TAG) even before the onset of nutrient limitation and a lipid productivity of 37 mg-TAG L-1 d-1 was measured in cultures incubated at an incident irradiance of 1,100 µmoles m-2 s-1 . The results of this study suggest that microalgae isolates from natural environments are well-suited for nutrient remediation and biomass production from wastewater containing diverse inorganic and organic nutrient species. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1336-1342, 2016.

Microbial composition and ecological features of phototrophic biofilms proliferating in the Moidons Caves (France): investigation at the single-cell level.

The authors investigated the microbial composition of phototrophic biofilms proliferating in a show cave using flow cytometry for the first time in such a context. Results are based on several biofilms sampled in the Moidons Caves (France) and concern both heterotrophic prokaryotes and autotrophic microorganisms. Heterotrophic microorganisms with low nucleic acid content were dominant in biofilms, as can be expected from the oligotrophic conditions prevailing within the cave. Analysis of the biofilm autotrophic components revealed the presence of several taxa, particularly the unicellular green algae Chlorella minutissima, specifically well adapted to this cave. Relationships between flow cytometry results and environmental variables determined in the cave were established and discussed so as to better understand biofilm proliferation processes in caves.

A two-stage microresistive pulse immunosensor for pathogen detection.

We present a two-stage immunosensor for pathogen detection in a mixed population. In this approach, antibody-conjugated microparticles were used to functionalize the surface of the capture chamber via a convenient magnetic method and a two-stage resistive pulse sensor was used to detect and quantify pathogen cells. We firstly tested the capture efficiency of the functionalized capture chamber. The specific capture efficiency of S. cerevisiae is greater than 94.8%, while the non-specific capture efficiency is 3.4%. We showed that the device can accurately measure pure S. cerevisiae at concentrations ranging from 1.0 to 8.0 × 10(3) cells per µL. We performed S. cerevisiae measurements in a mixture with Chlorella. Both cells have similar sizes. For S. cerevisiae to Chlorella ratios ranging from 1.0 to 2.0, the measurement error was less than 7%, while the error became 20% to 32% for lower ratios ranging from 0.1 to 0.5 caused by nonspecific attachment. We demonstrated that this device is able to isolate target cells and quantitatively measure the cell population in a short time. This device can be potentially used for pathogen detection in the food industry, biological research and clinical applications.

Microalgae removal with Moringa oleifera.

Moringa oleifera seed extract was tested for algae (Chlorella, Microcystis, Oocystis and Scenedesmus) removal by Jar-test technique. This coagulant can be used in drinking water treatment. Jar-test has been carried out in order to evaluate the efficiency of this natural coagulant agent inside real surface water matrix. The influence of variables has been studied in this process, including operating parameters such as coagulant dosage, initial algae concentration, pH, agitation time and water matrix. Removal capacity is verified for water with high contamination of algae while the process is not affected by the pH and water matrix. Coagulation process may be modelling through Langmuir and Freundlich adsorption hypothesis, so acceptable r2 coefficients are obtained.

Growth and lipid accumulation in three Chlorella strains from different regions in response to diurnal temperature fluctuations.

It was economically feasible to screen strains adaptive to wide temperature fluctuation for outdoor cultivation without temperature control. In this research, three Chlorella strains from arctic glacier, desert soil and temperate native lake were isolated and identified. The growth, biochemical composition, lipid content and fatty acid composition of each strain cultured under the mode of diurnal temperature fluctuations were compared. All the three Chlorella strains showed desirable abilities of accumulating lipid under diurnal temperature fluctuations and their fatty acid profiles were suitable for biodiesel production, although the growth and biochemical composition were seemed to be region-specific. The highest lipid content was at 51.83±2.49% DW, 42.80±2.97% DW and 36.13±2.27% DW under different temperature fluctuation of 11 °C, 25 °C, 7 °C, respectively. The results indicated that the three Chlorella strains could be promising biodiesel feedstock for outdoor cultivation by the cultural mode of diurnal temperature fluctuations.

[Isolation, Identification and Characteristic Analysis of an Oil-producing Chlorella sp. Tolerant to High-strength Anaerobic Digestion Effluent].

A Chlorella strain tolerant to high-strength anaerobic digestion effluent was isolated from the anaerobic digestion effluent with a long-term exposure to air. The strain was identified as a Chlorella by morphological and molecular biological methods, and named Chlorella sp. BWY-1, The anaerobic digestion effluent used in this study was from a biogas plant with the raw materials of swine wastewater after solid-liquid separation. The Chlorella regularis (FACHB-729) was used as the control strain. The comparative study showed that Chlorella sp, BWY-Ihad relatively higher growth rate, biomass accumulation capacity and pollutants removal rate in BG11. and different concentrations of anaerobic digestion effluent. Chlorella sp. BWY-1 had the highest growth rate and biomass productivity (324.40 mg.L-1) in BG11, but its lipid productivity and lipid content increased with the increase of anaerobic digestion effluent concentration, In undiluted anaerobic digestion effluent, the lipid productivity and lipid content of Chlorella sp. BWY-1 were up to 44. 43% and 108. 70 mg.L-1, respectively. Those results showed that the isolated algal strain bad some potential applications in livestock wastewater treatment and bioenergy production, it could be combined with a solid-liquid separation, anaerobic fermentation and other techniques for processing livestock wastewater and producing biodiesel.

Magnetic-Nanoflocculant-Assisted Water-Nonpolar Solvent Interface Sieve for Microalgae Harvesting.

Exploitation of magnetic flocculants is regarded as a very promising energy-saving approach to microalgae harvesting. However, its practical applicability remains limited, mainly because of the problem of the postharvest separation of magnetic flocculants from microalgal flocs, which is crucial both for magnetic-flocculant recycling and high-purity microalgal biomasses, but which is also a very challenging and energy-consuming step. In the present study, we designed magnetic nanoflocculants dually functionalizable by two different organosilane compounds, (3-aminopropyl)triethoxysilane (APTES) and octyltriethoxysilane (OTES), which flocculate negatively charged microalgae and are readily detachable at the water-nonpolar organic solvent (NOS) interface only by application of an external magnetic field. APTES functionalization imparts a positive zeta potential charge (29.6 mV) to magnetic nanoflocculants, thereby enabling microalgae flocculation with 98.5% harvesting efficiency (with a dosage of 1.6 g of dMNF/g of cells). OTES functionalization imparts lipophilicity to magnetic nanoflocculants to make them compatible with NOS, thus effecting efficient separation of magnetic flocculants passing through the water-NOS interface sieve from hydrophilic microalgae. Our new energy-saving approach to microalgae harvesting concentrates microalgal cultures (∼1.5 g/L) up to 60 g/L, which can be directly connected to the following process of NOS-assisted wet lipid extraction or biodiesel production, and therefore provides, by simplifying multiple downstream processes, a great potential cost reduction in microalgae-based biorefinement.

Effect of different organic matters on flocculation of Chlorella sorokiniana and optimization of flocculation conditions in swine manure wastewater.

In this study, flocculation of Chlorella sorokiniana cultivated in swine manure wastewater, BG-11 medium and BG-11 medium supplemented with different organic matters (glucose, urea and tryptone) was investigated. The results demonstrated that the minimum amount of Al(3+) required for complete flocculation in wastewater would increase substantially, and flocculation efficiency became highly sensitive to pH. Tryptone could cause similar extent of inhibition on flocculation as in wastewater. Meanwhile, glucose could increase concentrations of Algogenic Organic Matter (AOM), inhibiting flocculation strongly at higher pH, including flocculation induced by Al(3+) and autoflocculation. However, urea had little effect on flocculation of C. sorokiniana. Moreover, the major factors: dilution times, pH and flocculants dosage, which had significant impact on flocculation efficiency of C. sorokiniana in piggery wastewater, were optimized using response surface methodology (RSM). The optimal flocculation efficiency (100%) was achieved at pH 8.5, 7-folds of dilution and 52.14 mg L(-1) of Al(3+).

Kinetic Model of Photoautotrophic Growth of Chlorella sp. Microalga, Isolated from the Setúbal Lagoon.

In this work, a kinetic expression relating light availability in the culture medium with the rate of microalgal growth is obtained. This expression, which is valid for low illumination conditions, was derived from the reactions that take part in the light-dependent stage of photosynthesis. The kinetic expression obtained is a function of the biomass concentration in the culture, as well as of the local volumetric rate of absorption of photons, and only includes two adjustable parameters. To determine the value of these parameters and to test the validity of the hypotheses made, autotrophic cultures of the Chlorella sp. strain were carried out in a modified BBM medium at three CO2 concentrations in the gas stream, namely 0.034%, 0.34% and 3.4%. Moreover, the local volumetric rate of photon absorption was predicted based on a physical model of the interaction of the radiant energy with the suspended biomass, together with a Monte Carlo simulation algorithm. The proposed intrinsic expression of the biomass growth rate, together with the Monte Carlo radiation field simulator, are key to scale up photobioreactors when operating under low irradiation conditions, independently of the configuration of the reactor and of its light source.