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Three green algae – Palmellopsis texensis, Chlorococcum pinguideum and Chlorella minutissima – were isolated from the soil remnants among the plant roots of a herbarium specimen, kept 20 years in air-dry conditions without any chemical or freezing preservation treatment. The first two species are new for the algal flora of Bulgaria. Cytomorphology and reproduction of the isolate similar to Palmellopsis texensis were investigated in detail and data obtained were compared with the original species description of Groover & Bold (1969). Two differences from the original diagnosis were detected: presence of pulsating vacuoles in the vegetative cells of our isolate and its reproduction only by zoospores. All features of the architecture of zoospores coincide well with the original description. Further studies could verify if our isolate belongs to Palmellopsis texensis, as it is for the moment designated here, or is a distinct species.

A simple and high-throughput method for determining in situ intracellular neutral lipid accumulation in Chlorella ellipsoidea and Chlorococcum infusionum with flow cytometry and confocal microscopy was established by employing different solvents and a lipophilic dye, Nile red. Seven different organic solvents, acetic acid, dimethyl sulfoxide (DMSO), acetone, methanol, ethanol, n-hexane, and chloroform at different concentrations ranging from 0 to 80% (v/v) were tested. The fluorescence signal for neutral lipids was collected with a 586/42 emission filter (PE-A) and the maximum fluorescence intensity (% grandparent) was measured as 74.01 ± 4.82% for Chlorella and 70.1 ± 5.52% for Chlorococcum at 30% acetic acid (v/v). The statistical analysis of Nile red-stained cells showed a high coefficient of variation (CV), standard deviation (SD), mean, and median values in the acetic acid-based staining method, followed by DMSO, n-hexane and chloroform. Confocal microscopy revealed a high rate of accumulation of cytosolic neutral lipids when stained with Nile red and other organic solvents. Higher lipid accumulation in Fesupplemented conditions was also detected and a maximum lipid content of 57.36 ± 0.41% (4-fold) in Chlorella and 48.20 ± 0.43% (4-fold) in Chlorococcum were measured at 0.001 g/L of ferrous sulfate (FeSO4). High fluorescence intensity (75.16 ± 0.24% in Chlorella and 72.24 ± 1.07% in Chlorococcum) in Fe-treated cells confirmed the efficiency of the staining procedure.

Chlorococcum sp., grape marc, and Pinus radiata have been reacted in a semi-continuous (flow through) system at 425 °C under a flow of N 2 or H 2 and results compared to those from batch autoclave experiments. This paper concentrates on the structure of the condensate, which is more useful and more amenable to analysis. Under similar reaction conditions, the flow through system gave lower yields of dichloromethane soluble materials plus water plus gas for Chlorococcum sp. than a batch autoclave, higher yields for P. radiata , and similar yields of the same fraction for grape marc. Two distinct condensate phases (organic and aqueous) were produced, the composition and amount of which depended on the biomass type and the gas atmosphere (N 2 or H 2 ). Constituents of the original biomass were often better preserved in the flow-through than in batch autoclave reactions under similar conditions.

Cylindrospermopsin (CYN) is a toxic secondary metabolite produced by filamentous cyanobacteria which could work as an allelopathic substance, although its ecological role in cyanobacterial-algal assemblages is mostly unclear. The competition between the CYN-producing cyanobacterium Chrysosporum (Aphanizomenon) ovalisporum, and the benthic green alga Chlorococcum sp. was investigated in mixed cultures, and the effects of CYN-containing cyanobacterial crude extract on Chlorococcum sp. were tested by treatments with crude extracts containing total cell debris, and with cell debris free crude extracts, modelling the collapse of a cyanobacterial water bloom. The growth inhibition of Chlorococcum sp. increased with the increasing ratio of the cyanobacterium in mixed cultures (inhibition ranged from 26% to 87% compared to control). Interestingly, inhibition of the cyanobacterium growth also occurred in mixed cultures, and it was more pronounced than it was expected. The inhibitory effects of cyanobacterial crude extracts on Chlorococcum cultures were concentration-dependent. The presence of C. ovalisporum in mixed cultures did not cause significant differences in nutrient content compared to Chlorococcum control culture, so the growth inhibition of the green alga could be linked to the presence of CYN and/or other bioactive compounds.

A survey of 30 collections sites in Peninsular Malaysia, representing five types of habitats, namely, hot springs, eutrophicated freshwater lake, palm oil mill effluent ponds, brackish and marine water habitats, yielded 79 environmental samples. The total number of algal taxa and genera identified were 73 and 52 respectively; with 9 genera and 11 taxa of Cyanophyta; 25 genera and 33 taxa of Bacillariophyta; 13 genera and 16 taxa of Chlorophyta and 5 genera and 13 taxa of Euglenophyta. In terms of cell numbers, the samples from the oil palm mills had the highest cell density. Of the isolates obtained from the samples collected, only six genera, namely the Cyanophyte Cyanosarcina, the Chlorophytes Chlorella, Chlamydomonas, Chlorococcum,Scenedesmus and the Euglenophyte Euglena, are discussed in terms of their growth and biochemical profiles in this paper. Chlorococcum and Euglena had the highest specific growth rate, µ, followed by Chlorella andScenedesmus. Biomass productivity at day 12 was generally higher than that at day 8 for all genera. Highest biomass productivity was from Chlorella followed by Chlamydomonas. Highest lipid productivity was from Chlorella andChlamydomonas on day 12. Chlorella had the highest carbohydrate productivity followed by Chlamydomonas.Chlorella and Chlamydomonas had the highest protein productivity. In this short survey, some interestingalgae were identified and isolated, which proved to have potential for use as feedstocks for biofuel production. Keywords:microalgae, checklist, specific growth rate, carbohydrate, protein, lipid productivity

BACKGROUND The quality and quantity of lipids in microalgae mainly depend on nitrogen and phosphorous supply. In the present study, growth of newly isolated microalgal strain, Chlorococcum sp. IM-03 has been studied under nitrogen stressed conditions. Fluorescence microscopy and Fourier transform infrared spectroscopy have been used to observe changes in lipid and carbohydrate content. RESULTS Growth of selected species is monitored in terms of dry biomass and chlorophyll content under varying sodium nitrate concentration, viz. 0, 100 and 1500 mg L−1 (designated as N0, N100 and N1500, respectively). N100 was selected in the experimental plan as similar levels were observed in domestic wastewater, which can be used as possible culture media for mass cultivation. Maximum chlorophyll concentration (72.65 µg mg−1) was found at N100. It was also observed that cultivation at N100 resulted in highest lipid content of 17.8%. Carbohydrate content was 18% at N100 with moderate increase to 19.8% at N1500. CONCLUSIONS On the basis of the current study, it can be concluded that sodium nitrate concentration of 100 mg L−1 (N100) offers enhanced production of lipids in Chlorococcum sp. without compromising growth. © 2015 Society of Chemical Industry

Twelve indigenous microalgae, comprising of three fresh water and nine marine strains, were evaluated for their potential as source of dietary polyunsaturated fatty acids (PUFAs) under autotrophic growth conditions. The microalgal lipids showed higher content of omega-3 PUFAs as reflected in lower omega-6 to omega-3 ratios between 0.1 and 0.75. Alpha linolenic acid (ALA), was the predominant omega-3 PUFA in fresh water green algal strains. Scenedesmus dimorphus and Chlorococcum sp. showed higher content of ALA with an average productivity of 3 mgL−1 day−1. Long chain omega-3 PUFAs, eicosapentaenoic (EPA) and docosahexaenoic acid (DHA) were observed in marine strains. Nannochloropsis and Chlorella sp. had higher content of EPA with Chlorella sp. showing an average productivity of 2 mgL−1 day−1. Isochrysis sp. showed higher DHA content with average productivity of 0.37 mgL−1 day−1. Presence of EPA and DHA contributed to higher degree of unsaturation in lipids of marine strains. Among the marine strains, the growth and lipid profile of Nannochloropsis sp. and Chlorella sp. remained unaffected by growth medium, whereas strains like T. theli showed a differential response to media. Spirulina platensis SP6 from CFTRI algal culture repository was also included as a reference strain since its nutritional benefits have been well elucidated. Gamma Linolenic acid (GLA) was the predominant PUFA in Spirulina sp. with an average productivity of 0.73 mgL−1 day−1.

The aim of this work was to investigate the effect of zinc oxide nanoparticles (ZnO NPs) on freshwater and marine microalgae cultivated in different media. Freshwater species Chlorococcum sp. and Scenedesmus rubescens were cultivated in modified Blue-Green medium (BG-11) and Bold's Basal Medium (BBM), and marine species Dunaliella tertiolecta, and Tetraselmis suesica, cultured in salt modified BG-11 and f/2 medium. The microalgae species were exposed for 96 h with a daily reading of algal growth rate, to different ZnO NPs concentrations (0.081-810 mg/L). Significant differences were observed on microalgae growth rates, with the marine being more sensitive than the freshwater species, as revealed by their half inhibitory concentration values (IC50). The IC50 values in freshwater species were affected by the culture medium. The lowest IC50 values (2.57 mg/L) were observed in the marine species. S. rubescens showed the less toxic effect in cultures with modified BG-11, compared to BBM cultures, with IC50 values810 mg/L and 14.27 mg/L after 96 h exposure time, respectively. ZnO nanoparticles appeared to have toxic effects in all species tested, depended on the species type, the exposure time, the NPs concentration, and mainly the culture medium.

Identification of cost-effective cell disruption methods to facilitate lipid extraction from microalgae represents a crucial step in identifying promising biofuel-producing species. Various cell disruption methods including autoclaving, microwave, osmotic shock, and pasteurization were tested in the microalgae Chlorococcum sp. MCC30, Botryococcus sp. MCC31, Botryococcus sp. MCC32, and Chlorella sorokiniana MIC-G5. Lipid content (on dry weight basis) from the four cultures on day 7 ranged from 11.15 to 48.33%, and on day 14 from 11.42 to 44.26%. Among the methods tested, enhanced lipid extraction was achieved through osmotic shock (15% NaCl) for Botryococcus sp. MCC32, microwave (6 min) for Botryococcus sp. MCC31, osmotic shock (5% NaCl) for Chlorella sorokiniana MIC-G5 and microwave (2 min) for Chlorococcum sp. MCC30. The highest palmitate (16:0) contents (25.64% and 34.20%) were recorded with osmotic shock (15% NaCl) treatment for Botryococcus sp. MCC32 and microwave (6 min) for Botryococcus sp. MCC31, respectively. Two strains, along with their respective cell disruption methods, were identified as promising oil blends or nutraceuticals due to their high unsaturated fatty acid (UFA) content: Botryococcus sp. MCC31 (37.6% oleic acid content; 39.37% UFA) after autoclaving and Botryococcus sp. MCC32 after osmotic shock of 15% NaCl treatment (19.95% oleic acid content; 38.17% UFA).

The catalytic valorization of microalgae, a sustainable feedstock to alleviate dependence on fossil fuel and offset greenhouse gases emissions, is of great significance for production of biofuels and value-added chemicals from aquatic plants. Here, an interesting catalytic process is reported to convert microalgae (Chlorococcum sp.) into 1,2-propanediol (1,2-PDO) and ethylene glycol (EG) in water over nickel-based catalysts. The influences of reaction temperature, initial H2 pressure and reaction time on the product distribution were systematically investigated by using a batch reactor. Under optimal reaction conditions (at 250 °C for 3 h with 6.0 MPa of H2 pressure), microalgae were directly and efficiently converted over a Ni–MgO–ZnO catalyst and the total yield of polyols was up to 41.5%. The excellent catalytic activity was attributed to the smaller size and better dispersion of Ni particles on the MgO–ZnO supporter based on the characterization results as well as its tolerance to nitrogen-containing compounds. Besides, the reaction pathway was proposed based on the formation of reaction intermediates and the results of model compound conversion.

One-step and Two-step methods were studied for lipid extraction from wet and unbroken (water content is 65-67%) Chlorella sp. and Chlorococcum sp. (GN38) using deep eutectic solvent (DES) treated microalgae cells. Further we optimized the extraction process and studied on its underlying mechanism. Among all DES, Choline chloride-Acetic acid (Ch-Aa) DES treatment showed optimal conditions at the mass ratio of DES: methanol-H2SO4 (2.00%) mixture: algae biomass was 60:40:3 with reaction time was 60min, and the optimum temperature was 110°C (Chlorococcum sp.) and 130°C (Chlorella sp.) respectively. The total content of FAME by One-step method with DES treatment was improved by 30% compared with Two-step method. This process is effective on wet and unbroken paste of microalgae biomass, so the FAME extracted using one-step with DES process is feasible for microalgae based biodiesel production.

Water surface-floating microalgae have great potential for biofuel applications due to the ease of the harvesting process, which is one of the most problematic steps in conventional microalgal biofuel production. We have collected promising water surface-floating microalgae and characterized their capacity for biomass and lipid production. In this study, we performed chemical mutagenesis of two water surface-floating microalgae to elevate productivity. Floating microalgal strains AVFF007 and FFG039 (tentatively identified as Botryosphaerella sp. and Chlorococcum sp., respectively) were exposed to ethyl methane sulfonate (EMS) or 1-methyl-3-nitro-1-nitrosoguanidine (MNNG), and pale green mutants (PMs) were obtained. The most promising FFG039 PM formed robust biofilms on the surface of the culture medium, similar to those formed by wild type strains, and it exhibited 1.7-fold and 1.9-fold higher biomass and lipid productivities than those of the wild type. This study indicates that the chemical mutation strategy improves the lipid productivity of water surface-floating microalgae without inhibiting biofilm formation and floating ability.

Dairy effluent (DE) was evaluated for cultivation of the oleaginous micro alga Chlorococcum sp. RAP13 under mixotrophic and heterotrophic modes. The alga grew better and accumulated more lipids under heterotrophic cultivation. Supplementation of biodiesel industry waste glycerol (BDWG) to DE enhanced the biomass production as well as lipid accumulation. While the biomass yield was 0.8 g/L for mixotrophic cultivation, it was 1.48 g/L and 1.94 g/L respectively when cultivated with 4% or 6% BDWG. The cells accumulated 31% lipid when grown in mixotrophic mode, and heterotrophic cultivation with 4% or 6% BDWG resulted in a lipid accumulation of 39% and 42% respectively. Saturated fatty acids production was elevated in the DE, and the major fatty acid components of the algal oil were palmitic (16:0), oleic (18:1), stearic (18:0), linoleic (18:2) and linolenic (18:3) acids. DE quality improved with reduction in COD and BOD after algal cultivation.

Chlorococcum sp. RAP13—a fresh water microalga was evaluated for growth and lipid production in medium containing 50 % natural sea water under both phototrophic and heterotrophic cultivation modes. The algal cells adapted very well to the sea water and accumulated up to 38 % of their dry cell weight as lipids under heterotrophic cultivation. Biomass production and lipid accumulation were significantly higher than those in fresh water. Supplementation of biodiesel industry waste glycerol in sea water resulted in the highest yield of oil (330 mg L−1), and the biomass yield was 0.85 g L−1. The composition of oil produced under phototrophic and heterotrophic growth was significantly different, with the former enriched in polyunsaturated fatty acids while the latter contained more of medium chain saturated fatty acids and monounsaturated fatty acids suitable for biodiesel production. While the use of sea water reduces the requirement of mineral supplementation and saves fresh water, the ability to form oil from waste glycerol indicates the potential to recycle this waste. To the best of our knowledge, this is the first report on cultivation of the fresh water alga Chlorococcum sp. under heterotrophic mode in sea water.

The reactions of a strain of marine alga Chlorococcum sp. and of a polystrain containing a mixture of marine algae with CO/H2O have been studied. The yields and chemical composition of the products...

In this study, the performance of Chlorococcum sp. GD in synthetic medium with different glucose concentrations (ranging from 1 to10 g/L) was investigated. Moreover, transcriptome sequencing was conducted to clarify the response of the microalga to glucose concentrations. High concentration of glucose (6–10 g/L) not only did not provide a higher yield of biomass but also inhibited photosynthesis. Transcriptomic analysis revealed that the glucose metabolism mainly depended on the glycolysis and the pentose phosphate pathway (PPP) as the microalga was cultivated with 10 g/L glucose. Meanwhile the tricarboxylic acid (TCA) cycle, oxidative phosphorylation and photosynthesis were significantly inhibited. The significant change on carbon metabolic flux caused by the increase in glucose concentration affected the synthesis of reducing power and ATP, which ultimately influenced the growth of the microalga. Appropriate supplement of organic carbon not only enhances the biomass accumulation but also increases the utilization efficiency of organic carbon.

Carotenoids are lipophilic pigments found in plants and algae, as well as some bacteria, archaea, and fungi that serve two functions&mdash, (1) as light harvesting molecules&mdash, primary carotenoids, and (2) as antioxidants, acting against reactive oxygen species&ndash, secondary carotenoids. Because of their strong antioxidant properties, they are also valuable for the development of anti-aging and photo-protective cosmetic applications. Of particular interest is the carotenoid phytoene, for its colorless and UV absorption characteristics. In this study, we targeted a reduction of phytoene desaturase (PDS) activity with the pigment-inhibiting herbicide 1-methyl-3-phenyl-5-[3-(trifluoromethyl)phenyl]pyridin-4-one (fluridone), which leads to the over-accumulation of phytoene in the recently characterized microalgal strain Chlorococcum sp. (UTEX B 3056). After post-incubation with fluridone, phytoene levels were measured at ~33 ug/mg cell tissue, as opposed to non-detectable levels in control cultures. Hence, the novel microalga Chlorococcum sp. is a viable candidate for the production of the high-value carotenoid phytoene and subsequent applications in cosmeceuticals, as well as more obvious nutraceutical and pharmaceutical applications.

In the present study, ten microalgal strains found in fresh and saline waters were cultured, and used to conduct batch experiments in order to evaluate their potential contribution to nutrient removal and biofuel production. The growth rate of microalgae was inversely analogous to their initial concentration. Three freshwater strains were selected, based on their growth rate, and their behavior with synthetic wastewater was further investigated. The strains studied were the Scenedesmus rubescens (SAG 5.95), the Neochloris vigensis (SAG 80.80), and the Chlorococcum spec. (SAG 22.83), and higher growth rate was observed with S. rubescens. Total phosphorus removal at an initial phosphate concentration of 6-7 mg P/L in the synthetic wastewater, was 53%, 25% and 11% for N. vigensis, Chlorococcum spec., and S. rubescens, respectively. Finally, the lipid content was determined at 20th and 30th day of cultivation, and the highest amount was observed at the 20th day.

Algae-derived biofuels are increasingly seen as an alternative source of energy with potential to supplement the world's ever increasing demand. A great challenge exists today in energy input and costs to procure algal lipid from a cultivated suspension, which generally requires steps such as concentration, filtration, dewatering, grinding, and solvent extraction. Using well practiced sand filtration and ozonation processes, we sought to reduce processing steps and streamline the operations in one vessel. The specific Chlorococcum aquaticum suspension was acidified to pH 3.3 to promote agglomeration prior to biomass collection by sand filtration. The algae-loaded filter bed was drained of free water and added with methanol and ozonated for 2 min to rupture the cell membrane to accelerate release of cellular contents. The methanol solution containing the dissolved lipid product was collected by draining, while the filter bed was regenerated by further ozonation when needed. The results showed 95% collection of the algal biomass from the suspension and a 16% yield of lipid from the algae, as well as restoration of filtration velocity of the sand bed via ozonation. GCMS identification of the extract showed primary products in the forms of long-chain largely saturated hydrocarbons of 16 to 20 carbons. The new technique streamlines individual steps in the procurement of algal lipid from the suspension that is potentially an improvement over existing energy-intensive methods.

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.

Five indigenous microalgal strains of Scenedesmus, Chlorococcum, Coelastrum, and Ankistrodesmus genera, isolated from Indian fresh water habitats, were studied for carbon-dioxide tolerance and its effect on growth, lipid and fatty acid profile. Scenedesmus dimorphus strain showed maximum growth (1.5 g/L) and lipid content (17.83% w/w) under CO2 supplementation, hence selected for detailed evaluation. The selected strain was alkaline adapted but tolerated (i) wide range of pH (5–11); (ii) elevated salinity levels (up to 100 mM, NaCl) with low biomass yields and increased carotenoids (19.34 mg/g biomass); (iii) elevated CO2 levels up to 15% v/v with enhancement in specific growth rate (0.137 d−1), biomass yield (1.57 g/L), lipid content (19.6% w/w) and CO2 biofixation rate (0.174 g L−1 d−1). Unsaturated fatty acid content (alpha linolenic acid) increased with CO2 supplementation in the strain.

Three strains of green microalgae, Chlorococcum sp.C53, Chlorella sp. E53, and Chlorella sp.ED53 were studied for their antioxidant activities. Crude extracts of these microalgae in hot water and in ethanol were examined for their total phenolic contents and for their antioxidant capacities. In order to determine their phenolic contents, the Folin–Ciocalteu method was used. As for the determination of their antioxidant capacities, four different assays were used: (1) total antioxidant capacity determination; (2) DPPH radical scavenging assay; (3) ferrous ion chelating ability assay; and (4) inhibition of lipid peroxidation (using thiobarbituric acid reactive substance). For all the strains we have studied, their ethanolic extract showed more antioxidant activities than their hot water extract. Categorically, the ethanolic extract of Chlorella sp.E53 exhibited both the highest total phenolic content of 35.5 ± 0.14 mg gallic acid equivalent (GAE) g−1 dry weight and the highest DPPH radical scavenging of 68.18 ± 0.38 % at 1.4 mg mL−1 (IC50 0.81 mg mL−1), whereas Chlorella sp.ED53 showed both the highest ferrous ion chelation activity of 42.78 ± 1.48 % at 1 mg mL−1 (IC50 1.23 mg mL−1) and the highest inhibition of lipid peroxidation of 87.96 ± 0.59 % at 4 mg mL−1. This high level of inhibition is comparable to 94.42 ± 1.39 % of butylated hydroxytoluene, a commercial synthetic antioxidant, at the same concentration.

Cell disruption is an integral part of the downstream operation required to produce biodiesel from microalgae. This study investigated the use of ultrasonication and high-pressure homogenization (HPH) as cell disruption methods for two microalgal species, Tetraselmis suecica (TS) and Chlorococcum sp. (C sp.). The kinetics of cell disruption followed a first-order model (0.65

In chapter 1 we introduce microalgae, photosynthetic microorganisms with potential to replace commodities (such as food, feed, chemicals and fuels). Production costs are still high, reason why microalgae are still only economically feasible for niche markets. We suggest to borrow the concept of plant domestication to select industrial microalgae cells. Two approaches can be successfully used to domesticate microalgae: adaptive laboratory evolution (ALE) and fluorescence assisted cell sorting (FACS). ALE takes advantage of the natural adaptability of microorganisms to different environments, while FACS actually select cells with specific phenotypes. This thesis aimed to select cells of Chlorococcum littorale with improved phenotypes, assuming that these cells could establish new populations with increased industrial performance. In Chapter 2 we wanted to know what happened during time to biomass and lipid productivities of Chlorococcum littorale repeatedly subjected to N-starvation. We tested 2 different cycles of N-starvation, short (6 days) and long (12 days). Short cycles didn’t affect lipid productivity, highlighting the potential of C. littorale to be produced in semi-continuous cultivation. Repeated cycles of N-starvation could have caused adaptations of the strain. Hence, we also discussed the implications of using repeated N-starvation for adaptive laboratory evolution (ALE) experiments. Chapter 3 introduces a method to detect and to select microalgae cells with increased lipid content. The method requires only the fluorescence dye Bodipy505/515 dissolved in ethanol, and the method was designed to maintain cellular viability so the cells could be used to produce new inoculum. In chapter 4 we evaluated a question that emerged while deciding which criteria to use to sort lipid-rich cells: does cellular size affects lipid productivity of C. littorale? We hypothesized that cells with different diameters have different division rates, which could affect lipid productivity. Therefore, we assessed the influence of cell diameter, as a sorting parameter, on both biomass and lipid productivity of Chlorococcum littorale (comparing populations before and after sorting, based on different diameters). Results showed that the size of vegetative cells doesn’t affect the lipid productivity of C. littorale. In chapter 5 we present a strategy to sort cells of C. littorale with increased TAG productivity using the method developed at chapter 3. Both the original and the sorted population with the highest lipid productivity (namely, S5) were compared under simulated Dutch summer conditions. The results confirmed our data from experiments done under continuous light: S5 showed a double TAG productivity. Our results showed also that the selected phenotype was stable (1.5 year after sorting) and with potential to be used under industrial conditions. In chapter 6 we extrapolated our results (indoor and outdoor) to other climate conditions. We ran simulations changing the light conditions to four different locations worldwide (the Netherlands, Norway, Brazil and Spain) to estimate both biomass and TAG productivities. Results indicated that biomass yields were reduced at locations with higher light intensities (Brazil/Spain) when compared with locations with lower light intensities (Norway/Netherlands). Hence, the choice of location should not be based on light intensity, but on how stable irradiation is. Chapter 7 is the general discussion of the thesis, demonstrating that both ALE and FACS are effective approaches to select industrial microalgae cells. We also present our view on how ALE and FACS could further improve microalgae strains for industry.

Nitrogen deprivation (N-deprivation) is a proven strategy for inducing triacylglyceride accumulation in microalgae. However, its effect on the physical properties of cells and subsequently on product recovery processes is relatively unknown. In this study, the effect of N-deprivation on the cell size, cell wall thickness, and mechanical strength of three microalgae was investigated. As determined by analysis of micrographs from transmission electron microscopy, the average cell size and cell wall thickness for N-deprived Nannochloropsis sp. and Chlorococcum sp. were ca. 25% greater than the N-replete cells, and 20 and 70% greater, respectively, for N-deprived Chlorella sp. The average Young’s modulus of N-deprived Chlorococcum sp. cells was estimated using atomic force microscopy to be 775 kPa; 30% greater than the N-replete population. Although statistically significant, these microstructural changes did not appear to affect the overall susceptibility of cells to mechanical rupture by high pressure homogenisation. This is important as it suggests that subjecting these microalgae to nitrogen starvation to accumulate lipids does not adversely affect the recovery of intracellular lipids.

Algae biotechnologies offer one possible path to addressing the forecast challenges of human society which are the increased demands of food, fuel and water due to population growth and large scale lifestyle change. The development of renewable algae production technologies helps to establish a basis for sustainable development and provide options to decrease CO2 emissions to address climate change. Algae are increasingly recognised as a promising bioresource and the range of cultivated species and their products is expanding. Compared to terrestrial crops, microalgae are highly biodiverse and offer considerable versatility for a range of biotechnological applications including the production of animal feeds, fuels, high value products and waste-water treatment. Despite their versatility and capacity for high biomass productivity microalgae represent a relatively unexplored bioresource both for native and engineered strains. Success in this area requires (1) appropriate methods to source and isolate microalgae strains, (2) efficient maintenance and preservation of parental stocks, (3) rapid strain characterisation and correct matching of strains to applications, (4) ensuring productive and stable cultivation at scale, and (5) ongoing strain development (breeding, adaptation and engineering). In chapter 2 a streamlined process was developed for the isolation, identification and maintenance of over 150 local microalgae strains as a bioresource for ongoing strain development and biotechnological applications. 121 algal strains could be purified to the axenic level, whereas the rest were maintained as unialgal, non-axenic cultures. All algae used in the large scale trials were obtained as axenic cultures. A range of isolating techniques were explored. Fluorescence activated cell sorting (FACS) proved to be a useful method for high throughput isolation and purification of physically robust algal species, while micromanipulation was most beneficial to obtain a high species diversity. A separate survey by Wolf et al (2014) optimised the nutrient requirements and identified the highest biomass producing strains. Eight algae isolates identified as high production strains based on lab experiments, as well as an isolated invasive algal species and a polyculture of five morphological different strains were tested outdoors to identify key variables limiting optimised production at pilot-scale. The results showed that based on the maximum specific growth rates achieved in sterile laboratory systems, exposed pond systems achieved ~30-50%, indicating limitations imposed by environmental and biological iii factors. Highest daily growth rates in open pond trials single strain trials were obtained for M. pusillum (5_H4) (0.959 d-1), Chlorella sp. (11_H5) (0.719 d-1), Chlorococcum sp. (12_02) (0.755 d- 1), and the polyculture (0.743 d-1). M. pusillum (5_H4), Scenedesmus sp. (Pinjarra001). The polyculture trials achieved the highest optical densities (up to OD750=4). In addition to fast growth, other variables influencing performance included the ability to grow to high densities, autoflocculation, and resistance to predators proved important. Polyculture trials achieved the highest maximum areal and volumetric productivity (24.3 g m-2 day and 266.7 g m-3 day). However the polyculture productivity values are only about 6-10 % higher than those of the highest single strain cultivations and may have resulted from the different cultivation timings. The polyculture growth during spring was favoured by higher mean solar energy, higher absolute temperatures and greater temperature flux than cultivations during autumn and winter. Nevertheless the polyculture trials are considered to be a potential alternative to single species cultivation due to their increased robustness against invasive predatory influences. Contamination by invasive organisms is expected and observations revealed that their impact on the algal culture is dependent both on the cultivated species and the predatory organism. Grazing and competition for nutrients had negative impacts on algal growth and change of algal morphology (e.g. spines and flocculation). Positive impacts of certain grazers included reductions in bacterial and protozoa load, providing opportunities to use “predators” as a tool to sustain algal cultures and optimise the culture production. The flocculation of C. sorokiniana (8_C4) in the presence of a Tetrahymena like ciliate was one of the most promising leads for the optimisation of algae production systems in the form of a novel harvesting technology. Chapter 4 focused on the characterisation and optimisation of a novel bioflocculation method using the native isolated ciliate Tetrahymena. Tetrahymena is a common invader of microalgae cultures, typically feeding on bacteria rather than on the microalgae themselves. Tetrahymena thermophilia, a freshwater ciliate, is well studied for its ability of undergoing dramatic metabolic changes during starvation, its secretory granule biogenesis and exocytosis. Here a locally isolated and identified Tetrahymena culture was used as a controllable bioflocculation agent for the microalgae Chlorella sorokiniana (8_C4). The process was triggered by adding chemical substances to the starved ciliate leading to exocytosis of extracellular polymeric substances (EPS) which functioned as a binding substance between the algae cells. The ratio of ciliate to algae could be reduced to as little as 1:400 cells to initiate rapid bioflocculation. Furthermore stimulators of ryanodine receptors (caffeine and p-chlorocresol) were identified as useful triggers for exocytosis. Future experiments at larger scale iv can prove that the protocol is transferable to greater volumes which could have significant potential for industrial scale application. The work described in this chapter has been the basis for a patent application and has been included in a manuscript published in the journal Algal Research [1]. In conclusion, 150 native microalgae were successfully isolated, cryo-preservation applied (Bui et al., 2013) and their nutrient media optimised (Wolf et al., 2014). The performance of 9 strains was analysed in high rate ponds both in mono and polyculture and a novel Tetrahymena based harvesting system identified (paper published and patent filing submitted).

International audience; Macro- and microalgae have recently received much attention due to their valuable chemical constituents. In order to increase existing data, the authors studied nineteen microalgae species isolated from different reservoirs in the Fez region (northern Morocco), undertaking experiments to determine for each species the specific growth rate, their total amounts of proteins, carbohydrates and lipids and the influence of the growth phase on these chemical constituents. The growth rates of the nineteen studied species of microalgae showed a wide interspecies variation, ranging from 0.27 d-1 for Chlamydomonas nivalis to 3.64 d-1 for Chlorococcum wemmeri. Protein, carbohydrate and lipid contents also varied greatly between taxa and within genera. Ankistrodesmus falcatus, Chlamydomonas nivalis, Chlorococcum sp., Fragilaria ulna, Scenedesmus protuberans, and Synechocystis aquatilis tended to synthesize proteins, the concentrations exceeding 20% of dry weight (DW). Ankistrodesmus falcatus, Ankistrodesmus sp., Chlorococcum wemmeri, Coenocystis sp., Isocystis sp., Lyngbya bergei, Oscillatoria amphibia, Polytoma papillatum, Scenedesmus protuberans, Scenedesmus sp. and Synechocystis aquatilis showed a high capacity for lipid storage, higher than 20% DW. For carbohydrate contents, only Scenedesmus protuberans and Scenedesmus quadricauda showed an excessive level, compared to other scanned species with 29.21% and 24.76% DW, respectively. Green algae, Scenedesmus protuberans for example, generally tend to synthesize proteins, with a content reaching 45% DW, while blue-green algae, for example Lyngbya bergei, tend to synthesize lipids, with a content reaching 50% DW.

Chlorella ellipsoidea and Chlorococcum infusionum, promising microalgae for biodiesel feedstock production, were treated with ethylenediaminetetraacetate (EDTA) and phosphorous to induce stress which was then followed by flow cytometry to study the enhanced intracellular neutral lipid content. Treatment resulted in up to a threefold increase in total lipid content of Chlorella (41.8±1.9% at 16 days of incubation period) and more than twofold increases in Chlorococcum (31.3±1.0% at 18 days of incubation period) under phosphorous starvation in the culture. It was observed that maximum biomass yields in Chlorella and Chlorococcum were 1.56±0.06 and 2.17±0.12 g/L at 1.5 g/L of phosphorous after 20 and 18 days of incubation periods, respectively. The qualitative analyses of neutral lipid bodies under stress conditions were performed by confocal microscopy and revealed bright golden-yellow lipid droplets in stress exposed cells. Significant increase of monounsaturated fatty acids under the nutrient limited conditions was suitable to produce biodiesel. The maximum biomass (g/L) and lipid content (% dry cell weight) at different stresses showed significant results (p<0.05) by single-factor Analysis of Variance (ANOVA) followed by Duncan’s Multiple Range Test (DMRT).

Distribution in pigment deposition was studied in male Siamese fighting fish (Betta splendens Regan, 1910) of solid Blue and solid Red phenotypes. Growths were similar, and also indicated by similar activity ratio of trypsin to chymotrypsin. Growth strategy was different at maturing stage, whereas trypsin expression decreased in solid Blue while chymotrypsin expression decreased in solid Red (P ≤ 0.05). Ranking of total pigment contents was muscle > skin > caudal fin > anal fin > dorsal fin > pelvic fin > pectoral fin. Immature fish had similar total contents and concentrations of pigments, except solid Blue had higher total contents in dorsal fin than solid Red. At maturing stage, solid Blue showed higher total pigment contents than solid Red due to higher contents in the fins. Solid blue had higher concentration in pelvic fin, while solid Red had higher in pectoral and dorsal fins. Age, phenotype and their interaction influenced pigment deposition. Determinations of pigment levels, high temperature tolerant of pigments and in vitro digestibilities of pigments, protein and carbohydrate from twenty natural sources indicated that fermented red rice was the most suitable pigment source, following with Chlorococcum sp.

In recent years microalgae have been proved as the potential source for biodiesel production due to high oil content. In the present study three brackish water microalgal strains (Chlorococcum sp., Chlorella sp. and Scenedesmus sp.) of Odisha coast were screened for the suitability for biodiesel production. Among all, Scenedesmus sp. seems to be the best one for high lipid productivity (24.66mg/L/day) with high biomass yield of 0.9g/L at stationary phase. Also the Scenedesmus sp. possesses the most adequate fatty acid profile. The present study suggested that Scenedesmus sp. is appropriate for biodiesel production for its high lipid content; this strain was selected for higher scale studies.

The objective of this study was to identify the most promising nutritional mode of growth for enhanced biomass and lipid productivity in a set of twenty microalgal strains, grown under photoautotrophic and mixotrophic/heterotrophic conditions using 2% glucose as carbon source. These included four cyanobacterial strains (Cyanosarcina, Phormidium, Nostoc and Anabaena) and sixteen green algae belonging to six genera (five strains each of Chlorella and Chlorococcum, two of Scenedesmus and one each of Chlamydomonas, Kirchneria, Bracteacoccus and Ulothrix). Lipid productivity ranged from 2-13% under photoautotrophic conditions, 1.7-32% under mixotrophic conditions and 0.9-20% under heterotrophic conditions. MIC-G5 Chlorella sp. followed by MIC-G11 Chlorella sp. exhibited the highest cellular lipid content (355 and 271 μg/ml) and lipid productivity of 32% and 28% respectively in mixotrophic condition. In the glucose supplemented conditions (heterotrophic), a significant reduction in PUFA from 25.1 to 9.4, 29.2 to 12.4 and 44.7 to 10.2 was observed in MIC-G4, MIC-G5 and MIC-G11, respectively. A remarkable enhancement of 33-70% in SFA was recorded under mixotrophic conditions. As the quality of biodiesel is based on high SFA and low PUFA, our results illustrate the significance of glucose supplemented condition as a promising strategy for generating high value biodiesel from algae.

Coagulation is an important step in the harvesting of algal biomass. This paper presents experimental results for a variety of prospective marine microalgal species using several inorganic and organic coagulants. Tetraselmis suecica and Chlorococcum sp. are readily coagulated using alum or iron(III) sulphate without any pH adjustment; doses of 3–5 mg L−1 or 0.2 mmol m−2 of Al3+ or Fe3+ yielding cell recoveries above 90 % after only 5-min settling. Nannochloropsis salina, Dunaliella tertiolecta and Isochrysis galbana are harder to coagulate and require at least two times more coagulant to achieve similar recoveries. Several cationic polyacrylamides were investigated but were less effective than Al3+ or Fe3+. Addition of NaOH to control pH improved the coagulation efficiency of N. salina but not of D. tertiolecta. The high coagulant demand of N. salina is due in part to its small size and large surface area, while that of D. tertiolecta may be attributable to its high production of extracellular polymer. The implications of cell surface properties for coagulation efficiency are discussed. At the coagulant doses used herein, settled cells remain viable. Resuspension is a potential problem with some species, arising either from cell motility or from flotation of flocs by oxygen bubbles generated by photosynthesis. These effects can be eliminated by small additions of chlorine or by settling the algae in the dark.

Flocculation induced by pH increase for harvesting microalgae and reuse of flocculated medium were evaluated. Increasing the medium pH value induced the highest flocculation efficiency of up to 90% for freshwater microalgae (Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp.) with low/medium biomass concentrations and marine microalgae (Nannochloropsis oculata, Phaeodactylum tricornutum). The mechanism may be explained that Mg(2+) in the growth medium hydrolyzed to form magnesium hydroxide precipitate, which coagulated microalgal cells by sweeping flocculation and charge neutralization. Additionally, this study revealed that the microalgal biomass concentrations and released polysaccharide (RPS) from microalgae could influence the flocculation efficiencies. Furthermore, neutralizing pH and then supplementing nutrients allowed the flocculated medium to maintain an approximate growth yield to that of the fresh medium in algal cultivation. These results suggest that the method presented here is effective, and allows the reuse of the flocculated medium, thereby contributing to the economic production from algae to biodiesel.

The production of alternative fuels from microalgae involves lengthy processing steps. Cell disruption is an integral part of the downstream pool of unit operations as it facilitates the release of intracellular products essential for biofuel production. This study investigated the use of high-pressure homogenization, ultrasonication, bead beating, and sulfuric acid treatment as laboratory-scale disruption methods for microalgal cells. The performance of each cell disruption method was evaluated in terms of two key indicators: reduction in the intact cell count and reduction in the average colony diameter. The microalgal strain, Chlorococcum sp., was used throughout the study. The most effective disruption was obtained using high-pressure homogenization (average disruption = 73.8% of initial intact cells) followed by sulfuric acid treatment (average disruption = 33.2% of initial intact cells) and bead beating (average disruption = 17.5% of initial intact cells). Even though ultrasonication failed to disrupt the microalgal cells under the investigated conditions (average disruption = 4.5% of initial intact cells), it still managed to disintegrate cellular colonies.

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.

Microalgal biomass as feedstock for biofuel production is rapidly gaining appreciation in response to the increasing petroleum prices and the upsurge in global warming concerns. However, the process of creating concentrated biomass from microalgal cultures is limited by ineffective dewatering procedures. The economics of existing culture clarification unit operations make the process of creating biomass from microalgae unattractive for biofuel development. This work involves the comparison of the removal efficiency (recovery) of two marine microalgae species Chlorococcum sp. and Tetraselmis sp. by electrocoagulation (EC), a technique that has not been thoroughly explored in marine microalgae dewatering. High recovery efficiencies were obtained of up to 99 and 98% for Tetraselmis sp. and Chlorococcum sp., respectively. The effect of culture temperature and salinity on removal efficiency was also observed. A starting temperature of 60 °C resulted in optimal recovery values of 96 and 94% for Chlorococcum sp. and Tetraselmis sp., respectively. Whereas a starting temperature of 5 °C achieved optimal recovery of only 5 and 68% for Chlorococcum sp. and Tetraselmis sp., respectively. Increased salinity of microalgae culture showed increased microalgae recovery. Salinity of 20% gave optimal microalgae recovery values of only 6 and 9% for Chlorococcum sp. and Tetraselmis sp., respectively. Zeta potential (ZP) analysis was carried out to verify and further understand the charge neutralization mechanism due to Fe2+ cations.

Five species of microalgae Chlorella vulgaris, Scenedesmus quadricauda, Euglena gracilis, Ankistrodesmus convolutus and Chlorococcum oviforme, were screened for their ability to grow in treated landfill leachate (TL) using shake flask cultures. The treated leachate had undergone previous treatment through mechanical aeration in treatment ponds at the landfill site. The five algae, except for C. oviforme, were able to grow in medium containing up to 50% TL. Two high-rate algal ponds (HRAP) equipped with paddlewheel were used for the semi-continuous cultures. A mixture of the five algae was used to inoculate one of two HRAPs for secondary treatment of TL. The other HRAP was filled with natural lake water containing mixed populations of algae. A volume of 400 mL (1%) from both ponds were removed daily and replaced with TL. The leachate loading rate was increased to 2% (0.8 L day−1) on day 197 and then to 4% (1.6 L day−1) on day 309, providing hydraulic retention time of 100, 50 and 25 days, respectively. Although higher biomass was obtained in the HRAP containing the consortium of five algae, there was no significant difference in reduction of pollutants between the two ponds. The HRAPs produced algal biomass ranging from 2.00 to 5.54 g dry weight L−1 with significant reduction in chemical oxygen demand (91.0%), ammoniacal nitrogen (99.9%) and orthophosphate (86.0%) contents. The HRAP offers a potential treatment system for TL which is simple, low cost, flexible in use and requiring low maintenance.

Phototrophic biofilms seem to be suitable candidates for tertiary wastewater treatment due to their high uptake capacity for nutrients and other pollutants, also taking into account the time and cost savings derived from easy procedures for biomass harvesting. Biomass accrual, structure, and physiology of biofilms affect the efficiency of nutrient removal by its microbial community. Here, we construct a biofilm consisting of a cyanobacterium Synechocystis sp. and the green alga Chlorococcum sp. and determine the effect of combined variations of irradiance and temperature on the biofilm structure and function. The two species were isolated from phototrophic biofilms naturally developing in an Italian wastewater treatment plant and grown in a microcosm designed for biofilm investigations. Phototrophic biomass accumulation, percent species composition, photosynthetic response and the amount and composition of capsular polysaccharides (CPS), including anionic residues, are reported. The results showed that biofilm development required relatively moderate irradiances (60 μmol photons m−2 s−1) below which development was arrested. Both light and temperature had a strong effect on the composition of each species to the biofilm. The CPS compositions also changed with temperature, light and species composition. The CPS of the green-algal-dominated biofilm had the higher uronic acid content indicating a potential to exploit green algae in the treatment of waste contaminated with heavy metals. Given the knowledge of the response of certain species to light and temperature combinations, it may be possible to construct biofilms of known species and CPS composition to use them for specific applications.

Two conventional chemical coagulants (FeCl3 and Fe2(SO4)3) and five commercial polymeric flocculants (Drewfloc 447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300 and Chitosan) were comparatively evaluated for their ability to remove algal-bacterial biomass from the effluent of a photosynthetically oxygenated piggery wastewater biodegradation process. Chlorella sorokiniana, Scenedesmus obliquus, Chlorococcum sp. and a wild type Chlorella, in symbiosis with a bacterial consortium, were used as model algal-bacterial consortia. While the highest biomass removals (66-98%) for the ferric salts were achieved at concentrations of 150-250 mg L(-1), dosages of 25-50 mg L(-1) were required for the polymer flocculants to support comparable removal efficiencies. Process efficiency declined when the polymer flocculant was overdosed. Biomass concentration did not show a significant impact on flocculation within the concentration range tested. The high flocculant requirements herein recorded might be due to the competition of colloidal organic for the flocculants and the stationary phase conditions of biomass.

Published information on airborne algae and cyanobacteria worldwide and the related human health effects is scarce. Since 1844, a total of 353 morphological taxa (genera or species) have been identified in aerobiological studies. However, due to diverse methodologies and different microorganisms targeted in these studies, direct comparisons on the occurrences of airborne algae and cyanobacteria in various studies are rather dubious. Thirty-eight airborne algae and cyanobacteria were shown to induce allergy, skin irritation, hay fever, rhinitis, sclerosis and respiratory problems when aerosolized and inhaled. Another 14 airborne taxa are known toxin producers posing threat to human health. Most frequently associated with health effects are the genera Chlorella, Scenedesmus, Chlorococcum, Klebsormidium (Hormidium) and Lyngbya. In the air of the Mediterranean city of Thessaloniki, we found 63 algal and cyanobacterial taxa, with 21 reported for the first time in the air. Seven taxa were potentially harmful. Algae and cyanobacteria can contribute significantly in the total air particle load, rendering them as causative agents for health issues when inhaled.

Hydrotreating of bio-oil derived from Chlorococcum sp. in the absence of NiMo/Al-SBA-15 catalysts has been investigated. The silica alumina supports decreased product yields (DCM solubles and hydrocarbon gases) due to severe coking. Incorporation of NiMo increased the product yields to up to 65 wt.% and increased the proportion of products in the kerosene boiling point range (C12-C17).The superiority of the high acidity supports persisted after incorporation of NiMo.

This preliminary study aims to exploit the biomass of microalgae Chlorella vulgaris; Scenedesmus obliquus; and Chlorococcum sp. in the form of a mixed culture as raw material of alternative fuels. Microalgae were cultivated in the artificial growth medium of PHM (Provasoli Haematococcus Media) for 9 days to reach the exponential phase. Hydrolysis was carried out at a temperature variation of (oC) 25; 80; 100; 120; 140; 160 within 30 minutes by adding hydrochloric acid. Biomass fermentation by adding 50% (v/v) Saccharomyces cerevisiae for 5 day to produce alcohol compounds. The last stage is separation of the alcohol compounds from another compounds by distillation. The result showed that carbohydrate levels with color change indicator in luff schoorl solution at hydrolysis and fermentation stages severely were 12.20 mg/L. Carbohydrate levels in fermentation stage produced 17% (v/v) alcohol compounds while in the distillation stage, 98% (v/v) alcohol will be separated into intermediate compounds such as linoleic acid and methyl alcohol as a characteristic of bioethanol. At 80oC of hydrolysis temperature, 58% bioethanol was produced. Further research is needed, since the preliminary study proves the mixed culture of microalgae is potentially to be utilized in producing bioethanol.