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

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

Toxicity and bioaccumulation of iron in soil microalgae

Microalgae are extensively used in the remediation of heavy metals like iron. However, factors like toxicity, bioavailability and iron speciation play a major role in its removal by microalgae. Thus, in this study, toxicity of three different iron salts (FeSO₄, FeCl₃ and Fe(NO₃)₃) was evaluated towards three soil microalgal isolates, Chlorella sp. MM3, Chlamydomonas sp. MM7 and Chlorococcum sp. MM11. Interestingly, all the three iron salts gave different EC50 concentrations; however, ferric nitrate was found to be significantly more toxic followed by ferrous sulphate and ferric chloride. The EC₅₀ analysis revealed that Chlorella sp. was significantly resistant to iron compared to other microalgae. However, almost 900 μg g^?1 iron was accumulated by Chlamydomonas sp. grown with 12 mg L^?1 ferric nitrate as an iron source when compared to other algae and iron salts. The time-course bioaccumulation confirmed that all the three microalgae adsorb the ferric salts such as ferric nitrate and ferric chloride more rapidly than ferrous salt, whereas intracellular accumulation was found to be rapid for ferrous salts. However, the amount of iron accumulated or adsorbed by algae, irrespective of species, from ferrous sulphate medium is comparatively lower than ferric chloride and ferric nitrate medium. The Fourier transform infrared spectroscopy (FTIR) analysis shows that the oxygen atom and P?=?O group of polysaccharides present in the cell wall of algae played a major role in the bioaccumulation of iron ions by algae.     

Algal diversity in flowing waters at an acidic mine drainage “barrens” in central Pennsylvania,USA

Microscopic investigations were undertaken to decipher the diversity in the lotic algal communities from acidic waters (pH 2.4–3.2) flowing overland in sheets and channels at an acid mine drainage (AMD) barrens near Kylertown, PA, USA. Microscopic observations, supplemented with taxonomic keys, aided in identification of the dominant algae, and measurement of carbon from adjacent soils was undertaken. The unicellular protist Euglena sp. was most abundant in slower flowing waters (i.e., pool near point of emergence and surficial flow sheets), while Ulothrix sp. was most abundant in faster flowing water from the central stream channel. A diverse range of unicellular microalgae such as Chlorella, Cylindrocystis, Botryococcus, and Navicula and several filamentous forms identified as Microspora, Cladophora, and Binuclearia were also recorded. The observed high algal diversity may be related to the long duration of AMD flow at this site which has led to the development of adapted algal communities. The comparatively higher carbon content in soil materials adjacent to slower flowing water sampling locations provides evidence for the important role of algae as primary producers in this extreme environment.     

微藻光密度与细胞密度及生物质的关系

以四种常见微藻,小球藻(Chlorella sp.XQ-20044)、栅藻(Scenedesmus sp.SS-200716)、绿球藻(Chlorococcum sp.)和螺旋藻(Spirulina sp.CH-164)为实验材料,用梯度稀释法测定对数生长期不同浓度藻液的光密度(OD)、细胞密度和生物质干重(DW),在光自养分批培养模式下对4种微藻进行OD-波长(350—800 nm)扫描,同时测定细胞密度和生物质干重,分析藻液OD与细胞密度、生物质干重的关系。结果表明:在任何波长下,对数生长期的4种微藻细胞密度与OD值、生物质干重与OD值的变化都不成比例,波长不同其拟合曲线偏离直线的程度不同。但是,在435 nm处这种关系最接近直线,可以用直线方程近似描述(R20.98),其它波长处细胞密度-OD、干重-OD的关系都可以用二项式方程很好地描述(R20.99)。因此,光密度法适用于连续和半连续培养,可以用435 nm处测得的OD值计算细胞密度与干重。但是在分批培养模式下,4种微藻DW/OD比值随着培养时间均逐渐上升。小球藻DW/OD540为0.19—0.44 g/L,栅藻DW/OD540为0.36—0.53 g/L,绿球藻DW/OD540为0.48—0.75 g/L,螺旋藻DW/OD560为0.46—0.74 g/L,因此分批培养模式下采用测定藻液OD值反映细胞密度和生物质的方法不适用,只有直接测定细胞密度和生物质才是准确的。研究结果为正确使用分光光度法监测微藻生长提供依据。     

Evaluation of antioxidant capacities of green microalgae

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.     

Biomass and oil content of Chlorella sp., Haematococcus sp., Nannochloris sp. and Scenedesmus sp. under mixotrophic growth conditions in the presence of technical glycerol

The growth of algae strains Chlorella sp., Haematococcus sp., Nannochloris sp. and Scenedesmus sp. under mixotrophic conditions in the presence of different concentrations of technical glycerol was investigated with the aim of increasing biomass growth and algae oil content. The highest concentration of lipid obtained in media with 5 g L^?1 glycerol for Chlorella sp., Scenedesmus sp., Nannochloris sp. and Haematococcus sp. was 17.77, 22.34, 27.55 and 34.22 % larger than during the autotrophic growth of these species. Increases in triacylglycerols of up to ten times was observed for Scenedesmus sp. under mixotrophic conditions (using 10 g L^?1 glycerol), whereas an increase of 2.28 times was found for Haematococcus sp. The content of saturated fatty acids of Scenedesmus, Chlorella, Haematococcus and Nannochloris was 67.11, 34.63, 23.39 and 24.23 %, and the amount of unsaturated fatty acids was 32.9, 65.06, 79.61 and 75.78 % of total fatty acids, respectively. Growth on technical glycerol of these strains with light produced higher biomass concentrations and lipid content compared with autotrophic growth. The fatty acid content of oils from these species suggests their potential use as biodiesel feedstock.     

Transient expression of the GUS gene in a unicellular marine green alga,<Emphasis Type="Italic">Chlorella</Emphasis> sp.<Emphasis Type="Italic">MACC/C95</Emphasis>, via electroporation

A transient expression system for a unicellular marine green alga,Chlorella sp.MACC/C95, was developed using a reporter GUS gene coded for by plasmid pBI121. The results demonstrated a high transformation efficiency could be achieved by using electroporation to deliver DNA into intact cells and the CaMV35S promoter to drive the foreign gene expression inChlorella sp.MACC/C95. The use of a carrier DNA coupled with osmosis treatment improved the transformation efficiency, while linearization of the plasmid had minor effects. Investigation of the effects of DNA concentration and growth phases ofChlorella sp.MACC/C95 on transformation efficiency indicated that the highest level of transient expression was observed when 6 μg mL⁻¹ of plasmid DNA and cells 2–6 days old were used.     

The Effects of Plant Growth Substances and Mixed Cultures on Growth and Metabolite Production of Green Algae Chlorella sp.: A Review

Recent interest in the use of microalgae for the production of biofuels and bioproducts has stimulated an interest in methods to enhance the growth rate of microalgae. This review examines past work involving the stimulation of Chlorella sp. growth and metabolite production by plant growth substances as well as by mixed cultures of Chlorella sp. with bacteria. Plant growth substances known to regulate Chlorella sp. growth and metabolite production include auxins, cytokinins, abscisic acid, polyamines, brassinosteroids, jasmonic acid, salicylic acid, and combinations of two or three of the aforementioned substances. Mixed cultures of bacteria are examined, including both natural bacteria–algae consortia and artificially induced symbioses. For natural consortia, commonly occurring bacterial species, including the genera Brevundimonas and Sphingomonas, are discussed. For artificially induced symbioses, the use of the nitrogen-fixing bacterium Azospirillum is examined in detail. In particular, a variety of studies have involved the coimmobilization of Chlorella sp. with Azospirillum sp. in alginate beads, with the goal of using the mixed culture to treat wastewater. In summary, the use of plant growth substances and mixed cultures provides two methods to increase the growth of Chlorella sp., whether for the production of lipids for biofuels, the production of bioproducts, the treatment of wastewater, or a variety of other reasons.     

Lipid productivity and fatty acid composition-guided selection of Chlorella strains isolated from Malaysia for biodiesel production

The need to develop biomass-based domestic production of high-energy liquid fuels (biodiesel) for transportation can potentially be addressed by exploring microalgae with high lipid content. Selecting the strains with adequate oil yield and quality is of fundamental importance for a cost-efficient biofuel feedstock production based on microalgae. This work evaluated 29 strains of Chlorella isolated from Malaysia as feedstock for biodiesel based on volumetric lipid productivity and fatty acid profiles. Phylogenetic studies based on 18S rRNA gene revealed that majority of the strains belong to true Chlorella followed by Parachlorella. The strains were similarly separated into two groups based on fatty acid composition. Of the 18 true Chlorella strains, Chlorella UMACC187 had the highest palmitic acid (C16:0) content (71.3?±?4.2 % total fatty acids, TFA) followed by UMACC84 (70.1?±?0.7 %TFA), UMACC283 (63.8?±?0.7 %TFA), and UMACC001 (60.3?±?4.0 %TFA). Lipid productivity of the strains at exponential phase ranged from 34.53 to 230.38 mg L^?1 day^?1, with Chlorella UMACC050 attaining the highest lipid productivity. This study demonstrated that Chlorella UMACC050 is a promising candidate for biodiesel feedstock production.     

Nitrogen deprivation of microalgae: effect on cell size,cell wall thickness,cell strength,and resistance to mechanical disruption

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.     

Formation of N-Carboxymethyl Amino Acid from the Reaction of α-Amino Acid with Glyoxal

Enrichment cultures in a medium containing 0.1% methanol and 0.1% bicarbonate at pH 7.0 under anaerobic conditions in the light became mainly green in color. Forty-four enrichment cultures, which showed abundant growth, were obtained from 46 different sources and found to contain cells of methanol-utilizing bacteria and green algae as predominant members. From these enrichment cultures, two strains of bacteria and two strains of algae were isolated. The microorganisms isolated were designated as bacterium No. 7, bacterium No. 8, Chlorella sp. A-1 and Chlorella sp. B-1, respectively. Stable mixed cultures were easily formed by mixing the isolated cultures of bacteria and algae. Both methanol and bicarbonate were necessary for the growth of the mixed cultures under anaerobic-light conditions. Growth behavior of the mixed cultures was examined on a medium containing 0.1% methanol and 0.1 % bicarbonate at 30°C in the light (about 6000 lx). The maximum specific growth rate for the cultures, µ_max, was 0.092 hr^?1 (doubling time, 7.5 hr). The maximum cell yield was 0.87 g dry-cell weight per g of methanol used. The protein content of the biomass was 65%.     

Identification and quantification of indole-3-acetic acid in the kelp Laminaria japonica Areschoug and its effect on growth of marine microalgae

A method was established for the identification and quantification of indole-3-acetic acid (IAA) in extracts of the kelp Laminaria japonica. An IAA content of 90–95 μg kg⁻¹ fresh weight in kelp extract was determined by high performance liquid chromatography (HPLC). IAA identification was based on a combination of co-chromatography and comparative chromatography with a standard, analysis of UV spectra, and atmospheric pressure electrospray mass spectrometry (APESI-MS). IAA was isolated by silica gel chromatography and HPLC. The effect on the growth of four marine microalgae of the pure IAA isolated from kelp extract was investigated. Exogenously added IAA from kelp enhanced the growth of Chlorella sp., Dunaliella salina and Porphyridium cruentum, but not that of Chaetoceros muelleri. IAA from kelp significantly inhibited the accumulation of soluble cellular proteins in Chlorella sp. and P. cruentum, and had a very significant effect on chlorophyll biosynthesis in Chlorella sp. However, there was no obvious effect of IAA on the regulation of biosynthesis of cellular polysaccharides in these four marine microalgae.     

Cell disruption methods for improving lipid extraction efficiency in unicellular microalgae

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).     

Influence of algae species,substrata and culture conditions on attached microalgal culture

The objective of this study was to understand and optimize the formation of microalgae biofilms in specific culture conditions. Firstly, the adhesion of six freshwater algae species was compared. Chlorococcum sp. was selected because of the high adhesion biomass productivity (ABP) and adhesion rate achieved. Secondly, the adhesion of Chlorococcum sp. was compared with nine commonly used supporting materials, and glass fiber-reinforced plastic proved to be the optimal substrata. Thirdly, based on response surface methodology experiments, a second-order polynomial model was developed to examine the effect of culture period, initial total nitrogen concentration (ITNC) in manure wastewater, pH and culture volume of the growth chamber on the adhesion of Chlorococcum sp. using glass fiber-reinforced plastic. The experimental and modeling results showed that ITNC, pH and culture volume as well as the interactions between culture period and ITNC, culture period and culture volume were significant on ABP. Optimum culture conditions were predicted at a culture period of 11 days, ITNC of 70 mg L^?1, pH of 8 and culture volume of 340 mL, under which the predicted maximum ABP was 4.26 g m^?2 day^?1. The prediction was close to validation experimental results, indicating that the model could be used to guide and optimize the attached culture of Chlorococcum sp. using glass fiber-reinforced plastic.     

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

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

Hydrogen evolution by several algae

Summary Out of 33 strains of unicellular algae examined, H₂ evolution was observed only in species of Chlamydomonas, Chlorella and Scenedesmus. While the photoevolution of H₂ by these algae was generally stimulated both by an organic substrate and by the uncoupler CCCP¹, response to DCMU varied. On the basis of the response to DCMU, it was concluded that the mechanism of photoevolution of H₂ differed from one alga to another. The reaction in some algae appeared to be dependent on either the photooxidation of water or oxidative carbon metabolism for reductant; that in other algae was supported by reductant from both these sources.     

Antagonistic association of the chlorellavorus bacterium (“Bdellovibrio” chlorellavorus) withChlorella vulgaris

The chlorellavorus bacterium (Bdellovibrio chlorellavorus Gromov and Mamkaeva 1972) attaches to (but does not enter) cells of the unicellular green alga,Chlorella, which is killed and the cell contents of which are digested. The bacterium is pleomorphic (vibrios 0.3 μm wide; cocci 0.6 μm wide), and it has a Gram-negative cell wall structure pili, and a single, unsheathed, polar flagellum. Division may occur only in bacterial cells attached to algal cells, an attachment mediated by a pad (245×36 nm) of unknown composition. Bacterial growth occurs only in the presence of liveChlorella cells, and not on various bacteriological culture media, killedChlorella cells, 4 strains ofPrototheca, or 24 strains of Gram-negative bacteria. The chlorellavorus bacterium may not require algal protein synthesis, since the bacterium grows on algae in the presence of cycloheximide (30 μg/ml). Although the DNA base composition of the chlorellavorus bacterium (50 mol % G+C) is in the same range asBdellovibrio bacteriovorus, its ultrastructure, developmental cycle, host range, and format of its intermicrobial association all distinguish the chlorellavorus bacterium from members of the genusBdellovibrio.     

Enhancement of total lipid yield by nitrogen,carbon, and iron supplementation in isolated microalgae

The biochemical contents and biodiesel production ability of three microalgal strains grown under different sodium nitrate, sodium carbonate, and ferric ammonium citrate (iron) levels were investigated. The highest biomass and lipid contents were found in Scenedesmus sp., Chlorella sp., and Chlamydomonas sp. when grown in normal BG‐11 containing sodium carbonate concentration at 0.03 g · L^?1, and in normal BG‐11 containing iron concentration (IC) at 0.009 or 0.012 g · L^?1. Increasing the sodium nitrate level increased the biomass content, but decreased the lipid content in all three microalgae. Among the three microalgae, Scenedesmus sp. showed the highest total lipid yield of 0.69 g · L^?1 under the IC of 0.012 g · L^?1. Palmitic and oleic acids were the major fatty acids of Scenedesmus sp. and Chlamydomonas sp. lipids. On the other hand, Chlorella sp. lipids were rich in palmitic, oleic, and linolenic acids, and henceforth contributing to poor biodiesel properties below the standard limits. The three isolated strains had a potential for biodiesel production. Nevertheless, Scenedesmus sp. from stone quarry pond water was the most suitable source for biodiesel production with tolerance toward the high concentration of sodium carbonate without the loss of its biodiesel properties.     

Biotransformation of monoterpenes by immobilized microalgae

This paper reports the biotransformation of carvone, limonene, β-pinene, thymol, and linalool using whole-cell-immobilized microalgal strains isolated from paddy fields of Iran. The strains was recognized by morphological characterization and assigned according to amplified 16S/18S rRNA genes by PCR. Ten unialgal strains including Chlorella, Oocystis, Chlamydomonas, and Synechococcus were immobilized in calcium alginate beads. After a 24-h incubation with substrates, characterization and identification of biotransformation products were done by GC/MS. None of the isolated immobilized microalgae converted β-pinene. In contrast, most of these strains biotransformed carvone and limonene to the related compounds. Some strains only reduced the C = C double bond to yield the dihydrocarvone isomers while others reduced the ketone to give the dihydrocarveol. The transformation ratio showed that Oocystis sp. MCCS 033 and Synechococcus sp. MCCS 035 produced dihydrocarvone isomers with the highest efficiency. Furthermore, limonene was converted into a mixture of five corresponding products and the maximum yield was 52.1% for carvone, the bioconverted product. Only one strain, Synechococcus sp. MCCS 034, oxidized thymol, and the product obtained from thymol was thymoquinone. Also, linalooloxide isomers and dihydrolinalool were obtained from linalool, and finally dihydrolinalool was the main product. These results showed a novel conversion pathway of linalool-forming dihydrolinalool.