Mixotrophic Culture Conditions Research Articles

Enhanced Production of High-Value Porphyrin Compound Heme by Metabolic Engineering Modification and Mixotrophic Cultivation of Synechocystis sp. PCC6803.

Heme, as an essential cofactor and source of iron for cells, holds great promise in various areas, e.g., food and medicine. In this study, the model cyanobacteria Synechocystis sp. PCC6803 was used as a host for heme synthesis. The heme synthesis pathway and its competitive pathway were modified to obtain an engineered cyanobacteria with high heme production, and the total heme production of Synechocystis sp. PCC6803 was further enhanced by the optimization of the culture conditions and the enhancement of mixotrophic ability. The co-expression of hemC, hemF, hemH, and the knockout of pcyA, a key gene in the heme catabolic pathway, resulted in a 3.83-fold increase in the heme production of the wild type, while the knockout of chlH, a gene encoding a Mg-chelatase subunit and the key enzyme of the chlorophyll synthesis pathway, resulted in a 7.96-fold increase in the heme production of the wild type; further increased to 2.05 mg/L, its heme production was 10.25-fold that of the wild type under optimized mixotrophic culture conditions. Synechocystis sp. PCC6803 has shown great potential as a cell factory for photosynthetic carbon sequestration for heme production. This study provides novel engineering targets and research directions for constructing microbial cell factories for efficient heme production.

13C-metabolic flux analysis of Clostridium ljungdahlii illuminates its core metabolism under mixotrophic culture conditions

Carbon dioxide-fixing acetogenic bacteria (acetogens) utilizing the Wood-Ljungdahl Pathway (WLP) play an important role in CO2 fixation in the biosphere and in the development of biological processes – alone or in cocultures, under both autotrophic and mixotrophic conditions – for production of chemicals and fuels. To date, limited work has been reported in experimentally validating and quantifying reaction fluxes of their core metabolic pathways. Here, the core metabolic model of the acetogen Clostridium ljungdahlii was interrogated using 13C-metabolic flux analysis (13C-MFA), which required the development of a new defined culture medium. Autotrophic, heterotrophic, and mixotrophic growth in defined medium was possible by adding 1 mM methionine to replace yeast extract. Our 13C-MFA found an incomplete TCA cycle and inactive core pathways/reactions, notably those of the oxidative pentose phosphate pathway, Entner-Doudoroff pathway, and malate dehydrogenase. 13C-MFA during mixotrophic growth using the parallel tracers [1–13C]fructose, [1,2–13C]fructose, [1,2,3–13C]fructose, and [U–13C]asparagine found that externally supplied CO2 contributed the majority of carbon consumed. All internally-produced CO2 from the catabolism of asparagine and fructose was consumed by the WLP. While glycolysis of fructose was active, it was not a major contributor to overall production of ATP, NADH, and acetyl-CoA. Gluconeogenic reactions were active despite the availability of organic carbon. Asparagine was catabolized equally via conversion to threonine and subsequent cleavage to produce acetaldehyde and glycine, and via deamination to fumarate and then the anaplerotic conversion of malate to pyruvate. Both pathways for asparagine catabolism produced acetyl-CoA, either directly via pyruvate or indirectly via the WLP. Cofactor stoichiometry based on our data predicted an essentially zero flux through the ferredoxin-dependent transhydrogenase (Nfn) reaction. Instead, nearly all of NADPH generated from the hydrogenase reaction was consumed by the WLP. Reduced ferredoxin produced by the hydrogenase reaction and glycolysis was mostly used for ATP generation via the RNF/ATPase system, with the remainder consumed by the WLP. NADH produced by RNF/ATPase was entirely consumed via the WLP.

Nostoc flagelliforme capsular polysaccharides from different culture conditions improve hyperlipidemia and regulate intestinal flora in C57BL/6J mice to varying degrees

Two capsular polysaccharides (WL-CPS-1 and GLU-CPS-1) purified from Nostoc flagelliforme under normal and mixotrophic culture conditions were used to investigate the hypolipidemic activity and effect on intestinal flora in C57BL/6J mice respectively. Their molecular weight and monosaccharide composition have been determined in previous studies. They both improved the lipid level by affecting the expression of lipid metabolism genes. They down-regulated the TNF-α and IL-1β levels in serum and up-regulated the activity of antioxidant enzymes in liver thus decreased the atherosclerosis index and MDA content. They up-regulated the short chain fatty acids (SCFAs) synthesis. They decreased the abundance of pathogenic bacteria and increased the abundance of probiotics positively correlated with SCFAs. Compared with WL-CPS-1, GLU-CPS-1 exhibited higher in vivo activity and enriched Odoribacter and Alloprevotella correlating with the gene expression of lipid metabolism, suggesting that the bioactivity of polysaccharides could be regulated by culture conditions. These findings contributed to application of N. flagelliforme polysaccharides with higher activity in hypolipidemia by adjusting culture conditions.

Overexpression of the Transcription Factor AtLEC1 Significantly Improved the Lipid Content of Chlorella ellipsoidea.

Microalgae are considered to be a highly promising source for the production of biodiesel. However, the regulatory mechanism governing lipid biosynthesis has not been fully elucidated to date, and the improvement of lipid accumulation in microalgae is essential for the effective production of biodiesel. In this study, LEAFY COTYLEDON1 (LEC1) from Arabidopsis thaliana, a transcription factor (TF) that affects lipid content, was transferred into Chlorella ellipsoidea. Compared with wild-type (WT) strains, the total fatty acid content and total lipid content of AtLEC1 transgenic strains were significantly increased by 24.20–32.65 and 22.14–29.91%, respectively, under mixotrophic culture conditions and increased by 24.4–28.87 and 21.69–30.45%, respectively, under autotrophic conditions, while the protein content of the transgenic strains was significantly decreased by 18.23–21.44 and 12.28–18.66%, respectively, under mixotrophic and autotrophic conditions. Fortunately, the lipid and protein content variation did not affect the growth rate and biomass of transgenic strains under the two culture conditions. According to the transcriptomic data, the expression of 924 genes was significantly changed in the transgenic strain (LEC1-1). Of the 924 genes, 360 were upregulated, and 564 were downregulated. Based on qRT-PCR results, the expression profiles of key genes in the lipid synthesis pathway, such as ACCase, GPDH, PDAT1, and DGAT1, were significantly changed. By comparing the differentially expressed genes (DEGs) regulated by AtLEC1 in C. ellipsoidea and Arabidopsis, we observed that approximately 59% (95/160) of the genes related to lipid metabolism were upregulated in AtLEC1 transgenic Chlorella. Our research provides a means of increasing lipid content by introducing exogenous TF and presents a possible mechanism of AtLEC1 regulation of lipid accumulation in C. ellipsoidea.

The gene dosage effect of carbonic anhydrase on the biosynthesis of poly(3-hydroxybutyrate) under autotrophic and mixotrophic culture conditions

Ralstonia eutropha is a hydrogen-oxidizing chemolithotrophic bacterium that can synthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], which is used as a biodegradable plastic. Various genes coding for carbonic anhydrase (CA), which are involved in CO2 fixation and metabolism, are present in the genome of R. eutropha. Although the significance of CA for CO2 metabolism in R. eutropha is well known, CAs have never been deliberately employed to enhance P(3HB) production from CO2. Thus, we investigated the gene dosage effect of the β-carbonic anhydrase (can) gene on P(3HB) accumulation in R. eutropha H16 under autotrophic and mixotrophic conditions. The multicopy plasmid-based dosage of the can gene showed a 1.5-fold increase in P(3HB) content in cells grown under nitrogen-source-limited autotrophic conditions. A similar result was observed under nitrogen-source-limited mixotrophic conditions in the presence of glycerol. Thus, these findings suggest a viable strategy to increase P(3HB) yield under autotrophic and mixotrophic culture conditions. Ralstonia eutropha is a hydrogen-oxidizing chemolithotrophic bacterium that can synthesize poly[(R)-3-hydroxybutyrate] [P(3HB)], which can be used as a biodegradable plastic. Various genes for carbonic anhydrase involved in CO2 fixation are present in the genome of R. eutropha. In this study, we investigated the gene dosage effect of the β-carbonic anhydrase (can) gene on P(3HB) accumulation in R. eutropha under autotrophic and mixotrophic conditions.

Agro-industrial by-product in photoheterotrophic and mixotrophic culture of Tetradesmus obliquus: Production of \u03c93 and \u03c96 essential fatty acids with biotechnological importance

In recent years, researchers have highlighted the role of low cost-efficient agro-industrial by-products used as supplements in algal culture media. The aim of the study was to identify and characterize the basic metabolic pathways in Tetradesmus obliquus cells induced by supplementation with beet molasses in photoheterotrophic and mixotrophic culture conditions. To assess the impact of the nutritional strategy in unicellular algae, growth curves were plotted and lipid, carbohydrate, and protein levels were determined. Fourier Transform Infrared Spectroscopy was applied to measure the Tetradesmus obliquus cell composition. Additionally, the C16-C18 fatty acid profile of Tetradesmus obliquus was determined by gas chromatograph/mass spectrometry. The switch from autotrophy to photoheterotrophy and mixotrophy contributes to shortening of the adaptation growth phase. The highest protein content was obtained in the mixotrophic growth. This study has demonstrated high content of 18:1, cisΔ9, 18:2, cisΔ9,12, ω6, and 18:3, cisΔ9,12,15, ω3 in photoheterotrophic and mixotrophic culture conditions. High levels of proteins and essential fatty acids make Tetradesmus obliquus cell biomass important for human and animals health.

Evaluating differences in growth, photosynthetic efficiency, and transcriptome of Asterarcys sp. SCS-1881 under autotrophic, mixotrophic, and heterotrophic culturing conditions

Autotrophic microalgae can assimilate organic carbon sources for the growth of heterotrophy and mixotrophy. In particular, the physiological and biochemical response of different microalgae species on autotrophy, heterotrophy, and mixotrophy are quite different. The aim of the present study was to determine differences in growth, biochemical composition, photosynthetic characteristics, and metabolic pathway under autotrophic, heterotrophic and mixotrophic culture conditions in an oleaginous microalga, Asterarcys sp. SCS-1881. The results showed that Asterarcys sp. can use glucose for mixotrophic growth, but cannot under strict heterotrophic growth. Mixotrophic cultures can significantly increase the biomass concentration and cell count of Asterarcys sp. The microalga accumulated more protein under mixotrophic conditions, but more carbohydrates and total lipids under autotrophic conditions. The microalga under mixotrophic culture conditions showed significantly reduced pigment content, PSII activity, and photoprotective ability, but increased the activity of PSI when compared to those under autotrophic conditions. Under mixotrophic conditions, Asterarcys sp. also showed enhanced glycolysis, photosynthetic carbon fixation, and pentose phosphate pathway and thus more ATP, carbon skeleton structure, and NADPH. Additionally, the biosynthesis of triacylglycerol, carbohydrates, and pigments was reduced under mixotrophic conditions and the core protein complexes of PSI and PSII were attenuated. Similarly, photosynthetic electron transport and many other pathways related to protein synthesis were enhanced under mixotrophic conditions when compared to the microalga cultured under autotrophic conditions. These biochemical changes may work to co-operatively ensure a more efficient supply of energy and carbon skeleton for rapid growth of the microalga under mixotrophic conditions when compared to those raised under autotrophic conditions.

Effects of inorganic carbon and light on acetate assimilation by Nannochloropsis oceanica (Eustigmatophyceae) in mixotrophic cultivation

ABSTRACT Microalgae are capable of accumulating biomass and high value-added products in mixotrophic culture. Carbon sources and light intensity have significant impacts on mixotrophic algal growth and metabolites accumulation. To investigate the underlying mechanism of interaction between metabolism of inorganic carbon and organic carbon, growth of Nannochloropsis oceanica with two different carbon sources (organic sodium acetate and inorganic sodium bicarbonate) and under three different light conditions (low, medium and high) was examined. A combination of sodium acetate and sodium bicarbonate significantly enhanced algal growth with higher acetate assimilation, when compared with single carbon sources. With increasing light intensity from low to high, the algal growth rates decreased in both autotrophic and mixotrophic cultures. Proteome and 13C metabolic flux results showed that acetate utilization via the glyoxylate (GOX) cycle was enhanced, and both the tricarboxylic acid (TCA) cycle and putative C4 pathway were stimulated in the presence of sodium acetate and sodium bicarbonate. These data suggest that sodium bicarbonate could facilitate acetate assimilation and enhance the putative C4 pathway, GOX cycle and TCA cycle in N. oceanica under mixotrophic culture conditions.

Comparisons of Functional Properties of Polysaccharides from Nostoc flagelliforme under Three Culture Conditions.

Nostoc flagelliforme is an edible cyanobacterium with excellent food and herbal values. It has been used as food in China for more than 2000 years. Many studies have been focused on improving the yield and bioactivity of Nostoc flagelliforme polysaccharides although these have ignored the functional properties. In this study, we extracted and purified three polysaccharides (WL-CPS, NaCl-CPS and Glu-CPS) from Nostoc flagelliforme under normal, salt stress and mixotrophic culture conditions, respectively, in order to change the physicochemical properties of polysaccharides with the aim of obtaining better functional properties. Both salt stress and mixotrophic culture conditions increased the specific yield of polysaccharides. Their functional properties were comparatively investigated and the results showed that NaCl-CPS exhibited the highest emulsification activity and flocculation capability, which was also higher than that of some commercial products. In contrast, Glu-CPS exhibited the highest water and oil holding capacities, foaming property, intrinsic viscosity and bile acids binding capacity. Our results indicated that both NaCl-CPS and Glu-CPS could be considered to be functional polysaccharides according to their respective characteristics, which have great potential in numerous applications, such as food, pharmaceutical, cosmetic, chemical and mineral industries. These findings also demonstrated the potential application of the proper regulation of culture conditions in the development of polysaccharides with desired functional properties.

Effect of culture conditions on the physicochemical properties and antioxidant activities of polysaccharides from Nostoc flagelliforme

Three polysaccharides (WL-CPS-1, NaCl-CPS-1 and Glu-CPS-1) were extracted and purified from Nostoc flagelliforme under normal, salt stress and mixotrophic culture conditions respectively. Their physicochemical properties and antioxidant activities were investigated. WL-CPS-1, NaCl-CPS-1 and Glu-CPS-1 chemical composition differed in sugar and uronic acid contents, and they were composed of nine constituent monosaccharides and one uronic acid with different ratios, with the average molecular weights of 1.02 × 103, 1.12 × 103 and 1.33 × 103 kDa, respectively. They presented similar fourier transform infrared spectra, but different surface morphology, chain length and branching. Antioxidant assay showed that they all exhibited strong scavenging activity on ABTS+ and hydroxyl radicals and moderate activity on DPPH radical. Glu-CPS-1 exhibited the highest antioxidant activity suggested culture conditions could regulate the bioactivity through influencing the structure and properties. These findings demonstrated the potential application of proper regulation of culture conditions in the development of polysaccharides with high antioxidant activity.

Utilisation of Desmodesmus subspicatus LC172266 for simultaneous remediation of cassava wastewater and accumulation of lipids for biodiesel production

ABSTRACTIn view of the current demand for bioenergy and environmental sustainability, utilisation of Desmodesmus subspicatus LC172266 for simultaneous remediation of cassava wastewater and accumulation of lipids for biodiesel production was investigated. The microalga was grown under photoautotrophic, heterotrophic and mixotrophic culture conditions using Bold's Basal Medium (BBM) and cassava wastewater as substrates. Results showed that biomass accumulation under unoptimised autotrophic, heterotrophic and mixotrophic growths conditions were 0.217, 0.63 and 1.042 g/L, respectively. The lipid contents were 15.86, 21.40 and 24.70%, respectively. Fatty acid profiles revealed that although the most abundant compounds under autotrophic conditions were those recommended for biodiesel production, oleic acid eluted under mixotrophic (19.61%) and heterotrophic (10.25%) conditions exhibited more acceptable biodiesel quality. The physical properties of the fatty acid methyl esters (FAMEs) also meet the required biodiesel standards. The percentage reduction in total dissolved solids (TDS, 84.04), electrical conductivity (EC, 82.31), biochemical oxygen demand (BOD, 85.85) and chemical oxygen demand (COD, 89.04) under heterotrophic cultivation were more favourable than their respective mixotrophic values (80.56, 74.63, 62.22, and 51.39%). Although the lipid accumulation of the microalga under mixotrophic conditions was greater, bioremediation potential of cassava wastewater was more favourable under heterotrophy.

Physiological plasticity of a Chilean strain of the diatom Phaeodactylum tricornutum: the effect of culture conditions on the quantity and quality of lipid production

Microalgal biotechnology is always in demand for new fast-growing and oil-rich microalgal strains for different industries. A fusiform diatom strain with robust growth attributes was isolated from Coliumo’s Bay in the Biobio Region, Chile, in 2005. In order to determine the taxonomic identity of the strain, partial sequences of 18S rDNA and ITS region were obtained. Sequence analysis revealed 99% identity with strains of Phaeodactylum tricornutum Bohlin emend. The effect of autotrophic (combination of irradiances, nitrate, and phosphate concentrations) and mixotrophic (addition of sodium acetate under low light) culture conditions was evaluated on growth and lipid accumulation. Hierarchical analysis clustered the autotrophic culture conditions in three main groups of common growth and lipid accumulation responses. Independently on the irradiance, the lowest nitrate and phosphate concentrations induced the highest total lipid accumulation (26–29% lipids per biomass dry weight) and the maximum saturated fatty acid ratio (ca. 42% of total fatty acids), without negative effect on growth; conversely, the highest nitrate and phosphate concentrations stimulated growth and PUFA proportion (20–25% of total fatty acids), but not total lipid accumulation (ca. 15% lipids per biomass dry weight). On the other hand, mixotrophic conditions stimulated growth but not lipid accumulation. Saturated and monounsaturated fatty acid fractions increased as acetate was added while PUFA fraction decreased. Physiological plasticity demonstrated by the Chilean strain of P. tricornutum studied, when faced with different growing conditions, makes it a potential candidate for commercial oil production for different markets, such as biodiesel or feed/food.

Enhanced microalgal biomass and lipid production from a consortium of indigenous microalgae and bacteria present in municipal wastewater under gradually mixotrophic culture conditions

The goal of this study was to investigate the influences of gradually mixotrophic culture conditions on microalgal biomass and lipid production by a consortium of indigenous microalgae and bacteria present in raw municipal wastewater. Lab-scale photobioreactors containing the consortium were operated in repeated batch mode. Initial cultivation (phase I) was performed using only the municipal wastewater, then 10% and 25% of the reactor volumes were replaced with the effluent from a sewage sludge fermentation system producing volatile fatty acids (SSFV) at the beginnings of phase II and phase III, respectively. The highest biomass productivity (117.1±2.7mg/L/d) was attained during phase II, but the lipid productivity (17.2±0.2mg/L/d) was attained during phase III. The increase in the effluent from the SSFV influenced microalgal diversity with a preference for Chlorella sp., but bacterial diversity increased significantly during phase III.

Fatty acid and metabolomic profiling approaches differentiate heterotrophic and mixotrophic culture conditions in a microalgal food supplement 'Euglena'.

BackgroundMicroalgae have been recognized as a good food source of natural biologically active ingredients. Among them, the green microalga Euglena is a very promising food and nutritional supplements, providing high value-added poly-unsaturated fatty acids, paramylon and proteins. Different culture conditions could affect the chemical composition and food quality of microalgal cells. However, little information is available for distinguishing the different cellular changes especially the active ingredients including poly-saturated fatty acids and other metabolites under different culture conditions, such as light and dark.ResultsIn this study, together with fatty acid profiling, we applied a gas chromatography–mass spectrometry (GC-MS)-based metabolomics to differentiate hetrotrophic and mixotrophic culture conditions.ConclusionsThis study suggests metabolomics can shed light on understanding metabolomic changes under different culture conditions and provides a theoretical basis for industrial applications of microalgae, as food with better high-quality active ingredients.

Biomass and nutrient productivities of Tetraselmis chuii under mixotrophic culture conditions with various C:N ratios

Mass microalgal culture plays an irreplaceable role in aquaculture, but microalgal productivity is restricted by traditional autotrophic culture conditions. In the present study, a Tetraselmis chuii strain belonging to the phylum Chlorophyta was isolated from south Yellow Sea. The growth rate and biomass productivity of this strain was higher under mixotrophic conditions with different carbon:nitrogen (C:N) ratios than those under autotrophic conditions. When the C:N ratio was 16, the optical density and biomass productivity were 3.7- and 5-fold higher than their corresponding values under autotrophic culture conditions, respectively. Moreover, T. chuii synthesized more polysaccharides and total lipids under mixotrophic conditions. In addition, T. chuii cultured under mixotrophic conditions synthesized more types of fatty acids than autotrophic culture conditions. At a C:N ratio of 16, the percentage of C16:0 and C18:1 reached 30.08% and 24.65% of the total fatty acid (TFA) content, respectively. These findings may provide a basis for large-scale mixotrophic culture of T. chuii, as a potential bait-microalga.

Accumulation of α-tocopherol and β-carotene in Euglena gracilis Cells Under Autotrophic and Mixotrophic Culture Conditions

The aim of the work was to find the mode of cultivation of unicellular flagellate Euglena gracilis, favorable for the simultaneous accumulation of α-tocopherol and β-carotene. Cells were grown either in photoautotrophic or photoheterotrophic conditions in the presence of 100 mM ethanol (variant Et) or 40 mM glutamate (variant Gt), or their combination (variant EtGt). The exogenous substrates significantly stimulated light-dependent growth of E. gracilis. The largest increase of biomass was recorded on the 20th day in the variant EtGt and exceeded the autotrophic control by 7 times. The content of β-carotene and chlorophyll (Chl) per cell in mixotrophic cultures exceeded the control by 2-3 and 1.6-2 times, respectively. At the same time, α-tocopherol accumulation in autotrophic cells was greater than in the cells of mixotrophic cultures by 2-7 times. Total yield of tocopherol per unit volume of culture medium, which depended not only on its intracellular content, but also on the amount of accumulated biomass was highest in EtGt variant. A correlation between the accumulation of the antioxidants and the equilibrium concentration of oxygen in the growth medium, which depended on the intensities of photosynthesis and respiration has been analyzed.

Overexpression of the soybean transcription factor GmDof4 significantly enhances the lipid content of Chlorella ellipsoidea.

BackgroundThe lipid content of microalgae is regarded as an important indicator for biodiesel. Many attempts have been made to increase the lipid content of microalgae through biochemical and genetic engineering. Significant lipid accumulation in microalgae has been achieved using biochemical engineering, such as nitrogen starvation, but the cell growth was severely limited. However, enrichment of lipid content in microalgae by genetic engineering is anticipated. In this study, GmDof4 from soybean (Glycine max), a transcription factor affecting the lipid content in Arabidopsis, was transferred into Chlorella ellipsoidea. We then investigated the molecular mechanism underlying the enhancement of the lipid content of transformed C. ellipsoidea.ResultsWe constructed a plant expression vector, pGmDof4, and transformed GmDof4 into C. ellipsoidea by electroporation. The resulting expression of GmDof4 significantly enhanced the lipid content by 46.4 to 52.9%, but did not affect the growth rate of the host cells under mixotrophic culture conditions. Transcriptome profiles indicated that 1,076 transcripts were differentially regulated: of these, 754 genes were significantly upregulated and 322 genes were significantly downregulated in the transgenic strains under mixotrophic culture conditions. There are 22 significantly regulated genes (|log2 ratio| >1) involved in lipid and fatty acid metabolism. Quantitative real-time PCR and an enzyme activity assay revealed that GmDof4 significantly up-regulated the gene expression and enzyme activity of acetyl-coenzyme A carboxylase, a key enzyme for fatty acid synthesis, in transgenic C. ellipsoidea cells.ConclusionsThe hetero-expression of a transcription factor GmDof4 gene from soybean can significantly increase the lipid content but not affect the growth rate of C. ellipsoidea under mixotrophic culture conditions. The increase in lipid content could be attributed to the large number of genes with regulated expression. In particular, the acetyl-coenzyme A carboxylase gene expression and enzyme activity were significantly upregulated in the transgenic cells. Our research provides a new way to increase the lipid content of microalgae by introducing a specific transcription factor to microalgae strains that can be used for the biofuel and food industries.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-014-0128-4) contains supplementary material, which is available to authorized users.

Metabolic Engineering of Synechocystis sp. Strain PCC 6803 for Isobutanol Production

Global warming and decreasing fossil fuel reserves have prompted great interest in the synthesis of advanced biofuels from renewable resources. In an effort to address these concerns, we performed metabolic engineering of the cyanobacterium Synechocystis sp. strain PCC 6803 to develop a strain that can synthesize isobutanol under both autotrophic and mixotrophic conditions. With the expression of two heterologous genes from the Ehrlich pathway, the engineered strain can accumulate 90 mg/liter of isobutanol from 50 mM bicarbonate in a gas-tight shaking flask. The strain does not require any inducer (i.e., isopropyl β-d-1-thiogalactopyranoside [IPTG]) or antibiotics to maintain its isobutanol production. In the presence of glucose, isobutanol synthesis is only moderately promoted (titer = 114 mg/liter). Based on isotopomer analysis, we found that, compared to the wild-type strain, the mutant significantly reduced its glucose utilization and mainly employed autotrophic metabolism for biomass growth and isobutanol production. Since isobutanol is toxic to the cells and may also be degraded photochemically by hydroxyl radicals during the cultivation process, we employed in situ removal of the isobutanol using oleyl alcohol as a solvent trap. This resulted in a final net concentration of 298 mg/liter of isobutanol under mixotrophic culture conditions.

Enhanced algae growth in both phototrophic and mixotrophic culture under blue light

Biomass productivity and fatty acid methyl esters (FAME) derived from intracellular lipid of a Nannochloropsis sp. isolated from Singapore’s coastal waters were studied under different light wavelengths and intensities. Nannochloropsis sp., was grown in both phototrophic and mixotrophic (glycerol as the carbon source) culture conditions in three primary monochromatic light wavelengths, i.e., red, green and blue LEDs, and also in white LED. The maximum specific growth rate (μ) for LEDs was blue>white>green>red. Nannochloropsis sp. achieved a μ of 0.64 and 0.66d−1 in phototrophic and mixotrophic cultures under blue lighting, respectively. The intracellular fatty acid composition of Nannochloropsis sp. varied between cultures exposed to different wavelengths, although the absolute fatty acid content did differ significantly. Maximum FAME yield from Nannochloropsis sp. was 20.45% and 15.11% of dry biomass weight equivalent under photo- and mixotrophic culture conditions respectively for cultures exposed to green LED (550nm). However, maximum volumetric FAME yield was achieved for phototrophic and mixotrophic cultures (i.e., 55.13 and 111.96mg/l, respectively) upon cell exposure to blue LED (470nm) due to highest biomass productivity. It was calculated that incremental exposure of light intensity over the cell growth cycle saves almost 20% of the energy input relative to continuous illumination for a given light intensity.

Isolation of the non-fastidious microalga with astaxanthin-accumulating property and its potential for application to aquaculture

Astaxanthin has recently attracted considerable attention for its biological properties such as the antioxidant activity as well as a coloring agent used for farmed fish. However, its biological production, mainly by a green microalgae Haematococcus pluvialis, is costly because of its fastidious growth characters. Therefore, for future applications of biological astaxanthin production in aquaculture, non-fastidious microalgal strains were isolated from environmental samples, examined for their astaxanthin-accumulating activity, and characterized phylogenetically. While β-carotene was found in all tested isolates, one isolate, GK12, from activated sludge of a sewage treatment plant accumulated 2.5 ± 0.36 mg/g dry cell of free astaxanthin de novo under photoautotrophic culture condition, which was comparable to photoautotrophic cultures of other known astaxanthin-accumulating microorganisms. Mixotrophic culture conditions increased GK12 biomass, but astaxanthin content was decreased, suggesting that the simple photoautotrophic cultivation is more efficient way for GK12 to produce astaxanthin than mixotrophic cultivation. A phylogenetic study of SSU rDNA strongly suggested that GK12 is a novel species in the genus Monoraphidium, Chlorophyta. Besides making efforts to increase astaxanthin-productivity of known astaxanthin accumulators, it is also beneficial to study GK12 from the view point of applications to aquaculture.