Microplate assay for quantitation of neutral lipids in extracts from microalgae

Higgins, Brian; Thornton-Dunwoody, Alexander; Labavitch, John M.; VanderGheynst, Jean S.

doi:10.1016/j.ab.2014.07.020

Cited by 34 publications

(20 citation statements)

References 35 publications

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“…The lipid content was determined by the Nile Red method as described elsewhere (Chen et al, 2009;Higgins et al, 2014). Commercial corn oil from a local store was used for the preparation of the calibration curve.…”

Section: Methodsmentioning

confidence: 99%

Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides

Μάτσακας

Novak

Enman

et al. 2017

Bioresource Technology

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The aim of the current work was to convert an acetate-rich hemicellulose liquid fraction (LF) from hot-water extraction of Betula pendula to oils for biodiesel, with Rhodosporidium toruloides. The toxicity of acetate was circumvented by biological detoxification with an isolated alkali-tolerant and acetate-resistant Bacillus sp. strain. Removal of other lignocellulose-derived inhibitors, such as furfural and phenols, was evaluated by two strategies; an activated carbon (AC) treatment of the undiluted LF, and dilution of the LF by 25% (0.75LF) and 50%. (0.50LF). The bacterium consumed most of the acetic acid in 6-8days in the treated or diluted media, which were subsequently used for cultivation of the yeast, for conversion of sugars to oils. The oil concentration reached 2.8 and 1.8g/L, in the AC LF and 0.75LF medium, respectively. In comparison, the oil accumulation in the same media without prior cultivation of Bacillus sp. was 0.86 and 0.03g/L, respectively.

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Section: Methodsmentioning

confidence: 99%

Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides

Μάτσακας

Novak

Enman

et al. 2017

Bioresource Technology

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“…Lipid extracts were transesterified with 1 M methanolic HCl and analyzed by gas chromatography and mass spectrometry (GC‐MS) using methods previously described (Higgins et al, ). Briefly, internal standard (nonadecanoic acid) was added to lipid extracts that were then dried, re‐suspended in hexane, and transesterified.…”

Section: Methodsmentioning

confidence: 99%

“…In this study we used gas chromatography with mass spectrometry (GC‐MS), thin layer chromatography (TLC), and a neutral lipid assay (Higgins et al, ) to thoroughly describe the lipid content of axenic cultures and co‐cultures under autotrophic and mixotrophic conditions. Our objective was to determine the quantity of lipids conducive to biodiesel production and make inferences about potential biodiesel quality based on fatty acid data.…”

Section: Introductionmentioning

confidence: 99%

Co‐culturing Chlorella minutissima with Escherichia coli can increase neutral lipid production and improve biodiesel quality

Higgins

Labavitch

VanderGheynst

2015

Biotech & Bioengineering

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Lipid productivity and fatty acid composition are important metrics for the production of high quality biodiesel from algae. Our previous results showed that co-culturing the green alga Chlorella minutissima with Escherichia coli under high-substrate mixotrophic conditions enhanced both culture growth and crude lipid content. To investigate further, we analyzed neutral lipid content and fatty acid content and composition of axenic cultures and co-cultures produced under autotrophic and mixotrophic conditions. We found that co-culturing C. minutissima with E. coli under high substrate conditions (10 g/L) increased neutral lipid content 1.9- to 3.1-fold and fatty acid content 1.5- to 2.6-fold compared to equivalent axenic C. minutissima cultures. These same co-cultures also exhibited a significant fatty acid shift away from trienoic and toward monoenoic fatty acids thereby improving the quality of the synthesized fatty acids for biodiesel production. Further investigation suggested that E. coli facilitates substrate uptake by the algae and that the resulting growth enhancement induces a nitrogen-limited condition. Enhanced carbon uptake coupled with nitrogen limitation is the likely cause of the observed neutral lipid accumulation and fatty acid profile changes.

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“…The resulting lipid extract was then used to carry out a neutral lipid assay [21] with slight modifications: specifically 1 μg/mL Nile Red solution was used rather than 0.5 μg/mL as discussed in the publication. Canola oil was used as a standard.…”

Section: Lipid Analysismentioning

confidence: 99%

Impact of thiamine metabolites and spent medium from Chlorella sorokiniana on metabolism in the green algae Auxenochlorella prototheciodes

Higgins

Wang

et al. 2018

Algal Research

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Auxenochlorella protothecoides is a known thiamine auxotroph but our past work has shown that it can synthesize thiamine if provided with the precursor molecule 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP). Partial thiamine auxotrophy is common in microalgae with important ramifications for global phytoplankton productivity as well as engineering applications of algae. While thiamine deficiency can greatly depress algae growth and lipid content, the detailed metabolic impacts of thiamine deficiency are not well understood. We used metabolomics to study the response to thiamine-limited and replete conditions in mixotrophic A. protothecoides. We also investigated the impacts of exogenous HMP addition and the use of spent medium from another green algae, C. sorokiniana, as a source of thiamine metabolites. This is the first study, to our knowledge, that addresses metabolic impacts of thiamine deficiency and alleviation in green microalgae. Thiamine deficient cultures exhibited accumulation of pyruvate and α-ketoglutarate, indicating bottlenecks at the pyruvate dehydrogenase (PDH) and oxoglutarate dehydrogenase (OGDH) complexes. Both PDH and OGDH require thiamine pyrophosphate (TPP) as a cofactor. Transketolase also requires TPP but we only observed build-up of ribose-5-phosphate when glucose was supplied as a substrate. As expected, thiamine and HMP addition could alleviate these metabolic bottlenecks while greatly increasing algal growth, neutral lipid and starch content. Spent medium from C. sorokiniana only appeared to partially alleviate thiamine deficiency and resulted in build-up of isocitrate and glycolate, metabolites that appeared relatively unaffected by the presence or absence of thiamine. Interestingly, longer culture time of C. sorokiniana when preparing the spent medium led to much higher availability of thiamine metabolites. Thus, under the right conditions, it may be possible to co-culture mutually beneficial algae species and/or recycle spent cultivation medium to overcome auxotrophy in algae.

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Microplate assay for quantitation of neutral lipids in extracts from microalgae

Cited by 34 publications

References 35 publications

Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides

Acetate-detoxification of wood hydrolysates with alkali tolerant Bacillus sp. as a strategy to enhance the lipid production from Rhodosporidium toruloides

Co‐culturing Chlorella minutissima with Escherichia coli can increase neutral lipid production and improve biodiesel quality

Impact of thiamine metabolites and spent medium from Chlorella sorokiniana on metabolism in the green algae Auxenochlorella prototheciodes

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