MOESM1 of Time-resolved transcriptome analysis and lipid pathway reconstruction of the oleaginous green microalga Monoraphidium neglectum reveal a model for triacylglycerol and lipid hyperaccumulation

Additional file 1. Additional Methods, Results, Tables ST1–ST3 and Figures S1–S17

MOESM1 of Highly efficient methane generation from untreated microalgae biomass

Additional file 1: Figure S1. Methane concentration in the biogas, produced during the fermentation of replete-N and low-N algae biomass (replete-N BM and low-N BM, respectlively). Statistics: two-sample t-test with 95% confidence interval. Figure S2. Concentration of total carbon and nitrogen during the experimental time course in replete-N BM digester (A) and low-N BM digester (B). Concentration of total organic and inorganic carbon (TOC and TIC) is shown for replete-N BM digester (C) and low-N BM digester (D). Measurements were performed in three replicates; error bars represent standard deviation (SD). Figure S3. Concentration of volatile and total solids (VS and TS, respectively) during the experimental time course in replete-N BM digester (A) and low-N BM digester (B). Measurements were performed in at least three replicates; error bars represent standard deviation (SD). Figure S4. Concentration of chemical oxygen demand (COD) during the experimental time course in replete-N BM digester (A) and low-N BM digester (B). Measurements were performed in three technical replicates; error bars represent standard deviation (SD). Figure S5. Bacterial diversity dynamics as assessed by high-throughput 16S rRNA amplicon sequencing and represented at the OTU level. The reactors, fed with biomass cultivated in media with replete and low nitrogen content (replete-N BM and low-N BM) were exposed to increasing organic loading rates of 2 and 4 g VS L-1 d-1 (OLR 2 and OLR 4, respectively). The inoculum and the sampling periods at the end of each OLR were chosen for microbial community monitoring. Table S1. Analysis of the volatile fatty acid (VFA) content during the time course of the experiment. The identification and quantification of the intermediate fermentation products (mM) was determined via GC-FID. The indicated error (Âą) represents standard deviation (SD, n = 2). Table S2. Filtered sequences during amplicon processing. OTU=operational taxonomic unit, N=nitrogen, sd=standard deviation, OLR=organic loading rate, rep=replicate

Overview of the whole culture biomass composition of <i>B</i>. <i>braunii</i> CCAP807/2 during the proposed growth stages.

<p>Overview of the whole culture biomass composition of <i>B</i>. <i>braunii</i> CCAP807/2 during the proposed growth stages.</p

The overall intracellular metabolome profile of <i>Botryococcus braunii</i> CCAP 807/2, containing all identified metabolites during the proposed growth stages, referred as Phases II (linear phase), III (stationary phase) and IV (decline phase).

<p><b>a.</b> Non-targeted metabolome profile of primary metabolites showing the comparison of relative abundance level of metabolites, divided into three different categories based on the related metabolic pathways, thus (i) glycolysis intermediates, sugars and sugar alcohols, (ii) amino acids and other related metabolites and (iii) citric acid cycle intermediates, terpenoids, steroids and vitamins. <b>b.</b> Intracellular pigments with relative abundances of (i) chlorophylls and (ii) carotenoids. <b>c.</b> Gravimetrically determined total lipid content, containing polar (P lipids) and non-polar lipid (N-P lipids) and expressed as percentage of dry biomass weight. <b>d.</b> Polar lipid fraction with relative abundance level of fatty acids. <b>e.</b> Non-polar lipid fraction with relative abundance levels of (i) fatty acid and (ii) hydrocarbons. <b>f.</b> Comparison of hydrocarbons and fatty acids derived from the total non-polar lipid fraction on the basis of the relative abundance levels, considering Phase II as 100%. Metabolites were identified by ‘<i>a</i>’ comparison with the NIST 05 library and Golm Metabolome Database (Lib) and verified with purified standards; ‘<i>b</i>’ only via above mentioned databases with RSI value above 750. ‘<i>c</i>’ marks the identified hydrocarbons via mass spectra of GC-MS and available literature [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198976#pone.0198976.ref010" target="_blank">10</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0198976#pone.0198976.ref017" target="_blank">17</a>]; ‘#’ not detected. Error bars represent standard deviation (SD). Asterisks represent <i>p-values</i> as determined via Student’s t-test (* = < 0.05, ** = < 0.01).</p

Metabolic survey of <i>Botryococcus braunii</i>: Impact of the physiological state on product formation

<div><p>The microalga <i>Botryococcus braunii</i> is widely regarded as a potential renewable and sustainable source for industrial applications because of its capability to produce large amounts of metabolically expensive (exo-) polysaccharides and lipids, notably hydrocarbons. A comprehensive and systematic metabolic characterization of the <i>Botryococcus braunii</i> race A strain CCAP 807/2 was conducted within the present study, including the detailed analysis of growth-associated and physiological parameters. In addition, the intracellular metabolome was profiled for the first time and showed growth- and product-specific fluctuations in response to the different availability of medium resources during the cultivation course. Among the identified metabolites, a constant expression of raffinose was observed for the first time under standard conditions, which has until now only been described for higher plants. Overall, the multilayered analysis during the cultivation of strain CCAP 807/2 allowed the differentiation of four distinct physiological growth phases and revealed differences in the production profiles and content of liquid hydrocarbons and carbohydrates with up to 84% of organic dry weight (oDW). In the process, an enhanced production of carbohydrates with up to 63% of oDW (1.36±0.03 g L^-1) could be observed during the late linear growth phase, whereas the highest accumulation of extracellular hydrocarbons with up to 24% of oDW (0.66±0.12 g L^-1) occurred mainly during the stationary growth phase. Altogether, the knowledge obtained is potentially useful for the general understanding of the overall physiology of <i>Botryococcus braunii</i> and provide important insights into the growth behavior and product formation of this microalga, and is thus relevant for large scale biofuel production and industrial applications.</p></div

Extracellular product formation of <i>Botryococcus braunii</i> CCAP 807/2 in form of carbohydrates and hydrocarbons during the proposed growth stages referred as Phases I (lag phase), II (linear phase), III (stationary phase) and IV (decline phase).

<p><b>a.</b> Determination of organic dry weight (oDW) and C/N ratio of the whole culture broth (containing cells and supernatant) over the period of cultivation for 30 days. <b>b.</b> Determination of total carbohydrate concentration in the whole culture broth and the cell-free supernatant. <b>c.</b> Quantification of total extractable hydrocarbons via GC-FID at each time point during cultivation (except for day 12 –lost samples (##)). Error bars represent standard error (SE; n = 9 for <b>a</b>, n = 12 for <b>b</b>) and standard deviation (SD) for <b>c</b>.</p

Growth rate µ_max h⁻¹ of <i>Stm6Glc4</i> and <i>Stm6Glc4L01</i> at different cultivation conditions and biomass determination.

<p>(A) Growth rate at 450 µE m⁻² s⁻¹ under mixotrophic conditions at different culture depths using start OD₇₅₀∶0.1. (B) Growth rate at 450 µE m⁻² s⁻¹ and 35 µE m⁻² s⁻¹under photoautotrophic conditions at different culture depths using start OD₇₅₀∶0.3. Experimental data were compiled using triplicates. (C) OD₇₅₀ and biomass determination in g L⁻¹ under photoautotrophic conditions.</p

Schematic map of the transformation vector <i>pBDH-R</i>, relative abundance of LHC mRNAs and phenotypic cell distinction.

<p>(A) The RBCS promoter (Prbcs) with subsequent RBCS intron (rbcs int) and 35S terminator (T35S) flanking the RNAi expression cassette are marked. Sequences targeting the tryptophan synthase are indicated (Maa7 IR, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061375#pone.0061375-Rohr1" target="_blank">[38]</a>). Inverted repeat (IR) sequences used to target LHC genes (Lhcbm IR) and linker (Linker), which spaces the inverted repeats, are located in between two Maa7 inverted repeats (Maa7 IR). In this study ‘Lhcbm IR’ and ‘Linker’ were replaced with the sequences from the target <i>LHCBM</i> genes to minimize non-specific knock-down effects. <i>EcoR</i>I restriction sites used for cloning are marked. (B) mRNA levels of the three targeted LHCII genes (<i>LHCBM1</i> to <i>LHCBM3</i>) were determined in triplicate via quantitative real-time PCR and normalized to <i>CBLP</i> mRNA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061375#pone.0061375-Mus1" target="_blank">[36]</a>. Expression levels (<i>LHCBM1</i>∶20.6±0.27; <i>LHCBM2</i>∶81.2±0.037 and <i>LHCBM3</i>∶41.4±0.05) were displayed as a percentage of the expression level of the parental strain <i>Stm6Glc4</i> (which was set to 100%). (C) Optical transmission microscopy of <i>Stm6Glc4</i> (left panel) and <i>Stm6Glc4L01</i> cells (right panel). (D) Chlorophyll autofluorescence image of <i>Stm6Glc4</i> (left panel) and <i>Stm6Glc4L01</i> cells (right panel) taken in an inverted fluorescence microscope (Nicon Ti-U) with identical settings.</p

Mechanistic model of improved H₂ production in <i>Stm6Glc4L01</i>.

<p>(A) <i>Stm6Glc4</i> has a large PSII antenna system consisting of LHCBM1-9. LHCBM1-3 are reported to be most abundant. Large antenna size results in increased PSII mediated O₂ production and NPQ losses. NPQ losses reduce system efficiency; intracellular O₂ levels inhibit expression of HYDA until the system is sulfur deprived (sulfur required for the repair of the PSII-D1 subunit. <b>B: </b><i>Stm6Glc4L01</i> has a reduced antenna size which is figuratively shown, and leads to reduced O₂ production and early onset of H₂ production. The light green phenotype allows higher cell densities to be used leading to increased rates of H₂ production.</p

H₂ production of <i>Stm6Glc4L01</i> and <i>Stm6Glc4</i>.

<p>H₂ production rate in mL h⁻¹ L⁻¹ algae culture (A) and total H₂ production in mL L⁻¹ culture were determined (B). Experiments were performed under sulfur deprivation and with cultures adjusted to same chlorophyll content. Data were compiled using 3 replicates.</p