Schematic illustration of the chloroplast vector system.

<p>Destination vector and entry vector are recombined through L-R reaction yielding the final chloroplast transformation vector. Destination vector: Chloroplast homologous sequences (plastome DNA) with ribosomal (<i>rrn5</i>) and <i>psbA</i> (<i>psbA</i>) genes are indicated. <i>attR</i> sites (<i>aatR1</i>, <i>attR2</i>) supporting L-R reaction are indicated. Entry vector: <i>attL</i> sites (<i>attL1</i>, <i>attL2</i>) participating in L-R reaction are indicated as well as the expression cassette of the selection marker <i>aadA</i> and the <i>gfp</i> expression cassette. Important restriction sites used for cloning are indicated as well as the recombination reaction performed between the <i>attR</i> and <i>attL</i> sites (<i>L-R reaction</i>). Transformation vector: Schematic illustration of final transformation vector resulting from L-R reaction between destination and entry vector.</p

GFP fluorescence detection via Native PAGE.

<p>To identify GFP expressing mutants 1(wt), mutant 1 (mut1) and mutant 2 (mut2) algae were loaded and intrinsic GFP fluorescence was used for detection. 10, 5, 2 and 1 ng of GFP protein were used as standard.</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

Chlorophyll fluorescence and chlorophyll yield in <i>Stm6Glc4</i> vs. <i>Stm6Glc4L01</i>.

<p>(A) Graph derived from flow cytometry analysis showing relative fluorescence units (RFU) per cell. Over 2.7*10⁵ cells were analyzed for each cell line. RFU range used to determine the mean is indicated by the red and black bars. (B) Mean chlorophyll fluorescence in percentage normalized to <i>Stm6Glc4L01</i> (100%). (C) Chlorophyll a/b ratio of <i>Stm6Glc4</i> and <i>Stm6Glc4L01.</i> (D) Total chlorophyll content in microgram per milliliter culture. Chlorophyll measurements (C, D) show results of two independent experiments with 7 replicates in total.</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