Strains and Culture Conditions

The high H₂ producing C. reinhardtii strain Stm6Glc4 described in [30] and Stm6Glc4L01 were cultivated under mixotrophic (TAP (pH 7.2) [33] and photoautotrophic conditions (PCM (Photoautotrophic Chlamydomonas Medium) (pH 7, NH₄Cl [30 mM], CaCl₂*2H₂0 [850 µM], MgSO₄*3H₂0 [1.5 mM], KH₂PO₄ [10 mM], FeSO₄*7H₂O [1 µM], CuSO₄*5H₂O [6.4 µM], MnCl₂*4H₂O [25.8 µM], ZnSO₄*7H₂O [77 µM], H₃BO₃ [184 µM], (NH₄)₆Mo₇O₂₄*4H₂O [0.89 µM], CoCl₂*6H₂O [6.7 µM], Na₂SeO₃ [0.1 µM], VOSO₄*H₂O [0.009 µM], Na₂SiO₃*5H₂O [273 µM], Na₂EDTA [537.3 µM], Tris [100 mM], Vitamin B1 [52 µM], Vitamin B12 [0.1 µM]) under continuous white light (50 µE m⁻² s⁻¹), unless stated otherwise. Positive transformants were selected and cultivated under low light conditions (10 µE m⁻² s⁻¹) on TAP agar plates supplemented with 1.5 mM tryptophan, 60 µM 5-fluoroindole (5-FI) and hygromycin [30].

Growth Rate µ_max

To identify conditions yielding maximum microalgae growth rates in TAP and PCM, a number of factors were tested including light intensity (low light: 35 µE m⁻² s⁻¹ and high light: 450 µE m⁻² s⁻¹), culture volume (100, 112.5, 125, 137.5 and 150 µL trials corresponding to a culture depth of 2.7, 3, 3.5, 4 and 5 mm, respectively) and inoculation density (OD₇₅₀ = 0.1 and 0.3). These experiments were conducted in a specially designed robotic Tecan Freedom Evo system which was fitted with three microwell shakers each capable of holding six 96 well microwell plates, a bank of fluorescent lights positioned 1.2 m from the microwell surface and a CO₂ controller which maintained CO₂ concentrations at 1±0.2% CO₂ during photoautotrophic conditions. The lights consist of 12 cool white fluorescent lights (Phillips PL-L55W/840 Cool White, Phillips International B.V. Netherland) and 11 warm white fluorescent lights (Phillips PL-L55W/830 Warm White, Phillips International B.V. Netherland) which were installed in an alternating arrangement. The chamber temperature was 33±1°C. Algal cultures were adjusted to the desired OD₇₅₀ (OD₇₅₀ = 0.1 and 0.3, respectively) in TAP or PCM (supplemented with 1.5 mM tryptophan and 60 µM 5-FI for Stm6Glc5L01) and aliquoted into microwell plates. OD₇₅₀ measurements were recorded at 3 h intervals using a dedicated spectrophotometer (Tecan Infinite M200 PRO microwell plate reader). Growth curves were generated using Microsoft Excel and Graph Pad Prism software was used for growth curve fitting. An R² threshold of 0.85 was used to exclude false positives. The maximum value of the specific growth rate (µ_max) was determined using a linear fit to ln OD₇₅₀ vs. time, based on equation 1: µ = ln (ΔOD₇₅₀)/Δt.

To exclude that the chosen 5-FI concentration of 60 µM impaired growth of the mutant strain, growth rate was monitored as described above in 150 µL TAP supplemented with tryptophan and different 5-FI concentrations (0, 30, 60, 90, 120, 240, 360, 480 µM) at 100 µE m⁻² s⁻¹.

Biomass Determination

To determine direct biomass yields, Stm6Glc4 and Stm6Glc4L01 (grown in TAP medium) were adjusted to OD₇₅₀ of 0.3 in 144 mL PCM (OD₇₅₀ measurements were conducted using Tecan Freedom Evo system and a 96 well plate filled with 150 µL culture). Six 6 well microwell plates (total of 36 wells) for each strain were filled with 4 mL of adjusted cell suspension to a depth of 5 mm and incubated at a 1% CO₂ atmosphere for 43 h with 450 µE m⁻² s⁻¹ illumination. OD₇₅₀ was measured at the starting point and at 19 h and 43 h in the 6 well plates. After 43 h samples were harvested, pooled and washed in water to remove salt residues. Cells were dried at 60°C until no further mass change could be observed and obtained dry mass was calculated as g per Liter culture.

Sequence Studies

All genome sequences were obtained from NCBI (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/, accessed March 2013) and analyzed using the BLAST tool provided by the Chlamydomonas Center (http://www.chlamy.org/, accessed March 2013). The cross BLAST analysis of all mRNA sequences against each other and the identification of non-homologous parts of the sequence led to a map of unique gene regions of the LHCBM. Linker and sense sequence of the inverted repeat construct were picked from these regions; the anti-sense region was determined using the “reverse complement” function of Vector NTI and confirmed by BLAST alignment.

Plasmid Construction and Transformation

All nucleic acid work was conducted using standard procedures [34]. The LHCBM1 target sequence was synthesized based on a 95 bp sequence of LHCBM1 (bp 81–175), followed by an XbaI site. A subsequent 106 bp linker chosen from LHCBM1 3′UTR (bp 831–915) and the reverse orientated sequence of LHCBM1 (bp 81–190) completed the RNAi target sequence. The LHCBM2 target sequence was synthesized from two fragments. Fragment A consisted of a part of the LHCBM2 3′UTR (bp 1033–1132) followed by a SacI site. Fragment B contained the reverse orientated sequence to fragment A (bp 1033–1160) with subsequent EcoRI and SacI restriction sites. A linker region taken from LHCBM2 (bp 11–103) with a preceding SacI site and fragment B were amplified and joined via multiple template PCR. The linker and fragment B combination was cloned as a SacI fragment into SacI site downstream of fragment A to complete the inverted repeat sequence. The LHCBM3 target sequence was synthesized from bp 628–728 of the LHCBM3 5′UTR followed by a BamHI site, a linker of LHCBM3 5′UTR (bp 255–404) and the reverse orientated 5′UTR sequence (bp 255–404) was synthesized. Additional cloning details are provided in the supplementary materials (Figure S1).The inverted repeat sequences were flanked by EcoRI restriction sites to facilitate subsequent cloning into the transformation vector pBDH-R, and yielded vectors pMO52, pMO53 and pMO75, respectively. To create pBDH-R the Ribulose-1,5-bisphosphate-carboxylase/−oxygenase small subunit (RBCS) promoter, the intron fragment (with the start codon deleted) of RBCS was produced via fusion PCR and cloned into the XhoI and MluI site of pALKK1 [27] to replace its RBCS promoter/ATG/intron/BLE region. This deletes an undesired BLE gene fragment present in pALKK1. To produce the triple knock-down strains, equal amounts of each vector pMO52, pMO53 and pMO75 were mixed and transformed into the strain Stm6Glc4 [30] using biolistic bombardment [35].

RNA Isolation, cDNA Synthesis and Quantitative Real-time PCR (qRT-PCR)

Isolation of total RNA was performed using a PureLink™ RNA Mini kit (Invitrogen) following the protocol optimized for animal and plant cells according to manufacturer’s directions. As Stm6Glc4 is a cell wall deficient cell line the homogenization step was not carried out. DNase treatment was performed according to the PureLink™ RNA Mini kit manual using RQ1 RNase-free DNase (Promega). First strand cDNA synthesis was performed using iScript™ cDNA Synthesis kit (Bio-Rad Laboratories) and 1 µg total RNA according to manufacturer’s instructions. Quantitative real-time PCR analysis was carried out in triplicate using an Applied Biosystem 7500 Real Time PCR System with software SDS version 1.2.3 and the standard method ‘absolute quantification’ two step RT-PCR for thermal profile and dissociation stage (stage 1∶1 replication at 50°C, 1 min; stage 2∶1 replication at 95°C, 10 min; stage 3∶40 replications at 95°C, 15 sec; stage 4: dissociation; 1 replication at 95°C, 0.15 sec; 60°C, 1 min; 95°C, 0.15 sec). The experiment was carried out on a 96 well plate and each reaction contained 7.5 µl SYBR® Green Master Mix (Applied Bioscience), 5 µl of cDNA [1.6 ng µL⁻¹], 2 µL of each primer [2 µM] and 3.5 µL of water. Relative expression levels were normalized using the C. reinhardtii reference gene CBLP [36]. Primer sequences for LHCBM1, LHCBM2, LHCBM3 and CBLP were designed as reported previously [27], [36].

FACS Analysis and Chlorophyll Measurements

Cells were grown to late log phase in TAP liquid culture (OD₇₅₀ = ∼1.0), 1 mL of the culture was used for analysis in a FACSCanto II Flow Cytometer (Becton Dickson). Comparative chlorophyll fluorescence data was recorded using a 488 nm blue excitation laser and the 670 nm long pass detector to measure emission.

Total chlorophyll concentration was measured according to previously reported methods [37]. 1 mL of algae culture was sampled at late log phase (OD₇₅₀ = ∼1.0). The cells were then pelleted (500×g, 5 min, 20°C) and the clear supernatant discarded before being resuspended in 1 mL of ice cold 80% acetone. After vortexing the sample, the precipitate was pelleted (14100×g, 4 min) before measuring the absorption (A) of the supernatant. The A₇₅₀ was set to zero and A₆₆₄ and A₆₄₇ measured subsequently. Chlorophyll content was calculated as previously described [37].

Demonstration of Algal Fluorescence at Different Depths

Stm6Glc4L01 and Stm6Glc4 were adjusted to same cell density (7.5*10⁵ cells mL⁻¹) and transferred to a 100 mL glass cylinder positioned on a Safe Imager™ blue-light transilluminator (Invitrogen) fitted with light emitting diodes producing a narrow emission peak centered at ∼470 nm. Chlorophyll fluorescence was visualized by an ‘amber’ (530 nm long path) filter (Invitrogen).

H₂ Volume and Gas Composition Measurements

For H₂ production Stm6Glc4L01 and Stm6Glc4 were grown to late log phase in TAP medium. Cells were harvested, washed twice with sulfur-free TAP medium and resuspended in sulfur-free TAP medium containing glucose [1 mM] [30] and adjusted to the same chlorophyll concentration (14.5 µg mL⁻¹). H₂ measurements were performed under continuous white light (450 µE m⁻² s⁻¹) using a custom-built PBR system with a dedicated gas collection tube mount [32]. The evolved gas was sampled using a gas-tight Hamilton syringe and injected at regular intervals into a gas chromatograph (Agilent Micro GC3000) fitted with a PlotU pre-column (3 m x 0.32 mm) and MolSieve 5APlot column (10 m x 0.32 mm). Argon (32.5 psi, pound per square inch) was used as the carrier gas and H₂, O₂ and N₂ concentrations were monitored.

Identification of LHCBM Target Sequences

To develop specific LHCBM knock-down strains, the identification of highly specific target sequences is essential. As all LHCBM genes display a high degree of sequence similarity [27], genomic BLAST analysis of the three target mRNAs, encoding the major light harvesting complex proteins of PSII (LHCBM1, 2 and 3), was performed. This analysis aimed to identify target regions that had minimal similarities to other genes and so prevent their co-suppression. Based on this analysis, discrete regions for each single LHCBM target (between 95–100 bp) were chosen for the development of RNAi constructs (sequences see Figure S1). To minimize non-specific RNAi effects further, the LHCBM1-, 2- and 3-specific RNAi constructs were engineered so that the linkers positioned between the sense and anti-sense regions of the construct were specific to the respective target genes. The plasmid pALKK1 [27] was used to create the transformation vector pBDH-R (Figure 1A), additionally providing an RNAi target site for the endogenous tryptophan synthase. This allowed direct selection of positive transformants on media plates containing 5-fluoroindole (5-FI), as tryptophan synthase converts 5-FI into the toxic tryptophan analogue 5-fluorotryptophan [38].

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Figure 1. Schematic map of the transformation vector pBDH-R, relative abundance of LHC mRNAs and phenotypic cell distinction.

(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, [38]). 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 LHCBM genes to minimize non-specific knock-down effects. EcoRI restriction sites used for cloning are marked. (B) mRNA levels of the three targeted LHCII genes (LHCBM1 to LHCBM3) were determined in triplicate via quantitative real-time PCR and normalized to CBLP mRNA [36]. Expression levels (LHCBM1∶20.6±0.27; LHCBM2∶81.2±0.037 and LHCBM3∶41.4±0.05) were displayed as a percentage of the expression level of the parental strain Stm6Glc4 (which was set to 100%). (C) Optical transmission microscopy of Stm6Glc4 (left panel) and Stm6Glc4L01 cells (right panel). (D) Chlorophyll autofluorescence image of Stm6Glc4 (left panel) and Stm6Glc4L01 cells (right panel) taken in an inverted fluorescence microscope (Nicon Ti-U) with identical settings.

https://doi.org/10.1371/journal.pone.0061375.g001

Development and Selection of Positive Transformants

Single LHCBM knock-out or knock-down transformants have not to date shown any marked phenotype in terms of chlorophyll concentration. This suggests that the knock-down of one LHCBM may be compensated for by the over expression of the remaining LHCBM proteins [39], [40]. Consequently a triple LHCBM1/2/3 knock-down strategy was tested. LHCBM1, 2 and 3 were selected as they are reported to be the most abundant LHCII associated light harvesting proteins and so would be expected to achieve the greatest amount of antenna reduction [19], [20]. To develop triple knock-down mutants of LHCBM1, LHCBM2 and LHCBM3, equal amounts of the target vectors were mixed and used to transform the high H₂ producing C. reinhardtii strain Stm6Glc4 [30] using biolistic bombardment [35]. Positive transformants were selected on agar plates containing 5-FI since, as based on the vector design, it was expected that transformants with knocked-down tryptophan synthase, and thus resistance to 5-FI, would co-suppress the desired LHC protein. This strategy enabled the testing of all possible combinations of LHCBM1, LHCBM2 and LHCBM3 knock-downs, with the low-antenna (light green) phenotype as the secondary selection criterion. Consistent with our hypothesis that the simultaneous knock-down of these major LHCs could result in a marked reduction in chlorophyll concentration, a number of light green LCHBM1/2/3 multiple knock-down transformants were identified, of which the mutant with the palest phenotype (designated Stm6Glc4L01 to signify light harvesting mutant combination 01) was chosen for further characterization.

LHC mRNAs and Phenotypic Characterization

Total mRNA levels of LHCBM1, LHCBM2 and LHCBM3 in Stm6Glc4L01 were determined using quantitative real-time PCR (qRT-PCR) to establish which of the target LHCBM genes were primarily affected. To confirm that equal amounts of cDNA were used in this experiment, data were compared with those of 18S RNA and the mRNA for endogenous C. reinhardtii gene CBLP (Chlamydomonas ß-subunit-like polypeptide), which was previously shown to be stably expressed throughout growth and H₂ production [36]. Data normalized to CBLP (Figure 1B) showed that LHCBM1 has been down-regulated to 20.6% ±0.27% and LHCBM3 to 41.4% ±0.05% of their original levels, respectively. The down-regulation of LHCBM2 was less dramatic (81.2% ±0.037%) but importantly, LHCBM2 levels did not increase to compensate for the knock-down of LHCBM1 and LHCBM3. As gene dosage compensation has been suggested to be responsible for the unaffected phenotype of single antenna protein knock-outs [39]–[41], it is possible that the RNAi construct targeting LHCBM2 functions primarily to limit its expression level, preventing such compensation. Data with absolute expression levels in addition to the fold-differences relative to CBLP (Figure 1B) are provided (Table S1). The similarity of expression levels for 18S and CBLP genes between Stm6Glc4 and Stm6Glc4L01 suggests that the reduction in LHCBM1 and -3 levels is both meaningful and significant.

Light micrographs of Stm6Glc4 and Stm6Glc4L01 recorded under identical conditions (Figure 1C) showed that Stm6Glc4L01 cells contained considerably lower chlorophyll levels than the Stm6Glc4 parental control, consistent with the observed down-regulation of LHCBM1, LHCBM2 and LHCBM3 mRNA (Figure 1B) in a manner similar to that previously reported for other antenna mutants [16], [26], [27]. Auto-fluorescence images (Figure 1D), support the optical microscopy results and confirm that Stm6Glc4L01 accumulates less chlorophyll per cell, as fluorescence levels are significantly lower than in Stm6Glc4. The increased chlorophyll a/b ratio (Mean: Stm6Glc4∶2.29±0.05 and Stm6Glc4L01∶2.62±0.09; P-value <0.0021 [37] is indicative of a specific reduction in the ratio of ChlB-rich LHC proteins compared with the ChlA-rich PSII core complexes [42] confirming LHC antenna size reduction.

Fluorescence Properties and Chlorophyll Measurements

Flow cytometry (Figure 2A) was employed to quantify chlorophyll reduction in Stm6Glc4L01 compared to the parental strain Stm6Glc4. For this purpose over 10⁵ cells (2.9×10⁵ cells for Stm6Glc4; 2.79×10⁵ cells for Stm6Glc4L01, grown under 50 µE m⁻² s⁻¹ illumination to late log phase) were analyzed and chlorophyll fluorescence compared (670 nm long pass filtered). The relative fluorescence of the Stm6Glc4 culture peaked at 13488 relative fluorescence units (RFU) cell⁻¹ compared to the 8750 RFU cell⁻¹ of Stm6Glc4L01. Mean fluorescence was measured for Stm6Glc4 at 15606 RFU cell⁻¹ and for Stm6Glc4L01 at 8951 RFU cell⁻¹ (where the gate was set as indicated by the red and black lines in Figure 2A and displayed in Figure 2B as percentage of Stm6Glc4L01 (100%). This suggests that the control cell line Stm6Glc4 exhibited ∼175% higher levels of fluorescence losses than those observed in Stm6Glc4L01 mutant (100%). Furthermore as the red fluorescence emission observed is due to the transition from the lower energy state of chlorophyll to the ground state, a concomitant heat loss is also be expected due to the transition from the higher to the lower energy state transition. Therefore Stm6Glc4L01 is expected to display less heat losses than parental strain Stm6Glc4.

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Figure 2. Chlorophyll fluorescence and chlorophyll yield in Stm6Glc4 vs. Stm6Glc4L01.

(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 Stm6Glc4L01 (100%). (C) Chlorophyll a/b ratio of Stm6Glc4 and Stm6Glc4L01. (D) Total chlorophyll content in microgram per milliliter culture. Chlorophyll measurements (C, D) show results of two independent experiments with 7 replicates in total.

https://doi.org/10.1371/journal.pone.0061375.g002

Though in practice, fluorescence measured by flow cytometry may not be strictly linear with respect to chlorophyll content [43], it indicates that Stm6Glc4L01 exhibits a significant reduction compared to the parent strain. As the mean cell diameter of the two strains was similar (Figure 1C) a reduction in cell size cannot explain the reduced mean fluorescence in Stm6Glc4L01. The reduction in fluorescence in Stm6Glc4LO1 is however consistent with the increased chlorophyll a/b ratio (Stm6Glc4∶2.29±0.05; Stm6Glc4L01∶2.62±0.09, Figure 2C) which is indicative of a reduction in the ratio of ChlB-rich LHC proteins compared with the ChlA-rich PSII core complexes [42] as well as the total reduction in chlorophyll concentration per cell (Figure 2D). The observation that the ChlA/B ratio has only increased from 2.29 to 2.62 is completely consistent with many independent observations including the following. The first is the fact that purified thylakoid membranes containing PSII-LHCII and PSI-LHCI typically have a ChlA/B ratio of about 1.9–2.3. The second is that purified PSII-core complexes from higher plants binding CP29, CP26 and CP24 (all of which bind ChlB), but which are almost completely devoid of LHCII and LHCI, have a ChlA/B ratio of ∼7 [44]. The fact that the ChlA/B ratio of Stm6Glc4L01 cells is closer to 2.3 than 7 is therefore completely consistent with the fact that in addition to the ChlA/B binding proteins CP26 and CP29, the Stm6Glc4L01 cells also contain the full complement of LHCA Chla/b proteins of which there are ∼9 different types, as well as LHCBM4-9 and residual levels of LHCBM1-3. Collectively this explains why the ChlA/B ratio of Stm6Glc4L01 cells is significantly higher than that of the control (2.29+/−0.05 vs 2.62+/−0.09) but closer to 2.3 (wt thylokoids) than 7 (purified PSII core complexes).

Furthermore a determination of the total chlorophyll content [µg mL⁻¹] showed that at the same cell number, total chlorophyll content was reduced by 50% in Stm6Glc4L01 compared to Stm6Glc4 (Figure 2D) (total chlorophyll content: Stm6Glc4∶13.65 µg mL⁻¹, Stm6Glc4L01∶6.77 µg mL⁻¹, P-value <0.0006.

Light Penetration

To measure whether the decrease in LHCBM protein expression level increased light penetration through a liquid culture column, thereby making more light available for cells deeper in the culture, auto-fluorescence of Stm6Glc4 and Stm6Glc4L01 at the same cell density was measured (Figure 3A). As expected, Stm6Glc4 cultures were a darker green color compared to those of Stm6Glc4L01, due to their larger LHC antenna. The cultures were then placed onto a blue light illuminator and chlorophyll auto-fluorescence imaged using an orange filter to block the blue background light. As illumination was provided from below, the height of the fluorescent column was indicative of the light penetration distance. The fluorescence throughout the Stm6Glc4L01 culture column illustrates that light penetrated ∼4 times further than in Stm6Glc4 at the same cell density (Figure 3B).

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Figure 3. Light penetration into algal cultures with equal cell concentrations.

Comparison of Stm6Glc4 and Stm6Glc4L01 algal cultures adjusted to the same cell number per mL in white light (A) and during illumination with blue light from below (B). Light penetration depth is indicated by white arrows. Stm6Glc4L01 shows ∼4× more light penetration compared to parental strain Stm6Glc4.

https://doi.org/10.1371/journal.pone.0061375.g003

Growth Rates Under Mixotrophic and Photoautotrophic Conditions

As light is both necessary and damaging for photosynthesis [45] as it is required to drive charge separation but in excess causes photoinhibition, the maximum rate of photochemistry is achieved at the point at which the photosystems are just saturated, assuming that light is the limiting factor. To minimize the area required for microalgae production and thereby also PBR costs, in theory systems should operate at the maximum flux density (high light) and the system poised at this optimal point by adjusting culture depth and cell density. Theoretically small antenna cell lines could allow more cells to be packed into a given unit volume before light becomes limiting and decrease both cell shading and photoinhibition. To test this hypothesis biomass production (as a proxy for chemical energy/biofuels) in form of maximum growth rates (µ_max) of Stm6Glc4 and Stm6Glc4L01 cultures were measured under a range of culture depths and cell densities.

The growth rate of Stm6Glc4 and Stm6Glc4L01 was next determined under mixotrophic conditions (TAP medium) which are the best-established conditions for photobiological H₂ production. Experiments were conducted in microwell plates using three different volumes (100 µL, 125 µL and 150 µL) to simulate different culture depths (average depths of 2.7, 3.5 and 5 mm, respectively). Since an advantage of Stm6Glc4L01 was expected primarily in high light conditions, growth was measured under 450 µE m⁻² s⁻¹ illumination (optimal light intensity for the parental control and wt strains is 50–100 µE m⁻² s⁻¹) with an initial inoculation OD₇₅₀ of 0.1. Under all conditions Stm6Glc4L01 showed a higher growth rate than Stm6Glc4 (Figure 4A). In the shortest path length cultures (100 µL = 2.7 mm) exposed to the highest light intensity (450 µE m⁻² s⁻¹) Stm6Glc4L01 showed ∼3 times the growth rate of Stm6Glc4 based on OD₇₅₀ measurements. The increased growth rate could be due to reduced photoinhibition (due to the reduced LHC antenna size), improved light penetration and therefore increased total light-to-biomass conversion efficiency of the culture, or a combination of both factors. As the highest µ_max h⁻¹ values for Stm6Glc4L01 were observed in the shortest path length cultures (2.7 mm) and declined with increasing culture depth (Figure 4A) we concluded that path length and light distribution had a more dominant impact than photoinhibition. In contrast Stm6Glc4 showed no such trend. It was previously reported [31] that Stm6 (and hence Stm6Glc4) is light sensitive, demonstrated here by the poor growth displayed at very low cell densities, where no self-shading occurs at 450 µE m⁻² s⁻¹ (Figure 4A).

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Figure 4. Growth rate µ_max h⁻¹ of Stm6Glc4 and Stm6Glc4L01 at different cultivation conditions and biomass determination.

(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.

https://doi.org/10.1371/journal.pone.0061375.g004

To investigate the growth behavior of Stm6Glc4 and Stm6Glc4L01 under photoautotrophic conditions, we performed a parallel experiment (Figure 4B) to determine the effects of culture depth (100 µL = 2.7 mm, 112.5 µL = 3 mm, 125 µL = 3.5 mm, 137.5 µL = 4 mm and 150 µL = 5 mm) at low (35 µE m⁻² s⁻¹) and high light levels (450 µE m⁻² s⁻¹). Previous experiments in TP medium (TAP medium minus acetate) indicated that N and P were limiting and led to an early culture growth plateau. Consequently for this experiment, an improved in-house medium was developed (Photoautotrophic Chlamydomonas Medium, PCM) which supplied more nitrogen and phosphate, to ensure that these did not limit growth. All cultures were inoculated at a starting OD₇₅₀ of 0.3 to reduce light stress for Stm6Glc4 at 450 µE m⁻² s⁻¹.

At a culture depth of 2.7 mm and 450 µE m⁻² s⁻¹ illumination, Stm6Glc4L01 showed ∼120% of the maximum growth rate observed for the parental strain Stm6Glc4. Further increases in depth accentuated the advantage of the Stm6Glc4L01 phenotype, as the growth rate at 5 mm depth was >185% compared to parental strain Stm6Glc4. This result shows that a reduction in antenna size improved growth across the conditions tested but particularly in deeper and denser cultures. In contrast, under low light levels (35 µE m⁻² s⁻¹) growth rates of Stm6Glc4 and Stm6Glc4L01 were comparable, indicating that the reduced antenna did not disadvantage the cells.

To confirm the OD₇₅₀ measurements, direct biomass yields were also determined both for Stm6Glc4 and Stm6Glc4L01 grown in PCM under the best high light conditions tested (450 µE m⁻² s⁻¹, 5 mm culture depth, 1% CO₂ atmosphere). OD₇₅₀ was recorded at the starting point (0.113 for Stm6Glc4) and 0.169 forStm6Glc4L01) and at time intervals (19 h and 43 h) (Figure 4C). To produce sufficient biomass to allow accurately biomass determination, samples were harvested at 43 h (OD₇₅₀: Stm6Glc4∶0.692 g and Stm6Glc4L01∶0.803 g) and yielded 2.3 g L⁻¹ (Stm6Glc4) and 2.5 g L⁻¹ (Stm6Glc4L01) biomass dry weight, respectively. From this the biomass dry weight/OD_{750 nm} ratios of Stm6Glc4 and Stm6Glc4L01 were calculated to be 3.3 (2.3/0.692) and 3.1 (2.5/0.803) respectively. Based on these ratios, biomass yields at earlier time points were calculated Figure 4C. Although it appears initially that the differences in biomass yield at the 43 h time point are only approximately 10%, this was because the Stm6Glc4L01 growth rate had started to plateau presumably due to exhausted nutrient resources. In contrast at the point of maximum growth (19 h) biomass concentrations were by this method calculated to be 0.818 g L⁻¹ (Stm6Glc4) and 1.35 g L⁻¹ (Stm6Glc4L01). This represents a 165% increase for Stm6Glc4L01 over the control Stm6Glc4 level (100%).

Hydrogen Production

As photobiological H₂ production is the most efficient light-to-biofuel conversion strategy [46] the effect of specific LHC gene down-regulation in Stm6Glc4L01 on H₂ generation was investigated. For this purpose a time course measuring H₂ production rates (mL H₂ L⁻¹ h⁻¹) in Stm6Glc4 and Stm6Glc4L01 cultures, adjusted to the same chlorophyll concentration (14.5 µg mL⁻¹), was carried out in sulfur deprived media to establish anaerobiosis (Figure 5). At constant chlorophyll concentration and identical volumes, the cell number of Stm6Glc4L01 was approximately double the cell number of Stm6Glc4 (Stm6Glc4∶1×10⁷ cells mL⁻¹; Stm6Glc4L01∶2×10⁷ cells mL⁻¹). H₂ production in the Stm6Glc4L01 trials started almost immediately after transfer into sulfur deprived media in marked contrast to the ∼22 h lag time of the Stm6Glc4 control (Figure 5A). This suggests that intracellular O₂ concentrations were lower in Stm6Glc4L01 than in Stm6Glc4 and close to or below the induction threshold for HYDA expression. The reduced lag phase has marked operational benefits, and significantly increases the yield. The H₂ production rates of both strains peaked at 47 h after sulfur deprivation, with Stm6Glc4L01 producing 6.25 mL L⁻¹ h⁻¹ compared to 3.31 mL L⁻¹ h⁻¹ for Stm6Glc4 under the conditions tested. Both strains produced H₂ until 188 h after sulfur deprivation with a similar decline in both strains after peak production. Under the conditions tested Stm6Glc4L01 produced a total volume of 361 mL ±27.6 mL H₂ per liter culture compared to 198 mL ±21.2 mL H₂ per liter culture produced by Stm6Glc4. Stm6Glc4L01 therefore produced >180% more H₂ per unit chlorophyll than the Stm6Glc4 control (Figure 5B), which via gas chromatography was determined to be >95% pure. These results clearly show that Stm6Glc4L01 exhibited improved characteristics in terms of early onset and higher rates of H₂ production.

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Figure 5. H₂ production of Stm6Glc4L01 and Stm6Glc4.

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.

https://doi.org/10.1371/journal.pone.0061375.g005

Photosynthetic Efficiency

Light capture is the first step of all microalgae-based fuel production and its optimization is therefore of fundamental importance for the development of high-efficiency processes. Wild type microalgae antennae have evolved intricate mechanisms to adapt to natural fluctuations in light levels and quality to maximize light capture under low light conditions and dissipate excess light under high light conditions. While algae possess the ability to down-regulate their antenna, even 25% of full sunlight is sufficient to saturate the photosynthetic capacity of most algal species [17]. Under such over-saturated light conditions for a well-mixed culture in an open raceway or closed PBR, energy losses of up to 80% can occur [47]. This is because cells near the illuminated surface dissipate excess solar energy via NPQ which is then no longer available to cells deeper in the culture and which are therefore light-limited. Consequently, while these mechanisms are finely tuned for adaptation to their natural environment, they are far from optimized for industrial microalgae production, which to minimize area requirement and photobioreactor (PBR) construction costs should ideally be established for high flux density conditions. Photobioreactors can be designed to achieve light dilution (e.g. to 10% of 2000 µE m⁻² s⁻¹), but due to increased complexity and material costs this increases photobioreactor expense. Antenna engineering has the advantage that it can theoretically improve production efficiency and reduce capital costs both of which are major economic drivers for this technology [48].

Given the large number of LHC proteins in eukaryotic photosynthetic organisms, a diversity of function among family members is implied. The generation of Stm3LR3 [27] involved knocking-down all LHC classes and resulted in a low chlorophyll phenotype. It was however recognized during this initial antenna engineering step that some vital LHC functions might also be affected. This assumption was borne out by subsequent work [49] and the observation that Stm3LR3 is moderately light-sensitive at low cell densities. For example, CP29 (LHCB4) is essential for state transitions and for docking the LHCBM1, −2 and −3 trimers to PSI [49], while a role for CP26 (LHCB5) in preventing photoinhibition has also been described [13], [15], [49]–[51]. It is therefore likely that the pattern of LHC down-regulation displayed in Stm3LR3 does not preserve all of the functions required to support growth at high light levels [40].

While the high degree of homology in LHCs reflects a common ancestry, it also suggests that specific functions likely reside in the small non-conserved regions. Consequently, precise engineering of specific LHC proteins is important to avoid undesired effects due to co-suppression of other genes. As PSII is the main site of light-induced damage, there is a strong correlation between PSII antenna size and photosynthetic productivity [47], [52]–[54]. This supports the idea that the most abundant LHCII antenna proteins, LHCBM1, −2 and −3, are the most important targets for down-regulation. In this context it is of note that while the exact molecular mechanisms for photodamage of PSII are still under investigation (for review see [54]), it has been shown that reduced chlorophyll content plays a crucial role in preventing photodamage, and thus allows the cells to grow under higher light conditions [55], supporting our findings. The above experiments show that LHCBM1 (20.6% ±0.27%), LHCBM2 (81.2% ±0.037%) and LHCBM3 (41.4% ±0.05%) expression levels were effectively knocked-down (Figure 1B), resulting in the light green mutant Stm6Glc4L01 (Figure 1C), which had a reduced total chlorophyll content (50% of parental strain (Figure 2D)) and an increased ChlA/B ratio (Stm6Glc4∶2.29±0.05 vs. Stm6Glc4L01∶2.62±0.09) (Figure 2C), confirming LHCII depletion. Down-regulation of the LHCII antenna system was further supported by chlorophyll auto-fluorescence measurements obtained by flow cytometry (Figure 2A and B). Due to its reduced antenna size, cultures of Stm6Glc4L01 exhibited considerably improved light penetration (Figure 3B) over those of the control (Stm6Glc4) at the same cell density. Our data also indicates that the improved light penetration is in part due to reduced fluorescence and heat losses at the illuminated culture surface (Figure 2B) and in part due to the reduced chlorophyll content of each cell (Figure 2D) which improves light transmission through the culture (Figure 3B). Together these properties resulted in an increased fraction of cells in the culture being poised close to the optimum point of light saturation (achieving maximal rates of photochemistry, without causing photo-inhibition), rather than within the broad range of illumination levels typically experienced by cells in the Stm6Glc4 control culture. This improved poise was achieved at light levels of 450 µE m⁻² s⁻¹ instead of the more typical level of 100 µE m⁻² s⁻¹ at which Stm6Glc4 antenna cell line is saturated and led to growth rates of >185% of Stm6Glc4 under photoautotrophic conditions (Figure 4B). The importance of this is that under photoautotrophic conditions required for both biomass and ideally commercial scale H₂ production, at outside light levels (e.g. 2000 µE m⁻² s⁻¹), the light dilution factor could be reduced from 2000/100 = 20 to 2000/450 = ∼4.4 [52], offering the potential for significant savings in photobioreactor costs.

Molecular Mechanism of Improved H₂ Production

The results presented are summarized with the following mechanistic model (Figure 6). In wt cells, Stm6 and Stm6Glc4, light used by PSII is predominantly captured by LHCBM trimers bound to the PSII core complex. The major LHCII proteins are reportedly transcribed from 9 different genes LHCBM 1–9 which are numbered according to the relative expression levels initially observed (16, 17). These LHCBM proteins are reported to trimerize and their primary role is to capture solar energy and funnel it via the minor LHCII proteins, CP29 (LHCB4) and CP26 (LHCB5), to the PSII core (CP47, CP43, D1, D2, cytb559, PSBO and additional small and extrinsic subunits) (Figure 6). Down regulating LHCBM1-3 which are the most abundant of these, as expected, resulted in a marked reduction in antenna size. It should be noted that LHCBM1 has also been reported be to be involved in NPQ as well as the scavenging of radical oxygen species [40]. While the down regulation of these functions might be expected to have a detrimental effect at high light levels, our results indicate improved biomass and H₂ yields at 450 μΕ m⁻² s⁻¹ (>100 µE m⁻² s⁻¹ can saturate Stm6 and Stm6Glc4). This can be explained by the small antenna size of Stm6Glc4L01 resulting in lower levels of over excitation of PSII. This in turn is expected to result in the reduction of the formation of radical oxygen species and the requirement for the dissipation of excess energy via NPQ. Consequently the down regulation of the NPQ and ROS scavenging functions through the knock-down of LHCBM1 appears to be compensated for by the reduction of antenna size.

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Figure 6. Mechanistic model of improved H₂ production in Stm6Glc4L01.

(A) Stm6Glc4 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: Stm6Glc4L01 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.

https://doi.org/10.1371/journal.pone.0061375.g006

The light captured by the PSII antenna system drives the PSII-mediated oxidation of water to H⁺, e^- and O₂ (Figure 6A) [32]. Electron transport subsequently progresses via plastoquinone (PQ), cytochrome b₆f, plastocyanin to PSI where electrons are again energized by light, to reduce ferredoxin (Fd). Ferredoxin nucleotide reductase (FNR) couples the reoxidation of Fd to the reduction of NADP⁺ which under normal aerobic conditions is then used during carbon fixation to reduce CO₂ to sugars. During this process, protons are translocated to the lumen establishing the proton gradient which drives ATP production. Apart from water, products derived from starch metabolism can be used to provide electrons to PQ via the NADPH dehydrogenase (NDA2) [56]. Under illuminated anaerobic/micro-oxic conditions the derived H⁺ and e^- can also act as substrates for HYDA-mediated H₂ production. It is of note however that up to 80% of the H⁺ and e^- used by HYDA are reportedly produced by PSII [57], providing a direct pathway for the solar driven H₂ production from water.

H₂ production requires both illumination (to drive photosynthesis) and anaerobic conditions (to induce the expression of HYDA and prevent HYDA inhibition) [10]. Under anaerobic conditions, mitochondrial respiration largely ceases as the cell is deprived of O₂. The expression of HYDA however provides an alternative H⁺/e^- release mechanism which recombines H⁺ and e^- to produce H₂ gas that is excreted from the cell (i.e. instead of H₂O from mitochondria). This allows photosynthetic electron transport to continue to operate, providing an alternative pathway for the production of ATP and NADPH to enable cell survival (11).

In the wt, only small amounts of H₂ are typically produced as the large antenna phenotype increases oxygen production in illuminated cells, which is not removed quickly enough by wt respiration levels and so leads to a reduction in HYDA transcription and function. Typically, anaerobiosis can be established in wt algae cultures through sulfur depletion. This is because the Methionine containing D1 protein of PSII, which is rapidly photo-damaged under high light conditions, can only be repaired in the presence of sulfur. Consequently, by limiting sulfur, PSII-mediated O₂ evolution is gradually reduced over time. When it drops below the rate of O₂ uptake by mitochondrial respiration, the system becomes micro-oxic or anaerobic and induces HYDA expression [58].

The parental strains of Stm6Glc4L01 are moc1 mutants (i.e. Stm6 and Stm6Glc4 [30]–[32]). Both have an increased respiration rate due to upregulated AOX activity (high O₂ consumption), but also have a large wt like PSII antenna system (Figure 6A). As a result of their increased rate of respiration Stm6 and Stm6Glc4 exhibit a higher rate of oxygen consumption than the wt and so enter anaerobiosis faster under sulfur deprivation. This in turn results in earlier activation of HYDA expression [59] compared to the wt leading to the rapid onset of H₂ production. However the relatively large antenna systems of Stm6 and Stm6Glc4 still supports rates of O₂ production which exceed the respiration rate and so sulfur depletion is required to induce H₂ production.

In contrast in Stm6Glc4L01 the LHCBM1, LHCBM2 and LHCBM3 have been down-regulated within an upregulated AOX background (Figure 6B). The importance of this is that the simultaneous reduction of O₂ production and increased O₂ consumption is thought to reduce intracellular O₂ levels below the induction threshold for HYDA. This hypothesis is supported by three observations. First, Stm6Glc4L01 cultures showed an almost immediate onset of H₂ production upon sulfur deprivation (Figure5A) indicating that the intracellular O₂ levels in sulfur replete medium are already poised closed to HYDA threshold level. Second, during the early stages of H₂ production the contaminating O₂ concentrations in the gas produced by Stm6Glc4LO1 cultures were considerably lower than those produced by Stm6Glc4. Third, the rate of H₂ production by Stm6Glc4L01 was almost twice as high as that observed for Stm6Glc4 (Figure 5B) for a given culture volume adjusted to the same chlorophyll concentration, for reasons described above.

The finding that the Stm6Glc4L01 is so closely poised to HYDA expression opens up the exciting possibility of using this mutant for continuous H₂ production in sulfur replete medium. To date, to our knowledge continuous H₂ production in sulfur replete liquid medium has not been reported, presumably because under these conditions O₂ production usually exceeds O₂ consumption inhibition HYDA expression. For example the strains Stm3LR3 [27], Stm6Glc4T7 [26] and TLA1 [16], [60] have not been reported to increase H₂ yield in liquid culture which would be desirable for the development of continuous H₂ production in scale PBR systems. However, important steps along this development path include the use of sulfur microdosing approaches [61] and the use of alginate solid phase systems which reduced O₂ inhibition [60].

In summary, Stm6Glc4L01 exhibited an improved H₂ and biomass production efficiency (165–180% improvement over parental strain). Importantly, this was achieved at increased solar flux densities (450 µE m⁻² s⁻¹) and high cell densities which are best suited for microalgae production as light should ideally be the limiting factor. Our data suggests that the overall improved photon to H₂ conversion efficiency is due to: 1) reduced loss of absorbed energy by non-photochemical quenching (fluorescence and heat losses) near the photobioreactor surface; 2) improved light distribution in the reactor; 3) reduced photoinhibition; 4) early onset of HYDA expression, 5) reduction of O₂ induced inhibition of HYDA. The Stm6Glc4L01 phenotype therefore provides important insights for the development of high efficiency photobiological H₂ production systems.