Generation and characterization of reduced PSII antenna size mutants of Chlorella sorokiniana for improved biomass

Biofuel production from algal biomass is a fundamental component in developing sustainable energy sources that can replace fossil fuels. However, cost effectiveness needs to be taken into account as there is substantial difference between the higher cost of biofuel production and relatively low cost of fossil fuels. Studies on Chlorella species have focussed on improving algal biomass production capacity. One of the critical problems is inefficient use of light caused by its unequal distribution. The current study describes the development of photosynthetic Chlorella sorokiniana mutants by EMS mutagenesis. Mutagenesis and visual phenotypic selection procedures were applied and three C. sorokiniana chlorophyll mutants (CSCM) have been identified. The selected CSCM8, CSCM10 and CSCM21 mutant strains show diverse phenotypes with 33–47% reduced chlorophyll content. Further characterization reveal that these selected mutants had 23–44% reduced antenna size, improved effective quantum yield of PSII [Y(II)], reduced regulated (light-activated) energy dissipation Y(NPQ), and reduced non-photochemical quenching (NPQ). Moreover, the characterised mutants in the laboratory have shown 19–34% and 13–29% increased biomass productivity under low light and high light conditions (50 and 250 µmol photons m−2 s−1), respectively. This study indicates that genetic modification of C. sorokiniana with smaller antenna size can improve biomass content; further, these mutants can be used for strain improvement having higher lipid content.


Introduction
When compared to other resources, biodiesel production using biological feedstock has numerous advantages (Hu et al. 2010).The production of biodiesel from microalgae biomass is one of the most promising solutions for substituting fossil fuels and reducing greenhouse gas emissions (Rodolfi et al. 2009).Algae have been identified as a promising feedstock for biofuel production because they multiply rapidly and accumulate a large amount of biomass, including lipids, within the cells.Photosynthetic microalgae have gained attention because they can efficiently absorb energy from sunlight, lowering biodiesel production costs compared to other resources (Chisti 2007;Schenk et al. 2008).Microalgal biomass production has advantages over plant biomass production, including improved productivity, possibility to use non-arable land, and nutrient content retrieval from wastewater, effective carbon sequestration, and faster development of new domestic strains.
Photosynthesis is a process in which solar energy is converted into biochemically available electrochemical energy and PSI, PSII, cytochrome b 6 f and F-ATPase are four key protein super-complexes that carry out entire photosynthetic reactions in green plants, algae and cyanobacteria (Nelson and Ben-Shem 2004).PSI and PSII super-complexes protein molecules bind to chlorophyll and carotenoid molecules, allowing them to detect and absorb various light spectra and intensities (Nelson and Yocum 2006).A light-harvesting complex (LHC) allows light energy to be absorbed and directs it towards the reaction centre.The light-harvesting complex (LHC) also known as antenna system, is made up of wellorganized proteins, chlorophylls and carotenoids (Ruban 2015).These pigments improve the light-harvesting efficiency as a transformative change in a natural environment, where radiation from the sun frequently limits growth and competition for light with other organisms (Kirst and Melis 2014).Light captured by photosystem chlorophyll molecules and other pigments is transported to the photosynthetic reaction centre, where it further excites chlorophylls known as P680 and P700 to induce proton translocation (Nelson and Junge 2015).Although algae and higher plants have many common photosynthetic features, however there are significant differences in function and organization of several photosynthetic components, such as NPQ induction and state transitions components.In natural environment, light is captured more efficiently by larger photosystem II (PSII) light-harvesting antennae complexes, allowing photosynthetic organisms to compete with other photosynthetic organisms (Schenk et al. 2008).PSII super-complexes are formed by a primary reaction centre and a core antenna system with peripheral LHC trimers flanked by LHC monomers (Boekema et al. 1999;Kouřil et al. 2012;Tokutsu et al. 2012).These super-complexes prefer to cluster together to generate mega complexes, which are semicrystalline arrays (Kouřil et al. 2012).To shield PSII from excess irradiation, plants use the xanthophyll cycle to release excessive energy (Miyake et al. 2005;Yamori and Shikanai 2016).Most of the excess light that cells absorb in the natural environment triggers oxidative damage and photoinhibition, which is prevented by a photoprotective mechanism known as heat dissipation, also called non-photochemical quenching (NPQ) (Peers et al. 2009).However, NPQ drives the absorbed energy loss up to 80%, further decreasing the light use efficiency of cells.Those strains do not waste energy in low-light environments may be useful under Photo-Bio-Reactor (PBR) conditions which are heavily light-limited (Formighieri et al. 2012).Perin et al. (2015) stated that, strains having higher NPQ than the wild-type are probably not suitable for industrial purpose.
Because of the higher optical density of chlorophyll (Chl) content in cells, microalgal light usage efficacy in bioreactors is restricted by a sharp light gradient.Microalgae have evolved in their native habitat with limited light and inorganic sources, mainly iron, leading to low cell density; further, large antennae evolved around photosystems as a survival mechanism in order to enhance their capacity to absorb photons (Ort et al. 2011).As a result, wild-type algae present at surface layers absorb considerably more photons as they can employ for electron transport; moreover, due to higher optical density, the light penetration is restricted in the inner layers of PBR's (Powles 1984;Neale and Melis 1986;Münkel et al. 2013).Because of the non-homogeneous light penetration in PBRs, the system's productivity is low.These features suggest that choosing a mutant strain with lower pigment content per cell due to reduced antenna size or reduced density of photosynthetic units per cell can help light homogeneous penetration (Formighieri et al. 2012;Cazzaniga et al. 2014).As a result, cells in the outer layers absorb fewer photons while those in the interior layers receive more light, resulting in a faster growth rate (Polle et al. 2003).Development and characterization of the reduced antenna size strains by mutagenesis has been reported in several algal species, i.e.Chlamydomonas reinhardtii (Kirst and Melis 2014), Chlorella sorokiniana (Cazzaniga et al. 2014), Nannochloropsis (Peers et al. 2009) and Chlorella vulgaris (Shin et al. 2016).
In this study the algal cells were exposed to the chemical mutagen ethyl methanesulfonate (EMS) in order to induce random point mutations in the genome and generating mutants having lower pigment content.Pulse amplitude fluorimetry (PAM) was used to confirm the mutants' photosystem reduced antenna size and further check for biomass productivity.

EMS mutagenesis
Chlorella sorokiniana wild-type strain was obtained from the National Collection of Industrial Microorganisms (NCIM), CSIR-National Chemical Laboratary, Pune, India and grown in 50 mL TAP medium (Gorman and Levine 1965) at a light intensity of 50 µmol photons m −2 s −1 in 100 mL flasks at 25 ± 2 °C.A 1 mL sample log phase algal culture was harvested and washed three times with phosphate buffer (pH 7).The algal cells (1 mL) in TAP medium were treated with 50, 100, 150, 200, 250, 300 and 350 mM of ethyl methanesulfonate (EMS) mutagen and incubated at 25 ± 2 °C, 80 rpm for 2 h in the dark.The algal cells were centrifuged and rinsed three times with phosphate buffer before washing twice with TAP medium.The cultures were then kept in the dark for 3 h to avoid photoreactivation.The cells (100 µL) were plated on TAP agar medium and incubated in dark for 24 h before exposing to a 16:8 h light:dark photoperiod for 3 weeks.After mutant identification, individual colonies were selected and plated on a new TAP agar plate.The cell survival rate was estimated by comparing the proportion of macro colonies that survived against the mutagen dose to the percentage of not exposed colonies.

Screening for chlorophyll mutants
Reduced chlorophyll (pale phenotype) mutants were initially screened visually.Single colonies that appeared after three weeks were picked onto fresh TAP medium and allowed to grow in the light for two weeks.Strains still viable for phototrophic growth but significantly having lower chlorophyll content were selected for further analysis.To confirm whether the mutants had reduced antenna size, the colonies were tested initially for total Chl, Chl a, Chl b, carotenoid content and chlorophyll fluorescence.

Growth analysis, biomass and chlorophyll measurement
Both wild-type and mutant strains were grown in 100 mL of TAP broth at 25°Cunder 50 µmol photons m −2 s −1 irradiance with a 16:8 h light:dark photoperiod.After 24, 48, 72, and 96 h incubation, a Neubauer hemocytometer was used for cell counting.For biomass analysis, the cultures were grown in 1 L conical flasks with working volumes of 500 mL under 50, 100, 250, 500 µmol photons m −2 s −1 with a 16:8 h light:dark photoperiod.After 96 h the cultures were harvested by centrifuging at 3000 ×g for 5 min.The pellets were lyophilized (freeze dried) at 4°C for 8 h after being washed once with distilled water.The biomass (total dry weight) was determined by weighing them.
The Chl pigments were extracted in methanol using a 1 mL algal suspension culture that have been cultivated for four days and the pigments concentration was estimated by using the below equations (Arnon 1949;Porra et al. 1989); where A 663 , A 645 and A 470 are the absorbances at 663, 645 and 470 nm.

In-vivo chlorophyll fluorescence and photosynthetic parameters analysis by pulse amplitude modulation fluorometry
The algae were cultured in a 250 mL conical flask with a 100 mL working volume at 25 °C and 120 rpm rotation at 50 µmol photons m −2 s −1 irradiance with a 16:8 h light:dark photoperiod.The cell density was adjusted to 3 × 10 6 cells mL −1 and a Dual PAM 101 (Heinz Walz, Germany) was used to investigate the PSII functional antenna size, PSII functionality (actinic light ON and OFF), and PSII functionality at different photosynthetically active radiation (PAR).
The PSII antenna size was determined according to Cazzaniga et al. (2014).Briefly, 50 μM DCMU supplemented algal samples were dark adapted for 20 min and applied green light (15 µmol photons m −2 s −1 ) to induce variable fluorescence.The PSII functional antenna size was determined by using the reciprocal of time corresponding to two-thirds of the fluorescence rise (T 2/3 ) (Malkin et al. 1981).
To evaluate the PSII functionality, algal samples were dark adapted for 30 min and chlorophyll fluorescence was measured in vivo using a saturating light pulse (8000 µmol photons

Statistical analysis
Four to six biological replicates were used in each data presentation, and the values are expressed as mean ± standard error.One-way ANOVA was used in the statistical analysis.Statistical analyses used the software GraphPadInStat (GraphPad Software Inc., USA).

Isolation of Chlorella sorokiniana chlorophyll mutants (CSCM)
Chlorella sorokiniana wild-type strains were treated with different concentrations of EMS.The survival rates of C. sorokiniana cells were 98.12, 72.38, 9.21 and 3.14% at 50, 100, 150 and 200 mM of EMS concentrations, respectively.Colony formation was not observed at 250 mM and higher EMS concentrations.Thus 150 mM EMS concentration was selected for the generation of mutants.Approximately 3000 algal colonies were assessed based on the pigment phenotypic difference, wherein paler colonies were thought to have less chlorophyll content than the wild-type colonies.Several independent mutants with light green colour or paler were identified and sub-cultured in TAP agar medium for several generations and three mutants were selected and named as CSCM8, CSCM10 and CSCM21 (Fig. 1).These mutants were used for analysis of pigment content.The content of total chlorophyll was decreased by 33.34% in CSCM8, 46.93% in CSCM10 and 45.69% in CSCM21 compared to C. sorokiniana wildtype (Table 1).Furthermore, compared to wild-type, the Chl a/b ratio was increased by 46.76% in CSCM8 and 36.50% in CSCM10; however, it was decreased by 15.21% in CSCM21 mutants (Table 1).All mutants have a lower content of total carotenoids (Table 1).The quantum yield of PSII (F V /F M ) was not significantly (P < 0.05) affected in the mutants (Fig. 2).

Antenna size estimation in the selected pigment mutants
The PSII antenna size in the selected three mutants was determined using in vivo Chl a fluorescence.PSII functional antenna size was estimated by analysing the Chl a fluorescence rise in cell suspensions in the presence of DCMU (Fig. 2).The electron transport from PSII is blocked in DCMU treated cells and PSII reaction centres are saturated upon illuminating, leading to an increase in emitted fluorescence over time (Fig. 2).The fluorescence kinetics estimate the number of antenna complexes associated with each PSII because the time required to saturate all the reaction centres depends on the number of pigments capturing light and transmitting energy.The fluorescence rise was slower in the CSCM8, CSCM10 and CSCM21 mutants than in C. sorokiniana wild-type (Fig. 2).The T 2/3 of the in vivo Chl a fluorescence is inversely related to the functional antenna size of PSII, and the data suggest a reduction in Chl light-harvesting

Photosynthetic functionality of reduced antenna size mutants
The photosynthetic functionality of C. sorokiniana wildtype, CSCM8, CSCM10 and CSCM21 mutants was assessed using PAM fluorimetry at different photosynthetically active radiation irradiances (PAR).Chl fluorescence analysis was performed to assess the PSII efficiency to see if mutation affects the antenna system's ability to transmit absorbed energy to Reaction Centers (RCs).The PSII quantum yield Y(II) was not affected in mutants with reduced cellular pigments and reduced antenna size when compared to C. sorokiniana wild-type strain, implying that mutations were not detrimental to photosynthesis.The three mutants even displayed improved PSII effective quantum yield Y(II) compared to C. sorokiniana wild-type (Fig. 3A).We then analysed the photosynthetic electron transport rate (ETR) with increasing light intensity.With increasing irradiance all strains showed an increase in ETR up to 540 µmol photons m −2 s −1 , after which a further rise in irradiance did not affect ETR (Fig. 3B).The mutant strains not only acted the same way as wild-type strain but also had a higher ETR (Fig. 3B).Finally we measured non-photochemical quenching (NPQ) for heat dissipation of light energy received via the light-harvesting complex (LHC) and found that the mutants had significantly lower NPQ than the wild-type (Fig. 3C).Overall, the mutants demonstrated improved effective quantum yield Y(II) and decreased non-photochemical quenching (NPQ), which may contribute to improved biomass growth.

Photosynthetic regulation after the transition from dark to light
We examined changes in redox kinetics of the PSII primary electron donor (P680) upon transition from dark to actinic light (AL; 250 μmol photons m −2 s −1 ) (Fig. 4).After the transition from dark to AL for 10 s, the effective quantum yield of PSII (Y(II)) in the mutants were much higher than in the wild-type (Fig. 4A).Immediately after the onset of actinic illumination, the Y(II) values of the wild-type and mutants dropped from 0.8 to 0.1 and 0.25, respectively (Fig. 4A).Y(II) in the wild-type approached 0.4 at the end of the exposure interval, whereas the mutants had showed values close to 0.7; subsequently, Y(II) gradually reached to maximum (Fig. 4A).Y(NPQ) in the mutants was significantly lower than in the wild-type (Fig. 4B).Furthermore, having a lower Y(NPQ), mutants had lower quantum yield of non-regulated energy dissipation in PSII (Y(NO)) than the wild-type (Fig. 4C).

Characterization of mutants with reduced pigment and truncated antenna size for biomass production
We studied the growth of C. sorokiniana wild-type, CSCM8, CSCM10, and CSCM21 mutants to determine the beneficial effects of reduced antenna size.At 50 μmol photons m −2 s −1 the cell density was higher in the mutants than in the wild-type cultures (Fig. 5).Furthermore, the CSCM10, CSCM21, and CSCM8 mutants had 19.23, 25, and 34.61% higher DCW, respectively, than the wild-type at 50 μmol photons m −2 s −1 (Fig. 5).At 100 μmol photons m −2 s −1 the CSCM10, CSCM21, and CSCM8 mutants had 16.55, 23.44 and 32.75% higher DCW than the wild-type (Fig. 5).

Discussion
Autotrophic microalgae have been proposed as a potential substitute for plant-based bioenergy sources due to their efficient photosynthetic performance, rapid biomass turnover, and high carbohydrate and lipid contents (Milne et al. 1990;Shin et al. 2016).The non-homogeneous light penetration or steep light gradient caused by high optical density of the near-molar concentration of chlorophylls in cells which limits the light usage efficiency of microalgae in PBR's, and the inner layers are practically completely black (Melis 2009).
In search of novel mutant strains with improved optimum optical properties, we selected C. sorokiniana, a robust species with a strong market interest.It is still not possible to reliably manipulate specific genes in this species, such as TLA (Kirst et al. 2012) or ARSA1 (Formighieri et al. 2013) which have been connected to antenna size.A fluorescencebased video-imaging approach make the screening process effortless, allowing thousands of antenna mutants to be screened in less time (Cazzaniga et al. 2014;Perin et al. 2015).The screening of antenna mutants becomes timeconsuming without a fluorescence-based video-imaging technology.Due to the non-availability of such advanced technology in our laboratory, we adopted a manual method to screen pale green mutants and sub-cultured single colonies on agar TAP media and further grown in liquid media to estimate the chlorophyll content.We observed a range of paler green phenotypes in EMS-generated C. sorokiniana mutant colonies.We took advantage of the pale phenotype, presumed as first screening marker for the decreased antenna size as reported for C. reinhardtii tla mutants (Kirst and Melis 2014).Previous studies on C. sorokiniana, C. vulgaris, N. gaditana and C. saccharophila also used ethyl methanesulfonate (EMS)-mediated random mutagenesis followed by selection of reduced chlorophyll content and chlorophyll fluorescence for the optimal conversion of sunlight to algal biomass, and they have reported increased biomass productivity (Cazzaniga et al. 2014;Perin et al. 2015;Shin et al. 2016;Patil et al. 2020).
Ethyl methanesulfonate (CH 3 SO 2 OC 2 H 5 ) is an effective and widely used mutagen known to induce random mutations in the genome, majorly GC to AT transition point mutations (70-99%) (Sega 1984;Till et al. 2007) and sometimes loss of chromosome segments (Alcantara et al. 1996).We assessed different EMS dosages on C. sorokiniana wild-type and determined 150 mM mutagen concentration for the development of mutants (Tillich et al. 2012).Following EMS mutagenesis, several distinct colonies were selected based on visual screening.Of these, three mutant colonies with consistently reproducible phenotypes were chosen for characterization.The CSCM10, CSCM8 and CSCM21 mutants with pale green phenotype exhibited 47, 33 and 46% lower total chlorophyll content (decrease in both Chl a and b) compared to C. sorokiniana wild-type, respectively.Based on the analysis of Chl a/b and Chl/car ratio we assumed that CSCM8 and CSCM10 strains were considered to be the most suitable mutants.According to Kirst et al. (2012), strains with higher Chl a/b ratio have decreased peripheral LHC complexes.
The three potential mutants with reduced chlorophyll content were further analysed for photosynthesis functionality using a PAM fluorimeter.The measurement of chlorophyll fluorescence induction in algal cells in the presence of DCMU confirms that CSCM8, CSCM10, and CSCM21 mutants have a significantly reduced PSII antenna size than the wild-type C. sorokiniana.Unlike C. sorokiniana wild-type, the reduced antenna size containing CSCM8, CSCM10, and CSCM21 mutant strains have efficiently allowed homogeneous light distribution and safe dissipation of absorbed energy in dense cultures.Although reducing the size of PSII antenna is beneficial for increasing photosynthetic performance, which also results in increased photosensitivity (Cazzaniga et al. 2014).
The three mutants with lower cellular chlorophyll content and smaller antennas size exhibited an increased PSII maximum quantum yield compared to the wild-type, suggesting that the mutations were not detrimental to photosynthesis (Fig. 3A).The three mutants also had higher ETR than the wild-type (Fig. 3B).Another critical characteristic of photosynthetic organisms is their potential to dissipate light energy as heat, which has a significant impact on algal light use efficiency (Muller et al. 2001).The NPQ was lower in mutants than in the wild-type when measured over a range of irradiances (Fig. 3C).It is worth noting that NPQ competes with photochemistry, hence less dissipation of energy via NPQ in mutants is expected to benefit photosynthetic efficiency and may also be contributing to improve cellular growth conditions, especially when light intensity was non-saturating.
Further, we measured Y(NPQ) [quantum yield of light-induced non-photochemical fluorescence quenching], Y(II) [effective photochemical quantum yield of PS II], and Y(NO) [quantum yield of non-light-induced nonphotochemical fluorescence quenching] under transition conditions (Fig. 4).These three processes compete with each other, and the sum of these three parameters was one [Y(II) + Y(NPQ) + Y(NO) = 1] (Pfündel et al. 2008).Since the sum of photochemistry, dissipated heat or re-emitted fluorescence rate constants are constant, any increase in one process will reduce the yield of another (Maxwell et al. 2000).The Y(II) values of C. sorokiniana wild-type and mutants approached 0.4 and 0.7, respectively, immediately after the onset of actinic illumination, and this data clearly indicates that mutants had showed higher Y(II) values (Fig. 4A).Pfündel et al. (2008) have also reported similar phenomena like noticeably higher PSII photochemical yields [Y(II)] in Chl b less barley mutants.At the start of illumination, C. sorokiniana wild-type exhibit 0.8 Y(NO), and mutants had showed nearly 0.6 (Fig. 4C).When a total trans-thylakoid pH is not yet formed at the onset of illumination, the higher Y(NO) (produced by a higher fraction of closed PS II centres) is responsible for lower Y(II) and as a result, fluctuations in the pH-dependent Y(NPQ) dominate Y(NPQ) pattern Y(II) (Pfündel et al. 2008).The mutants characterized by reduced PSII antennae size have considerably low in non-photochemical energy dissipation.It was noted that Y(NPQ) and Y(II), which are mainly influenced by mutation in mutants, have significantly higher variability than Y(NO).Variations in regulated states may result from different LHCII acclimation states formed in response to subtle differences in samples and pre-treatment conditions (Pfündel et al. 2008).
The isolated novel reduced antenna size mutants of C. sorokiniana wild-type, have higher cell density, and higher dried cell weight (P < 0.05) (DCW or biomass) (Fig. 5).After 24, 48, 72 and 96 h incubation, the CSCM8, CSCM10 and CSCM 21 mutants had showed higher cell density (cell number), indicating that it outperformed the wild-type under artificial culture conditions.The DCW was measured particularly at later growth phase (96 h).These findings imply that generating mutants with smaller antennae can improve biomass conversion efficiency in C. sorokiniana.Several previous studies have also reported that improved biomass yield by optimizing optical features of algal cultures using genetic engineering or mutagenesis techniques (Polle et al. 2003;Cazzaniga et al. 2014;Shin et al. 2016;Patil et al. 2020) supporting the hypothesis that lower antenna size mutants can offer significant control over light harvesting and application in high-density cultures in artificial laboratory conditions.

Conclusion
Optimizing photon use efficiency is one of the primary objectives for increasing the productivity of algal biomass for biofuel production.EMS mutagenesis plays a crucial role in achieving the objective.In this paper, we describe the process of generation of C. sorokiniana mutants with reduced chlorophyll and reduced antenna size.The three mutant strains with lower chlorophyll content studied showed improved photosynthetic activity and higher biomass which can be attributed to reduced antenna size and reduction in non-photochemical quenching under laboratory condition where low light environment is provided.Furthermore, the identified mutants displayed a diverse range of phenotypes and had mutations in the photosynthetic apparatus.These mutants can be used to learn more about photosynthesis regulation in C. sorokiniana.

Fig. 1
Fig. 1 Visible phenotypic differences between CSCM8, CSCM10, and CRCM21 mutants of C. sorokiniana wild-type.A The samples were inoculated in TAP media and grown for seven days in continuous light (50 µmol photons m −2 s −1 ), after which they were equally

Fig. 5
Fig. 5 C. sorokiniana wild-type and their mutants grown at 25 °C and pH 7 under fluorescent light.(A) Cell density (cell number); (B to E) Dried cell weight (DCW) at different light conditions; Data

Table 1
Pigment content, PSII maximum quantum yield (F V /F M ) and functional antenna size of C.